U.S. patent application number 11/226892 was filed with the patent office on 2010-10-07 for method and apparatus for aligning microbeads in order to interrogate the same.
Invention is credited to Richard L. Lemoine, Martin A. Putnam.
Application Number | 20100255603 11/226892 |
Document ID | / |
Family ID | 46322633 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255603 |
Kind Code |
A9 |
Putnam; Martin A. ; et
al. |
October 7, 2010 |
Method and apparatus for aligning microbeads in order to
interrogate the same
Abstract
A method and apparatus are provided for aligning optical
elements or microbeads 8, wherein each microbead has an elongated
body with a code embedded therein along a longitudinal axis thereof
to be read by a code reading device. The microbeads 8 are aligned
with a positioning device (or cell) 500 having a plate or platform
200, 1252 with grooves 205, 1258 so the longitudinal axis of the
microbeads is positioned in a fixed orientation relative to the
code reading device. The microbeads 8 are typically cylindrically
shaped glass beads having a diffraction grating-based code embedded
in the bead 8 disposed along an axis, which requires a
predetermined alignment between the incident code readout laser
beam and the code readout detector in two of three rotational axes.
The geometry of the grooves 205 are designed to allow for easy
loading and unloading of beads from a cell, and the grooves 205 may
be straight or curved. Also, the cell may be segmented into regions
each associated with a different reaction or used for a different
identification process/application, and may have many different
geometries depending on the application.
Inventors: |
Putnam; Martin A.;
(Cheshire, CT) ; Lemoine; Richard L.; (Canton,
CT) |
Correspondence
Address: |
THE SMALL PATENT LAW GROUP LLP
225 S. MERAMEC, STE. 725T
ST. LOUIS
MO
63105
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20060063271 A1 |
March 23, 2006 |
|
|
Family ID: |
46322633 |
Appl. No.: |
11/226892 |
Filed: |
September 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10661836 |
Sep 12, 2003 |
7399643 |
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11226892 |
Sep 13, 2005 |
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10645689 |
Aug 20, 2003 |
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10661836 |
Sep 12, 2003 |
|
|
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11063665 |
Feb 22, 2005 |
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11226892 |
Sep 13, 2005 |
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60609583 |
Sep 13, 2004 |
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60610910 |
Sep 17, 2004 |
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60610833 |
Sep 17, 2004 |
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60410541 |
Sep 12, 2002 |
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60546435 |
Feb 19, 2004 |
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Current U.S.
Class: |
436/174 |
Current CPC
Class: |
G03H 2210/53 20130101;
B01L 2200/0668 20130101; B01L 2300/0654 20130101; B01J 2219/00466
20130101; B01J 2219/00702 20130101; G01N 15/147 20130101; B01J
2219/00459 20130101; G01N 2035/00574 20130101; B01J 2219/00545
20130101; B01J 2219/00463 20130101; G01N 35/00732 20130101; B01L
2200/0636 20130101; B01J 2219/00659 20130101; B01L 3/502761
20130101; G01N 2035/00782 20130101; G03H 1/0272 20130101; B01J
2219/005 20130101; Y10T 436/2575 20150115; B01J 2219/00648
20130101; B01J 2219/00657 20130101; B01L 2400/0409 20130101; G03H
2230/10 20130101; B01J 2219/00547 20130101; B01J 2219/0047
20130101; B01L 3/5085 20130101; B01L 2400/0457 20130101; Y10T
436/25 20150115; G03H 2270/20 20130101; B01J 2219/00576 20130101;
B01L 2400/0487 20130101; G03H 2270/24 20130101 |
Class at
Publication: |
436/174 |
International
Class: |
G01N 1/00 20060101
G01N001/00 |
Claims
1. A method for aligning microbeads to be read by a code reading or
other detection device, comprising the step of: providing
microbeads to a positioning device, each having an elongated body
with a code embedded therein along a longitudinal axis thereof;
aligning the microbeads with the positioning device so the
longitudinal axis of the microbeads is in a fixed orientation
relative to the code reading or other detection device.
2. A method according to claim 1, wherein the positioning device is
a plate having a multiplicity of grooves therein.
3. A method according to claim 1, wherein the method includes
agitating the plate to encourage the alignment of the microbeads in
the grooves.
4. A method according to claim 1, wherein the microbeads are
cylindrically shaped glass beads between 25 and 250 microns in
diameter and between 100 and 500 microns long.
5. A method according to claim 1, wherein the microbeads have a
holographic code embedded in a central region thereof.
6. A method according to claim 1, wherein the code is used to
correlate a chemical content on each bead with a measured
fluorescence signal.
7. A method according to claim 1, wherein each microbead is
substantially aligned in relation to its pitch and yaw rotational
axes.
8. A method according to claim 1, wherein the plate has a series of
parallel grooves having one of several different shapes, including
square, v-shaped or semi-circular.
9. A method according to claim 1, wherein the plate is an optically
transparent medium including boro-silicate glass, fused silica or
plastic, and the grooves are formed therein.
10. A method according to claim 1, wherein the grooves have a depth
that is dimensioned to be at least the diameter of the microbeads,
including at least 110% of the diameter of the microbead.
11. Apparatus for aligning microbeads to be read by a code reading
device, comprising: a positioning device for aligning microbeads,
each microbead having an elongated body with a code embedded
therein along a longitudinal axis thereof, so the longitudinal axis
of the microbeads is positioned in a fixed orientation relative to
the code reading device.
12. Apparatus according to claim 11, wherein the positioning device
is a plate having a multiplicity of grooves therein.
13. Apparatus according to claim 1, wherein the apparatus includes
means for agitating the plate to encourage the alignment of the
microbeads in the grooves.
14. Apparatus according to claim 1, wherein the microbeads are
cylindrically shaped glass beads between 25 and 250 microns in
diameter and between 100 and 500 microns long.
15. Apparatus for aligning an optical identification element,
comprising: the optical identification element having an optical
substrate having at least a portion thereof with at least one
diffraction grating disposed therein, the grating having at least
one refractive index pitch superimposed at a common location, the
grating providing an output optical signal when illuminated by an
incident light signal, the optical output signal being indicative
of a code, and the optical identification element being an
elongated object with a longitudinal axis; and an alignment device
which aligns the optical identification element such that said
output optical signal is indicative of the code.
16. Apparatus according to claim 15, wherein the alignment device
is a plate having a multiplicity of grooves therein.
17. Apparatus according to claim 15, wherein the plate is a disk
and the multiplicity of grooves are concentric circles or a
spiral.
18. Apparatus according to claim 15, wherein the alignment device
is a tube having a bore for receiving the optical identification
element.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims the benefit of: U.S.
Provisional Application Ser. No. 60/609,583, filed Sep. 13, 2004,
entitled "Improved Method and Apparatus for Aligning Microbeads in
Order to Interrogate Same" (Docket No. CV-0082 PR); Ser. No.
60/610,910, filed Sep. 17, 2004, entitled "Method and Apparatus for
Aligning Microbeads in Order to Interrogate Same" (Docket No.
CV-0086 PR); and Ser. No. 60/610,833, filed Sep. 17, 2004, entitled
"Method and Apparatus for Transporting and Kitting Microbeads"
(Docket No. CV-0087 PR); and is a continuation-in-part of: U.S.
patent application Ser. No. 10/661,836, filed Sep. 12, 2003,
entitled "Method And Apparatus For Aligning Microbeads In Order To
Interrogate The Same" (Docket No. CV-0042), and Ser. No.
11/063,665, filed Feb. 22, 2005, entitled "Multi-well Plate with
Alignment Grooves for Encoded Microparticles" (Docket No. CV-0053
US), all the above of which are incorporated herein by reference in
their entirety.
[0002] The following cases contain subject matter related to that
disclosed herein and are all incorporated herein by reference in
their entirety: U.S. patent application Ser. No. 10/661,234, filed
Sep. 12, 2003, entitled "Diffraction Grating-Based Optical
Identification Element", (Docket No. CV-0038A); Ser. No.
10/661,031, filed Sep. 12, 2003, entitled "Diffraction
Grating-Based Encoded Micro-particles for Multiplexed Experiments",
(Docket No. CV-0039A); Ser. No. 10/661,082, filed Sep. 12, 2003,
entitled "Method and Apparatus for Labeling Using Diffraction
Grating based Encoded Optical Identification Elements" (Docket No.
CV-0040); U.S. patent application Ser. No. 10/661,115, filed Sep.
12, 2003, entitled "Assay Stick" (Docket No. CV-0041); Ser. No.
10/661,254 filed Sep. 12, 2003, entitled "Chemical Synthesis Using
Diffraction Grating-Based Encoded Optical Elements" (Docket No.
CV-0043); U.S. patent application Ser. No. 10/661,116, filed Sep.
12, 2003, entitled "Method Of Manufacturing Of A Diffraction
Grating-Based Identification Element" (Docket No. CV-0044); and
U.S. patent application Ser. No. 10/763,995, filed Jan. 22, 2004,
entitled, "Hybrid Random Bead/Chip Based Microarray" (Docket No.
CV-0054); and U.S. patent application Ser. No. 10/956,791, filed
Oct. 1, 2004, entitled "Optical Reader for Diffraction
Grating-Based Encoded Optical Identification Elements" (Docket No.
CV-0092 US).
BACKGROUND OF THE INVENTION
[0003] 1. Technical Field
[0004] The present invention generally relates to a method and
apparatus for processing information contained on microbeads, each
microbead having an elongated body with a code embedded therein
along a longitudinal axis thereof to be read by a code reading
device; and more particularly to a method and apparatus for
aligning the microbeads so the longitudinal axis thereof is in a
fixed orientation relative to the code reading or other device.
[0005] This invention also relates to transporting beads, and more
particularly to transporting microbeads from one location to
another.
[0006] 2. Description of Related Art
[0007] Many industries have a need for uniquely identifiable
objects or for the ability to uniquely identify objects, for
sorting, tracking, and/or identification/tagging. Existing
technologies, such as bar codes, electronic
microchips/transponders, radio-frequency identification (RFID), and
fluorescence and other optical techniques, are often inadequate.
For example, existing technologies may be too large for certain
applications, may not provide enough different codes, or cannot
withstand harsh temperature, chemical, nuclear and/or
electromagnetic environments.
[0008] Therefore, it would be desirable to obtain a coding element
or platform that provides the capability of providing many codes
(e.g., greater than 1 million codes), that can be made very small
and/or that can withstand harsh environments.
[0009] Moreover, it would be desirable to provide a method and
apparatus to position and align such encoded elements so as to
identify the code to determine information about the process or
application to which it is related and/or to better sense the
chemical content on the elements and correlate it in relation to
such process or application.
[0010] It is also well known that microbeads or microparticles may
be used for various types of multiplexed chemical experiments or
assays or for identifying, authenticating or sorting items. One
challenge in transporting microbeads is being able to move them
reliably from one location to another reliably and/or being able to
move a predetermined number of beads.
[0011] Accordingly, it would be desirable to provide a reliable
technique for transporting microbeads from one location to
another.
SUMMARY OF THE INVENTION
[0012] In its broadest sense, the present invention provides a new
and unique method and apparatus for aligning new and unique coding
elements or microbeads, wherein each microbead has an elongated
body with a code embedded therein along a longitudinal axis thereof
to be read by a code reading or other detection device. The method
features the step of aligning the microbeads with a positioning
device so the longitudinal axis of the microbeads is positioned in
a fixed orientation relative to the code reading or other detection
device.
[0013] The new and unique microbeads are not spherical, but instead
have an elongated shape and may be cylindrical, cubic, rectangular,
or any other elongated shape. The microbeads are typically composed
of silica glass with some germanium and/or boron doped region or
regions that are photosensitive to ultraviolet light. Coded
microbeads are individually identifiable via a single or series of
spatially overlapping pitches written into them. The microbeads may
be used in many different processes. After such processing, the
microbeads have a resulting chemical content on the surface of each
bead that is sensed and correlated in relation to the code
contained with the microbead to determine information about the
process.
[0014] When used in an assay process, the microbeads are typically
cylindrically (i.e. tubular) shaped glass beads and between 25 and
250 .mu.m in diameter and between 100 and 500 .mu.m long. Other
sizes may be used if desired. They have a holographic code embedded
in the central region of the bead, which is used to identify it
from the rest of the beads in a batch of beads with many different
DNA or other chemical probes. A cross reference is used to
determine which probe is attached to which bead, thus allowing the
researcher to correlate the chemical content on each bead with the
measured fluorescence signal. Because the code consists of a
diffraction grating 12 typically disposed along an axis of the
microbead, there is a particular alignment required between the
incident readout laser beam and the readout detector in two of the
three rotational axes. In aeronautical terms, the two of the three
rotational axes include the pitch of the microbead in the
front-to-back direction and the yaw of the microbead in a
side-to-side direction. The third axis, rotation about the center
axis of the cylinder, is azimuthally symmetric and therefore does
not require alignment. The third axis is analogous to the roll
axis.
[0015] The invention provides a method for aligning the microbeads
in the two rotational axes to a fixed orientation relative to an
incident laser beam and a readout camera, otherwise known as the
code camera. The invention further provides a method for rapidly
aligning a large number of microbeads, between 1,000 and 1,000,000
microbeads or more, economically, and with the necessary
tolerances. The method is flexible as it relates to the size of the
microbeads and can be integrated into a fully automated system,
which prepares the microbeads for rapid readout by an automated
code-reading machine.
[0016] In one embodiment of the present invention, the positioning
device includes a plate with a series of parallel grooves (or
channels), which could have one of several different shapes,
including square, rectangular, v-shaped, semi-circular, etc., as
well as a flat bottom groove with tapered walls. The grooves are
formed into an optically transparent medium such as boro-silicate
glass, fused silica, or other glasses, or plastic or other
transparent support materials. The depth of the grooves will depend
on the diameter of the microbead but generally they will be between
10 and 125 .mu.m, but may be larger as discussed hereinafter,
depending on the application. The spacing of the grooves is most
optimal when it is between 1 and 2 times the diameter of the
microbead, providing for both maximum packing density as well as
maximum probability that a microbead will fall into a groove. The
width of grooves is most optimal when the gap between the microbead
and the walls of the grooves is sufficiently small to prevent the
microbeads from rotating within the grooves by more than a few
degrees. The bottom of the groove must also be maintained flat
enough to prevent the microbeads from rotating, by more than a few
tenths of a degree, relative to the incident laser beam. Another
critical aspect of the grooved plate is the optical quality of the
grooves. To prevent excess scatter of the readout laser beam, which
could lead to low contrast between the code signal and the
background scatter, it is important that the grooves exhibit high
optical quality. The beads can be read in the groove plate from
below, on top of, or the side of the plate, depending on the
application and type of microbead used.
[0017] Some advantages of the groove plate approach include:
[0018] Rapid simultaneous alignment of microbeads. Alignment rates
.about.1000's per second.
[0019] Once the microbeads are aligned, they can be read as many
times as necessary to get a good reading or improve statistics.
[0020] Microbeads naturally fall into groove (presumably by
capillary forces) at very high packing densities.
[0021] Microbeads can be mixed after reading then re-read to
enhance the statistics of readout process.
[0022] In an alternative embodiment of the present invention, the
positioning device may includes a tube having a bore for receiving,
aligning and reading the microbeads.
[0023] Moreover, the present invention also provides an apparatus
for aligning an optical identification element. The optical
identification element having an optical substrate having at least
a portion thereof with at least one diffraction grating disposed
therein, the grating having at least one refractive index pitch
superimposed at a common location, the grating providing an output
optical signal when illuminated by an incident light signal, the
optical output signal being indicative of a code, and the optical
identification element being an elongated object with a
longitudinal axis. The apparatus also having an alignment device
which aligns the optical identification element such that said
output optical signal is indicative of the code.
[0024] The present invention also provides an optical element
capable of having many optically readable codes. The element has a
substrate containing an optically readable composite diffraction
grating having one or more collocated index spacing or pitches
.LAMBDA.. The invention allows for a high number of uniquely
identifiable codes (e.g., millions, billions, or more). The codes
may be digital binary codes and thus are digitally readable or may
be other numerical bases if desired.
[0025] Also, the elements may be very small "microbeads" (or
microelements or microparticles or encoded particles) for small
applications (about 1-1000 microns), or larger "macroelements" for
larger applications (e.g., 1-1000 mm or much larger). The elements
may also be referred to as encoded particles or encoded threads.
Also, the element may be embedded within or part of a larger
substrate or object.
[0026] The code in the element is interrogated using free-space
optics and can be made alignment insensitive.
[0027] The gratings (or codes) are embedded inside (including on or
near the surface) of the substrate and may be permanent
non-removable codes that can operate in harsh environments
(chemical, temperature, nuclear, electromagnetic, etc.).
[0028] The code is not affected by spot imperfections, scratches,
cracks or breaks in the substrate. In addition, the codes are
spatially invariant. Thus, splitting or slicing an element axially
produces more elements with the same code. Accordingly, when a bead
is axially split-up, the code is not lost, but instead replicated
in each piece.
[0029] The invention is a significant improvement over prior art
bead movement techniques in being able to repeatably move a
predetermined number of beads from one location (or container or
well) to another location (or container or well). Also, the
invention provides for the reliable and repeatable transportation
of all beads from one container or well to another or from one well
to multiple wells using a "telegraph" technique. The invention is
useful for creating multiplexed bead kits having a required number
of beads of each code in a kit. The present invention may also be
used to move the beads from a container to a reader to allow for
the bead codes and/or chemistry on the beads to be read. The
invention may be used in any assay or multiplexed experiment,
combinatorial chemistry or biochemistry assay process, or in a
taggant application, or any other application where beads are in a
liquid solution and need to be transported, kitted and/or read.
[0030] Advantages of the "telegraph" technique of the present
invention are that it is low cost, fast, effective/reliable for
moving beads, and low precision is required. Advantages of the
pipetting techniques of the present invention is that the pippeter
is a standard off the shelf product, it is flexible to be used with
any type of well or container (e.g., sizes, shapes and other
characteristics), or other fluid configurations, and does not
require any sealing or physical connections to the wells.
[0031] The foregoing and other objects, features and advantages of
the present invention will become more apparent in light of the
following detailed description of exemplary embodiments
thereof.
BRIEF DESCRIPTION OF THE DRAWING
[0032] The drawing is not drawn to scale and includes the following
Figures:
[0033] FIG. 1 shows the steps of a microbead platform assay
process.
[0034] FIG. 2 is a side view of an optical identification element,
in accordance with the present invention.
[0035] FIG. 3 is a top level optical schematic for reading a code
in an optical identification element, in accordance with the
present invention.
[0036] FIG. 4 is a perspective view of a grooved plate for use with
an optical identification element, in accordance with the present
invention.
[0037] FIG. 5 is a diagram of the flat grooves and an example of
the dimensionality thereof in accordance with the present
invention.
[0038] FIG. 6 is a perspective view of a plate with holes for use
with an optical identification element, in accordance with the
present invention.
[0039] FIG. 7 is a perspective view of a grooved plate for use with
an optical identification element, in accordance with the present
invention.
[0040] FIG. 8 is a diagram of a microbead mapper reading, in
accordance with the present invention.
[0041] FIG. 8a is a diagram of a system for both detecting a
material on and reading a code in a microbead, in accordance with
the present invention.
[0042] FIG. 9 is a diagram of a plate having microbeads thereon in
relation to an open plate format for detection and reading of the
microbead in accordance with the invention.
[0043] FIG. 10 is a diagram of a starting point for handling
microbeads for readout in a cuvette process in accordance with the
invention.
[0044] FIG. 11 is a diagram of a second step in the readout process
in accordance with the invention.
[0045] FIG. 12 is a diagram of the readout step in accordance with
the invention.
[0046] FIG. 13 is a diagram of final steps in the cuvette process
in accordance with the invention.
[0047] FIG. 14 is a diagram of an example of the cuvette showing
its mount on a kinematic plate in accordance with the
invention.
[0048] FIG. 15 is a diagram of an alternative embodiment of a
cuvette showing a port for fluid filling/emptying using a pipette
in accordance with the invention.
[0049] FIG. 16 is a diagram of an alternative embodiment of a
cuvette showing an alternative port for fluid filling/emptying
using a pipette in accordance with the invention.
[0050] FIG. 17 is a diagram of a two zone cuvette showing a free
region and a trapped region in accordance with the invention.
[0051] FIG. 18(a) is a diagram of steps for a conventional flow
cytometer reader in a single pass cytometer process in accordance
with the invention.
[0052] FIG. 18(b) is a diagram of steps for a disk cytometer reader
in a multipass cytometer process in accordance with the
invention.
[0053] FIGS. 19(a), (b) and (c) show embodiments of a disk
cytometer in accordance with the invention.
[0054] FIG. 20(a) show an embodiment of a disk cytometer having
radial channels for spin drying in accordance with the
invention.
[0055] FIG. 20(b) show an alternative embodiment of a disk
cytometer having a mechanical iris for providing a variable
aperture for bead access to grooves in accordance with the
invention.
[0056] FIG. 21 show an embodiment of a SU8 groove plate having 450
in accordance with the invention.
[0057] FIG. 21 show an embodiment of a SU8 cylindrical grooved
plate having 450.times.65 microns beads in accordance with the
invention.
[0058] FIG. 22 show an embodiment of an alignment tube in
accordance with the invention.
[0059] FIG. 23 show an alternative embodiment of an alignment tube
having a receiving flange in accordance with the invention.
[0060] FIG. 24 is an optical schematic for reading a code in an
optical identification element, in accordance with the present
invention.
[0061] FIG. 25(a) is an image of a code on a CCD camera from an
optical identification element, in accordance with the present
invention.
[0062] FIG. 25(b) is a graph showing an digital representation of
bits in a code in an optical identification element, in accordance
with the present invention.
[0063] FIG. 26 illustrations (a)-(c) show images of digital codes
on a CCD camera, in accordance with the present invention.
[0064] FIG. 27 illustrations (a)-(d) show graphs of different
refractive index pitches and a summation graph, in accordance with
the present invention.
[0065] FIG. 28 is an alternative optical schematic for reading a
code in an optical identification element, in accordance with the
present invention.
[0066] FIG. 29 illustrations (a)-(b) are graphs of reflection and
transmission wavelength spectrum for an optical identification
element, in accordance with the present invention.
[0067] FIGS. 30-31 are side views of a thin grating for an optical
identification element, in accordance with the present
invention.
[0068] FIG. 32 is a perspective view showing azimuthal multiplexing
of a thin grating for an optical identification element, in
accordance with the present invention.
[0069] FIG. 33 is side view of a blazed grating for an optical
identification element, in accordance with the present
invention.
[0070] FIG. 34 is a graph of a plurality of states for each bit in
a code for an optical identification element, in accordance with
the present invention.
[0071] FIG. 35 is a side view of an optical identification element
where light is incident on an end face, in accordance with the
present invention.
[0072] FIGS. 36-37 are side views of an optical identification
element where light is incident on an end face, in accordance with
the present invention.
[0073] FIG. 38, illustrations (a)-(c), are side views of an optical
identification element having a blazed grating, in accordance with
the present invention.
[0074] FIG. 39 is a side view of an optical identification element
having a coating, in accordance with the present invention.
[0075] FIG. 40 is a side view of whole and partitioned optical
identification element, in accordance with the present
invention.
[0076] FIG. 41 is a side view of an optical identification element
having a grating across an entire dimension, in accordance with the
present invention.
[0077] FIG. 42, illustrations (a)-(c), are perspective views of
alternative embodiments for an optical identification element, in
accordance with the present invention.
[0078] FIG. 43, illustrations (a)-(b), are perspective views of an
optical identification element having multiple grating locations,
in accordance with the present invention.
[0079] FIG. 44, is a perspective view of an alternative embodiment
for an optical identification element, in accordance with the
present invention.
[0080] FIG. 45 is a view an optical identification element having a
plurality of gratings located rotationally around the optical
identification element, in accordance with the present
invention.
[0081] FIG. 46, illustrations (a)-(e), show various geometries of
an optical identification element that may have holes therein, in
accordance with the present invention.
[0082] FIG. 47, illustrations (a)-(c), show various geometries of
an optical identification element that may have teeth thereon, in
accordance with the present invention.
[0083] FIG. 48, illustrations (a)-(c), show various geometries of
an optical identification element, in accordance with the present
invention.
[0084] FIG. 49 is a side view an optical identification element
having a reflective coating thereon, in accordance with the present
invention.
[0085] FIG. 50, illustrations (a)-(b), are side views of an optical
identification element polarized along an electric or magnetic
field, in accordance with the present invention.
[0086] FIGS. 51 and 52 are diagrams of bead reads from flat
retro-reflector trays, in accordance with the present
invention.
[0087] FIGS. 53 and 54 are diagrams of beads read thru V-grooves,
in accordance with the present invention.
[0088] FIGS. 55-83 are various alternative embodiments of the
present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0089] FIG. 1 shows, by way of example, steps of a microbead assay
process which uses the microbead technology of the present
invention. The steps of the assay process shown in FIG. 1 include a
first step in which the microbeads are used in a solution; a second
step in which the microbeads are aligned in a desired manner; a
third step in which the code and florescence in and/or on the
microbeads are read-out; and a fourth step in which the information
related to the code and florescence is processed in relation to
data management and bioinformatics. The present invention primarily
relates to step 2 wherein the microbeads are uniquely aligned so
the longitudinal axis of the microbeads is positioned in a fixed
orientation relative to the code and florescence reading device, as
well as relating to a lesser extent to step 3. It is important to
note that the scope of the present invention is not intended to be
limited to any particular type or kind of assay process or other
process in which the microbead technology is used. The scope of the
invention is intended to include embodiments in which the microbead
technology of the present invention is used in many different
processes.
[0090] Other processes/applications where the present invention may
be used include use of the beads in taggant applications, where the
encoded beads are used to identify, track, and/or authenticate,
items such as is discussed in aforementioned copending U.S. patent
application Ser. No. 10/661,082, filed Sep. 12, 2003, entitled
"Method and Apparatus for Labeling Using Diffraction Grating-Based
Encoded Optical Identification Elements", (CyVera Docket No.
CV-0040).
FIG. 2: The Microbead Element 8
[0091] FIG. 2 shows a diffraction grating-based optical
identification element 8 (or encoded element or coded element) that
comprises a known optical substrate 10, having an optical
diffraction grating 12 disposed (or written, impressed, embedded,
imprinted, etched, grown, deposited or otherwise formed) in the
volume of or on a surface of the substrate 10 along the length or
longitudinal axis L of the element 8, which is otherwise known
hereinafter as the microbead. The grating 12 is a periodic or
aperiodic variation in the effective refractive index and/or
effective optical absorption of at least a portion of the substrate
10.
[0092] The optical identification element 8 described herein is
same as that described in Copending U.S. patent application Ser.
No. 10/661,234, filed Sep. 12, 2003, entitled "Diffraction
Grating-Based Optical Identification Element", which is
incorporated herein by reference in its entirety.
[0093] In particular, the substrate 10 has an inner region 20 where
the grating 12 is located. The inner region 20 may be
photosensitive to allow the writing or impressing of the grating
12. The substrate 10 has an outer region 18, which does not have
the grating 12 therein.
[0094] The grating 12 is a combination of one or more individual
spatial periodic sinusoidal variations (or components) in the
refractive index that are collocated at substantially the same
location on the substrate 10 along the length of the grating region
20, each having a spatial period (or pitch) .LAMBDA.. The resultant
combination of these individual pitches is the grating 12,
comprising spatial periods (.LAMBDA.1-.LAMBDA.n) each representing
a bit in the code. Thus, the grating 12 represents a unique
optically readable code, made up of bits, where a bit corresponds
to a unique pitch .LAMBDA. within the grating 12. Accordingly, for
a digital binary (0-1) code, the code is determined by which
spatial periods (.LAMBDA.1-.LAMBDA.n) exist (or do not exist) in a
given composite grating 12. The code or bits may also be determined
by additional parameters (or additional degrees of multiplexing),
and other numerical bases for the code may be used, as discussed
herein and/or in the aforementioned patent application.
[0095] The grating 12 may also be referred to herein as a composite
or collocated grating. Also, the grating 12 may be referred to as a
"hologram", as the grating 12 transforms, translates, or filters an
input optical signal to a predetermined desired optical output
pattern or signal.
[0096] The substrate 10 has an outer diameter D1 and comprises
silica glass (SiO.sub.2) having the appropriate chemical
composition to allow the grating 12 to be disposed therein or
thereon. Other materials for the optical substrate 10 may be used
if desired. For example, the substrate 10 may be made of any glass,
e.g., silica, phosphate glass, borosilicate glass, or other
glasses, or made of glass and plastic, or solely plastic. For high
temperature or harsh chemical applications, the optical substrate
10 made of a glass material is desirable. If a flexible substrate
is needed, plastic, rubber or polymer-based substrate may be used.
The optical substrate 10 may be any material capable of having the
grating 12 disposed in the grating region 20 and that allows light
to pass through it to allow the code to be optically read.
[0097] The optical substrate 10 with the grating 12 has a length L
and an outer diameter D1, and the inner region 20 diameter D. The
length L can range from very small "microbeads" (or microelements,
micro-particles, or encoded particles), about 1-1000 microns or
smaller, to larger "macro beads" or "macroelements" for larger
applications (about 1.0-1000 mm or greater). In addition, the outer
dimension D1 can range from small (less than 1000 microns) to large
(1.0-1000 mm and greater). Other dimensions and lengths for the
substrate 10 and the grating 12 may be used.
[0098] The grating 12 may have a length Lg of about the length L of
the substrate 10. Alternatively, the length Lg of the grating 12
may be shorter than the total length L of the substrate 10.
[0099] The outer region 18 is made of pure silica (SiO.sub.2) and
has a refractive index n2 of about 1.458 (at a wavelength of about
1553 nm), and the inner grating region 20 of the substrate 10 has
dopants, such as germanium and/or boron, to provide a refractive
index n1 of about 1.453, which is less than that of outer region 18
by about 0.005. Other indices of refraction n1,n2 for the grating
region 20 and the outer region 18, respectively, may be used, if
desired, provided the grating 12 can be impressed in the desired
grating region 20. For example, the grating region 20 may have an
index of refraction that is larger than that of the outer region 18
or grating region 20 may have the same index of refraction as the
outer region 18 if desired.
FIG. 3: The Code Reader or Detector 29
[0100] FIG. 3 shows a configuration for reading or detecting the
code in the microbead 8 using a code reader or other detector
device 29, which is used in step 3 of the process shown in FIG. 1.
In operation, an incident light 24 of a wavelength .lamda., e.g.,
532 nm from a known frequency doubled Nd:YAG laser or 632 nm from a
known Helium-Neon laser, is incident on the grating 12 in the
substrate 10. Any other input wavelength .lamda. can be used if
desired provided .lamda. is within the optical transmission range
of the substrate (discussed more herein and/or in the
aforementioned patent application). A portion of the input light 24
passes straight through the grating 12, as indicated by a line 25.
The remainder of the input light 24 is reflected by the grating 12,
as indicated by a line 27 and provided to a detector 29. The output
light 27 may be a plurality of beams, each having the same
wavelength .lamda. as the input wavelength .lamda. and each having
a different output angle indicative of the pitches
(.LAMBDA.1-.LAMBDA.n) existing in the grating 12. Alternatively,
the input light 24 may be a plurality of wavelengths and the output
light 27 may have a plurality of wavelengths indicative of the
pitches (.LAMBDA.1-.LAMBDA.n) existing in the grating 12.
Alternatively, the output light may be a combination of wavelengths
and output angles. The above techniques are discussed in more
detail herein and/or in the aforementioned patent application.
[0101] The code reader or detector 29 has the necessary optics,
electronics, software and/or firmware to perform the functions
described herein. In particular, the detector reads the optical
signal 27 diffracted or reflected from the grating 12 and
determines the code based on the pitches present or the optical
pattern, as discussed more herein or in the aforementioned patent
application. An output signal indicative of the code is provided on
a line 31.
[0102] The dimensions, geometries, materials, and material
properties of the substrate 10 are selected such that the desired
optical and material properties are met for a given application.
The resolution and range for the optical codes are scalable by
controlling these parameters as discussed herein and/or in the
aforementioned patent application. Also, the beads 8 may be made of
any of the materials, geometries, and coatings described in
copending U.S. patent application Ser. No. (Docket No.
CV-0038A).
[0103] We have used the present invention with cylindrical beads
having size of about 65 micron diameter and 400 microns long and
about 28 microns diameter and about 250 microns long. However,
other bead sizes may be used.
FIG. 4: The Grooved Tray or Plate
[0104] FIG. 4 shows one embodiment of a positioning device 200 for
aligning the microbeads 8 so the longitudinal axis of the
microbeads is in a fixed orientation relative to the code reading
or other detection device. The positioning device 200 is shown in
the form of a tray or plate 200 having v-grooves 205 for align the
microbeads 8 and is used in step 2 of the process shown in FIG.
1.
[0105] As shown, the microbead elements 8 are placed in the tray
200 with v-grooves 205 to allow the elements 8 to be aligned in a
predetermined direction for illumination and reading/detection as
discussed herein. Alternatively, the grooves 205 may have holes 210
that provide suction to keep the elements 8 in position.
Forming the Grooves in the Groove Plate
[0106] The grooves in the groove plate may be made in many
different ways, including being formed by SU8 photoresistant
material, mechanically machining; deep reactive ion etching; or
injection molding. One advantage of the injection molding approach
is that the plate can be manufactured in volume at relatively low
cost, and disposed of after the information about the beads is
gathered in the assay process. The groove plate may be made of
glass, including fused silica, low fluorescence glass, boro
silicate glass, or other transparent glasses or plastic. Silicon is
used because it is reflective so a reflective coating is typically
not needed. Alternative, a mirror coating can be applied to the
plate material to achieve the desired reflectivity.
FIG. 5: Flat Grooves
[0107] The scope of the invention is not intended to be limited to
any particular groove shape. For example, FIG. 5 shows a diagram a
plate 300 having flat grooves 302 instead of V-grooves as shown in
FIG. 3. Some characteristics of the groove according to the present
invention are as follows:
[0108] The groove width (w) should be at least as wide as the
diameter of the bead (D) but not larger than D+15 .mu.m.
[0109] The thickness of the depth of the groove (T) should be at
least 0.5 times the diameter of the bead so that it sufficiently
traps a bead once it falls into the groove even when it is
subjected to mechanical agitation. The depth should not exceed 1.5
times the diameter of the bead so as to prevent more than one bead
from falling into the same groove location.
[0110] Groove plates have been made using a thick photoresist
called SU8 and is available from Microchem. The resist is both
chemically inert and mechanically robust once fully cured. The
groove walls are formed by the resist material, which is deposited
onto a glass or substrate. Advantages of this process include the
ability to tailor the depth of groove by controlling the thickness
of the resist material, and virtually every other geometric
attribute through the design of the photo mask. Because it is
photolithographic process, essentially any shape profile can be
made. For example grooves can be made in simple rows, concentric
circles, or spirals. Other features such as discrete wells, spots
and cross hatches can be made as fiducial marks for tracking and
positional registration purposes.
[0111] The scope of the invention is also intended to include the
grooves having a flat bottom as shown in FIG. 5 with outwardly
tapered walls.
FIG. 6: The Holey Plate 674
[0112] FIG. 6 shows an alternative embodiment, wherein alignment
may be achieved by using a plate 674 having holes 676 slightly
larger than the elements 8 if the light 24 (FIGS. 2 and 4) is
incident along the grating axis 207. The incident light indicated
as 670 is reflected off the grating and exits through the end as a
light 672 and the remaining light passes through the grating and
the plate 674 as a line 678. Alternatively, if a blazed grating is
used, incident light 670 may be reflected out the side of the plate
(or any other desired angle), as indicated by a line 680.
Alternatively, input light may be incident from the side of the
plate 674 and reflected out the top of the plate 674 as indicated
by a line 684. The light 670 may be a plurality of separate light
beams or a single light beam 686 that illuminates the entire tray
674 if desired.
FIG. 7: V-Groove Plate 200 with End Illumination
[0113] FIG. 7 shows an alternative embodiment, wherein the v-groove
plate discussed hereinbefore with FIG. 4 may be used for the end
illumination/readout condition. In this case, the grating 12 may
have a blaze angle such that light incident along the axial grating
axis will be reflected upward, downward, or at a predetermined
angle for code detection. Similarly, the input light may be
incident on the grating in a downward, upward, or at a
predetermined angle and the grating 12 may reflect light along the
axial grating axis for code detection.
FIG. 8: Microbead Mapper Readings
[0114] FIG. 8 shows microbeads 8 arranged on a plate 200 having
grooves 205. As shown, the microbeads 8 have different codes (e.g.
"41101", "20502", "41125") using 16-bit, binary symbology), which
may be read or detected using the reader or detector configuration
described in relation to FIG. 3. The codes in the beads are used to
provide a cross reference to determine which probe is attached to
which bead, thus allowing the researcher to correlate the chemical
content on each bead with the measured fluorescence signal in Step
3 of the process shown in FIG. 1.
[0115] FIG. 8a shows a code reader and detector for obtaining
information from the microbead 8 in FIG. 8. The codes in the
microbeads 8 are detected when illuminated by incident light 24
which produces a diffracted or output light signal 27 to a reader
820, which includes the optics and electronics necessary to read
the codes in each bead 8, as described herein and/or in the
aforementioned copending patent application. The reader 820
provides a signal on a line 822 indicative of the code in each of
the bead 8. The incident light 24 may be directed transversely from
the side of the grooved plate 200 (or from an end or any other
angle) with a narrow band (single wavelength) and/or multiple
wavelength source, in which case the code is represented by a
spatial distribution of light and/or a wavelength spectrum,
respectively, as described hereinafter and in the aforementioned
copending patent application. Other illumination, readout
techniques, types of gratings, geometries, materials, etc. may be
used for the microbeads 8, as discussed hereinafter and in the
aforementioned patent application.
[0116] For assays that use fluorescent molecule markers to label or
tag chemicals, an optical excitation signal 800 is incident on the
microbeads 8 on the grooved plate 200 and a fluorescent optical
output signal 802 emanates from the beads 8 that have the
fluorescent molecule attached. The fluorescent optical output
signal 802 passes through a lens 804, which provides focused light
802 to a known optical fluorescence detector 808. Instead of or in
addition to the lens 802, other imaging optics may be used to
provide the desired characteristics of the optical image/signal
onto the fluorescence detector 808. The detector 808 provides an
output signal on a line 810 indicative of the amount of
fluorescence on a given bead 8, which can then be interpreted to
determine what type of chemical is attached to the bead 10.
[0117] Consistent with that discussed herein, the grooved plate 200
may be made of glass or plastic or any material that is transparent
to the code reading incident beam 24 and code reading output light
beams 27 as well as the fluorescent excitation beam 800 and the
output fluorescent optical signal 802, and is properly suited for
the desired application or experiment, e.g., temperature range,
harsh chemicals, or other application specific requirements.
[0118] The code signal 822 from the bead code reader 820 and the
fluorescent signal 810 from the fluorescence detector are provided
to a known computer 812. The computer 812 reads the code associated
with each bead and determines the chemical probe that was attached
thereto from a predetermined table that correlates a predetermined
relationship between the bead code and the attached probed. In
addition, the computer 812 and reads the fluorescence associated
with each bead and determines the sample or analyte that is
attached to the bead from a predetermined table that correlates a
predetermined relationship between the fluorescence tag and the
analyte attached thereto. The computer 812 then determines
information about the analyte and/or the probe as well as about the
bonding of the analyte to the probe, and provides such information
on a display, printout, storage medium or other interface to an
operator, scientist or database for review and/or analysis,
consistent with shown in step 4 of FIG. 1. The sources 801, 803 the
code reader 820, the fluorescence optics 804 and detector 808 and
the computer 812 may all be part of an assay stick reader 824.
[0119] Alternatively, instead of having the code excitation source
801 and the fluorescence excitation source 803, the reader 24 may
have only one source beam which provides both the reflected optical
signal 27 for determining the code and the fluorescence signal 802
for reading the tagged analyte attached to the beads 8. In that
case the input optical signal is a common wavelength that performs
both functions simultaneously, or sequentially, if desired.
[0120] The microbeads 8 may be coated with the desired probe
compound, chemical, or molecule prior to being placed in the
grooved plate 200. Alternatively, the beads 8 may be coated with
the probe after being placed in the grooved plate 200. As discussed
hereinbefore, the probe material may be an Oligo, cDNA, polymer, or
any other desired probe compound, chemical, cell, or molecule for
performing an assay.
[0121] The scope of the invention is not intended to be limited to
using or detecting fluorescent molecule markers during the assay
process. For example, embodiments of the invention are envisioned
using and detection other types of molecular markers in other types
of processes.
Modes of Microbead Alignment
[0122] There are at least two possible modes or approaches of use
for the groove plate.
FIG. 9: Open Format Approach
[0123] FIG. 9 shows the first, or open plate format, meaning there
is no top to cover the microbeads 8 and the v-grooves 205. In this
mode, the microbeads 8 are dispensed onto the plate 200 using, for
example, a pipette tip or syringe tip, although the scope of the
invention is not intended to be limited to the manner of depositing
the microbeads on the plate. The microbeads 8 may be then agitated
by a sonic transducer (not shown), or manipulated with a mechanical
wiper (not shown) or some form of spray nozzle (not shown) to
encourage all the microbeads 8 to line up in the grooves 205. It
has been observed that substantially all the microbeads naturally
line up in the grooves 205 without the need for encouragement.
However, there are always some microbeads, such as microbead 8a,
8b, that do not fall naturally into the grooves, and these must
either be removed from the plate 200 or forced to fall into a
groove 205. The open format approach has the advantages that
grooves plate consists just of the plate and no other complicated
features such as walls and a top, and possibly other chambers or
channels to allow fluid flow and bubble removal. It also has the
advantage that it can easily be made with a standard microscope
slide, which is designed to fit conventional micro array readers or
microscopes. However, the open format approach would most likely
require the microbeads to be dried out prior to reading, to prevent
non-uniform and unpredictable optical aberrations caused by the
uneven evaporation of the buffer solution.
FIGS. 10-17: The Closed Format Approach
[0124] FIGS. 10-17 show the second mode which is called a closed
format, that consists of not only of a groove plate but also a top
and at least three walls to hold the solution and the microbeads in
a cuvette-like device (or cell or chamber) generally indicated as
500 shown, for example, in FIG. 10.
[0125] In summary, the closed format approach provides a method for
effectively distributing and aligning microbeads during the readout
process, as described below:
[0126] The basic process for handling microbeads with a curvette
for readout consists of the following steps:
[0127] (1) FIG. 10 shows a starting point for handling microbeads
for a readout. The microbeads start in a test tube. Typical
test-tube volumes are 1.5 ml. The microbeads will generally be in a
liquid (usually water with a small amount of other buffer chemicals
to adjust pH and possibly a small amount [.about.0.01%] of
detergent.) As shown, a bead tube 502 contains the microbeads in a
solution, which forms part of step 1 of the process shown in FIG.
1.
[0128] (2) FIG. 11 shows the bead tube 502 is coupled to a flange
504 of the cuvette 500 is inverted and the beads flow onto the
groove plate. The cuvette consists of two round flanges that accept
test-tubes, a transparent window, and an opposing groove plate.
FIG. 14 shows a drawing of a prototype cuvette. The groove plate
outer dimensions can be any size, but typical microscope slide
dimensions are convenient (1''.times.3''). The grooves are
mechanically or laser cut lengthwise, and have dimensions that are
chosen for the exact size of cylindrical microbead. For instance,
for a 125 .mu.m diameter bead, grooves of approximately 150 .mu.m
wide by 150 .mu.m deep are used. One tube carries the microbeads
and a small amount of carrier fluid. The second tube may be larger
and hold more fluid. The purpose of the second tube is to guarantee
a certain fluid level in the next step.
[0129] (3) After the cuvette is inverted and the microbeads flow
out onto the groove plate side of the cuvette, the microbeads
naturally align in the grooves via a small amount of rocking or
agitation, which forms part of step 2 of the process shown in FIG.
1.
[0130] (4) FIG. 12 shows the readout step, in which, after the
beads are all (or nearly all) aligned in the groove plate, the
entire plate is moved (or the readout laser beam is scanned) in
order to read the codes of each beam, which forms part of step 3 of
the process shown in FIG. 1. In effect, once the microbeads are in
the grooves, the entire cuvette is moved back and forth across a
readout beam. The readout beam is transmitted through the cuvette
and contains the code bits encoded on the scattering angles.
[0131] (5) FIG. 13 shows a final step, in which the cuvette is
inverted to its original position and the beads flow back into the
original tube 502, which forms part of step 3 of the process shown
in FIG. 1. In other words, after the readout process, the cuvette
is re-inverted and the microbeads flow back into the original test
tube.
[0132] FIG. 14 shows an example of a cuvette generally indicated as
700 that is mounted on a kinematic base plate 710. As shown, the
cuvette 700 has a tube 702 for holding the solution with the beads
and a top window 704 that is a 1 mm thick glass plate having
dimensions of about 1'' by 3''. The cuvette also has a bottom plate
that is a transparent groove plate. The location pins 712 and lever
arm 714 hold the cuvette 700 in place on the kinematic plate
710.
[0133] One of the key advantages of using the cuvette device is
that the potential to nearly index match the glass microbeads with
a buffer solution thereby reducing the divergence of the laser beam
caused by the lensing effect of the microbeads, and minimizing
scatter form the groove plate itself.
[0134] Another advantage involves the potential to prevent
microbeads from ever stacking up on top of each other, by limiting
the space between the bottom and the top plate to be less than
twice the diameter of the microbeads.
[0135] Another advantage is that the cover keeps the fluid from
evaporating.
FIGS. 15-16
[0136] FIGS. 15-16 show alternative embodiments of the cuvette
shown in FIGS. 10-14. As shown, the microbeads are injected into
the cuvette by placing them near the edge of the opening and
allowing the surface tension, or an induced fluid flow, to pull the
microbeads into the cuvette, where, because of the limited height
between the floor and the ceiling of the cuvette, they are confined
to move around in a plane, albeit with all the rotational degrees
of freedom unconstrained. Once in the cuvette the microbeads are
quickly and sufficiently constrained by the grooves as the
microbeads fall into them. As in the case of the open format there
is still the finite probability that some number of microbeads will
not fall into the grooves and must be coaxed in by some form of
agitation (ultrasonic, shaking, rocking, etc.).
FIG. 17: Two Region Approach
[0137] FIG. 17 shows an alternative embodiment of the closed
approach, which involves sectioning the closed region into two
regions, one where the microbeads are free to move about in a
plane, either in a groove or not, and a second region where the
microbeads are trapped in a groove and can only move along the axes
of a groove. Trapping the microbeads in a groove is accomplished by
further reducing the height of the chamber to the extent that the
microbeads can no longer hop out of a groove. In this embodiment,
the free region is used to pre-align the microbeads into a groove,
facilitating the introduction of microbeads into the trapped
section. By tilting this type of cuvette up gravity can be used to
pull the microbeads along a groove from the free region to the
trapped region. Once in the trapped region the microbeads move to
the end of the groove where they stop. Subsequent microbeads will
begin to stack up until the groove is completely full of
microbeads, which are stacked head to tail. This has the advantage
of packing a large number of microbeads into a small area and
prevents the microbeads from ever jumping out of the grooves. This
approach could also be used to align the microbeads prior to
injection into some form of flow cytometer, or a dispensing
apparatus.
FIGS. 18-23: The Cytometer
[0138] FIGS. 18-23 show method and apparatus related to using a
cytometer.
[0139] FIG. 18(a) shows steps for a method related to a
conventional (single pass) flow cytometer reader and FIG. 18(b)
shows a method related to a disk cytometer reader (multipass).
[0140] In FIG. 18(a), the method generally indicated as 900 has a
step for providing beads and a solution similar to step 1 in FIG.
1; and a step for reading information from the beads similar to
steps 2 and 3 in FIG. 1.
[0141] In FIG. 18(b), the method generally indicated as 1000 has a
step for providing beads and solution similar to step 1 in FIG. 1;
and a step for spinning and reading information from the beads
similar to steps 2 and 3 in FIG. 1.
[0142] In the methods shown in FIGS. 18(a) and (b), a rotating disk
(see FIGS. 19(a), (b) and (c) and 20) is used for aligning the
microbeads consistent with step 2 of the process shown in FIG.
1.
[0143] FIG. 19(a) shows an embodiment of a cytometer bead reader
having a rotating disk generally indicated as 1250, having a disk
platform 1252 with circumferential, concentric, grooves 1254 for
aligning microbeads 8. As shown, the rotating disk 1250 has various
sectors for processing the microbeads, including a bead loading
zone 1256, a bead removal zone 1258 and a readout zone 1260, as
well as a barrier 1259 for preventing the microbeads from flying
off the plate. As shown, a window 1262 for reading the beads is in
contact with the fluid containing the beads.
[0144] FIG. 19(b) shows an alternative embodiment of a rotating
disk generally indicated as 1200, having a disk platform 1202 with
planar groove plates 1204a, b, c, d, e, f that are shown with
grooves oriented in any one or more different ways. One or more of
the planar groove plates 1204a, b, c, d, e, f may have an optional
channel for fluid run-off, as shown, and a barrier (FIG. 19(a)) for
preventing the microbeads from flying off the plate. All other
attributes may be the same as described in FIG. 19(a). A window
1263 may be used for loading and/or reading the beads on the groove
plates 1204a, b, c, d, e, f.
[0145] FIG. 19(c) shows an alternative embodiment of a rotating
disk generally indicated as 1280, having a disk platform 1282 with
radial grooves 1284a, 1284b. The disk platform 1282 has a bead
loading zone 1286 in the center of the disk. One advantage of this
embodiment is that the opening of the bead loading zone 1286 will
also serve to allow the release of air bubbles that will naturally
collect in the center of the disk due the reduced density of the
fluid, which results from the centrifugal force pushing the fluid
radially outwardly. The rotating disk 1280 has tight bead packing
due to the centrifugal forces due to the spinning action of the
disk. The rotating disk 1280 has a wedge shape spacer 1288 that
keeps the channel at a constant gap width and a wall 1290.
[0146] FIG. 20(a) shows an alternative embodiment of a rotating
disk generally indicated as 1300 having narrow radial channels 1302
for spin drying so liquid is forced out of the circumferential
grooves through the radial channels. The plate 1300 may have a
mechanical catcher 1320 coupled thereto for moving radially
outwardly in direction 1320a if desired, for recirculating loose
beads.
[0147] FIG. 20(b) show an alternative embodiment of a disk
cytometer 1400 having a mechanical iris 1402 for providing a
variable aperture for bead access to grooves in accordance with the
invention.
[0148] FIG. 21 shows a rotating groove plate having 450 by 65
microns beads arranged in the rotating SU8 circumferential
channels.
[0149] For any of the circular groove plates shown herein, the disk
may rotate as discussed above and/or the reader excitation
laser(s)/detector(s) may rotate to read the code and/or the
fluorescence on the beads 8.
Continuous Mode--Process Steps
[0150] The following are the processing steps for a continuous mode
of operation:
[0151] 1. Dispense batch of microbeads onto plate.
[0152] 2. Spin slowly while agitating the plate theta x and y to
get microbeads into grooves. The agitation can be performed using
rocking, ultrasound, airflow, etc.
[0153] 3. Once sufficient number of microbeads are in grooves, spin
up plate to remove excess microbeads (microbeads that did not go
into a groove).
[0154] 4. Spin disk to read code and fluorescence.
[0155] 5. To remove microbeads, purge with high velocity aqueous
solution (enough to knock microbeads out of groove) and vacuum up,
or spin microbeads off plate while they are not in a groove.
[0156] 6. Inspect disk (probably with code camera) to verify that
all microbeads have been removed.
[0157] 7. Inject next batch of microbeads.
FIGS. 22-23: The Alignment Tube 502
[0158] In FIG. 22, instead of a flat grooved plate 200 (FIG. 3),
the microbeads may be aligned in a tube 502 that has a diameter
that is only slightly larger than the substrate 10, e.g., about
1-50 microns, and that is substantially transparent to the incident
light 24. In that case, the incident light 24 may pass through the
tube 502 as indicated by the light 500 or be reflected back due to
a reflective coating on the tube 502 or the substrate as shown by
return light 504. Other techniques can be used for alignment if
desired.
[0159] FIG. 23 shows the tube 502 has an opening flange 512 for
receiving the microbeads. FIG. 23 also shows an excitation laser
550, a diode laser 552 and a CCD camera 554 for gathering
information from the bead 8 consistent with that discussed above.
If desired, the beads 8 may be aligned and flowed through the tube
502 (similar to that discussed with FIG. 18(a) flow cytometer). In
that case, fluid (liquid and/or gas) may flow through the tube 508
to move the beads 8 along the tube 502, using a flow cytometer
approach.
FIGS. 24-44: Reading the Microbead Code and Alternative
Embodiments
[0160] FIGS. 24-44 provide a method and apparatus for reading the
code in the microbeads 8, as well as a more detailed description of
the microbeads 8 and certain alternative embodiments therefore. The
scope of the invention is not intended to be limited in any way to
the manner in which the code is read, or the method of doing the
same.
[0161] Referring to FIG. 24, The reflected light 27, comprises a
plurality of beams 26-36 that pass through a lens 37, which
provides focused light beams 46-56, respectively, which are imaged
onto a CCD camera 60. The lens 37 and the camera 60, and any other
necessary electronics or optics for performing the functions
described herein, make up the reader 29. Instead of or in addition
to the lens 37, other imaging optics may be used to provide the
desired characteristics of the optical image/signal onto the camera
60 (e.g., spots, lines, circles, ovals, etc.), depending on the
shape of the substrate 10 and input optical signals. Also, instead
of a CCD camera other devices may be used to read/capture the
output light.
[0162] Referring to FIG. 25, the image on the CCD camera 60 is a
series of illuminated stripes indicating ones and zeros of a
digital pattern or code of the grating 12 in the element 8.
Referring to FIG. 26, lines 68 on a graph 70 are indicative of a
digitized version of the image of FIG. 25 as indicated in spatial
periods (.LAMBDA.1-.LAMBDA.n).
[0163] Each of the individual spatial periods (.LAMBDA.1-.LAMBDA.n)
in the grating 12 is slightly different, thus producing an array of
N unique diffraction conditions (or diffraction angles) discussed
more hereinafter. When the element 8 is illuminated from the side,
in the region of the grating 12, at an appropriate input angle,
e.g., about 30 degrees, with a single input wavelength .lamda.
(monochromatic) source, the diffracted (or reflected) beams 26-36
are generated. Other input angles .theta.i may be used if desired,
depending on various design parameters as discussed herein and/or
in the aforementioned patent application, and provided that a known
diffraction equation (Eq. 1 below) is satisfied:
sin(.theta..sub.i)+sin(.theta..sub.o)=m.lamda./n.LAMBDA. Eq. 1
where Eq. 1 is diffraction (or reflection or scatter) relationship
between input wavelength .lamda., input incident angle .theta.i,
output incident angle .theta.o, and the spatial period .LAMBDA. of
the grating 12. Further, m is the "order" of the reflection being
observed, and n is the refractive index of the substrate 10. The
value of m=1 or first order reflection is acceptable for
illustrative purposes. Eq. 1 applies to light incident on outer
surfaces of the substrate 10 which are parallel to the longitudinal
axis of the grating (or the k.sub.B vector). Because the angles
.theta.i,.theta.o are defined outside the substrate 10 and because
the effective refractive index of the substrate 10 is substantially
a common value, the value of n in Eq. 1 cancels out of this
equation.
[0164] Thus, for a given input wavelength .lamda., grating spacing
.LAMBDA., and incident angle of the input light .theta.i, the angle
.theta.o of the reflected output light may be determined. Solving
Eq. 1 for .theta.o and plugging in m=1, gives:
.theta.o=sin.sup.-1(.lamda./.LAMBDA.-sin(.theta.i)) Eq. 2 For
example, for an input wavelength .lamda.=532 nm, a grating spacing
.LAMBDA.=0.532 microns (or 532 nm), and an input angle of incidence
.theta.i=30 degrees, the output angle of reflection will be
.theta.o=30 degrees. Alternatively, for an input wavelength
.lamda.=632 nm, a grating spacing .LAMBDA.=0.532 microns (or 532
nm), and an input angle .theta.i of 30 degrees, the output angle of
reflection .theta.o will be at 43.47 degrees, or for an input angle
.theta.i=37 degrees, the output angle of reflection will be
.theta.o=37 degrees. Any input angle that satisfies the design
requirements discussed herein and/or in the aforementioned patent
application may be used.
[0165] In addition, to have sufficient optical output power and
signal to noise ratio, the output light 27 should fall within an
acceptable portion of the Bragg envelope (or normalized reflection
efficiency envelope) curve 200, as indicated by points 204,206,
also defined as a Bragg envelope angle .theta.B, as also discussed
herein and/or in the aforementioned patent application. The curve
200 may be defined as: I .function. ( ki , ko ) .apprxeq. [ KD ] 2
.times. sin .times. .times. c 2 .function. [ ( ki - ko ) .times. D
2 ] Eq . .times. 3 ##EQU1## where K=2.pi..delta.n/.lamda., where,
.delta.n is the local refractive index modulation amplitude of the
grating and .lamda. is the input wavelength, sinc(x)=sin(x)/x, and
the vectors k.sub.i=2.pi.cos(.theta..sub.i)/.lamda. and
k.sub.o=2.pi.cos (.theta..sub.o)/.lamda. are the projections of the
incident light and the output (or reflected) light, respectively,
onto the line 203 normal to the axial direction of the grating 12
(or the grating vector k.sub.B), D is the thickness or depth of the
grating 12 as measured along the line 203 (normal to the axial
direction of the grating 12). Other substrate shapes than a
cylinder may be used and will exhibit a similar peaked
characteristic of the Bragg envelope. We have found that a value
for .delta.n of about 10.sup.-4 in the grating region of the
substrate is acceptable; however, other values may be used if
desired.
[0166] Rewriting Eq. 3 gives the reflection efficiency profile of
the Bragg envelope as: I .function. ( ki , ko ) .apprxeq. [ 2
.times. .pi. .delta. .times. .times. n D .lamda. ] 2 .function. [
Sin .function. ( x ) x ] 2 .times. .times. where: .times. .times. x
= ( ki - ko ) .times. D / 2 = ( .pi. .times. .times. D / .lamda. )
* ( cos .times. .times. .theta. .times. .times. i - cos .times.
.times. .theta. .times. .times. o ) Eq . .times. 4 ##EQU2##
[0167] Thus, when the input angle .theta.i is equal to the output
(or reflected) angle .theta..sub.o (i.e., .theta.i=.theta..sub.o),
the reflection efficiency I (Eqs. 3 & 4) is maximized, which is
at the center or peak of the Bragg envelope. When
.theta.i=.theta.o, the input light angle is referred to as the
Bragg angle as is known. The efficiency decreases for other input
and output angles (i.e., .theta.i # .theta.), as defined by Eqs. 3
& 4. Thus, for maximum reflection efficiency and thus output
light power, for a given grating pitch .LAMBDA. and input
wavelength, the angle .theta.i of the input light 24 should be set
so that the angle .theta.o of the reflected output light equals the
input angle .theta.i.
[0168] Also, as the thickness or diameter D of the grating
decreases, the width of the sin(x)/x function (and thus the width
of the Bragg envelope) increases and, the coefficient to or
amplitude of the sinc.sup.2 (or (sin(x)/x).sup.2 function (and thus
the efficiency level across the Bragg envelope) also increases, and
vice versa. Further, as the wavelength .lamda. increases, the
half-width of the Bragg envelope as well as the efficiency level
across the Bragg envelope both decrease. Thus, there is a trade-off
between the brightness of an individual bit and the number of bits
available under the Bragg envelope. Ideally, .delta.n should be
made as large as possible to maximize the brightness, which allows
D to be made smaller.
[0169] From Eq. 3 and 4, the half-angle of the Bragg envelope
.theta..sub.B is defined as: .theta. B = .eta. .times. .times.
.lamda. .pi. .times. .times. D .times. .times. sin .function. (
.theta. i ) Eq . .times. 5 ##EQU3## where .eta. is a reflection
efficiency factor which is the value for x in the sinc.sup.2(x)
function where the value of sinc.sup.2(x) has decreased to a
predetermined value from the maximum amplitude as indicated by
points 204,206 on the curve 200.
[0170] We have found that the reflection efficiency is acceptable
when .eta..ltoreq.1.39. This value for .eta. corresponds to when
the amplitude of the reflected beam (i.e., from the sinc.sup.2(x)
function of Eqs. 3 & 4) has decayed to about 50% of its peak
value. In particular, when x=1.39=.eta., sinc.sup.2(x)=0.5.
However, other values for efficiency thresholds or factor in the
Bragg envelope may be used if desired.
[0171] The beams 26-36 are imaged onto the CCD camera 60 to produce
the pattern of light and dark regions 120-132 representing a
digital (or binary) code, where light=1 and dark=0 (or vice versa).
The digital code may be generated by selectively creating
individual index variations (or individual gratings) with the
desired spatial periods .LAMBDA.1-.LAMBDA.n. Other illumination,
readout techniques, types of gratings, geometries, materials, etc.
may be used as discussed in the aforementioned patent
application.
[0172] Referring to FIG. 26, illustrations (a)-(c), for the grating
12 in a cylindrical substrate 10 having a sample spectral 17 bit
code (i.e., 17 different pitches .LAMBDA.1-.LAMBDA.17), the
corresponding image on the CCD (Charge Coupled Device) camera 60 is
shown for a digital pattern of 7 bits turned on
(10110010001001001); 9 bits turned on of (1000101010100111); all 17
bits turned on of (11111111111111111).
[0173] For the images in FIG. 26, the length of the substrate 10
was 450 microns, the outer diameter D1 was 65 microns, the inner
diameter D was 14 microns, .delta.n for the grating 12 was about
10.sup.-4, n1 in portion 20 was about 1.458 (at a wavelength of
about 1550 nm), n2 in portion 18 was about 1.453, the average pitch
spacing .LAMBDA. for the grating 12 was about 0.542 microns, and
the spacing between pitches .DELTA..LAMBDA. was about 0.36% of the
adjacent pitches .LAMBDA..
[0174] Referring to FIG. 27, illustration (a), the pitch .LAMBDA.
of an individual grating is the axial spatial period of the
sinusoidal variation in the refractive index n1 in the region 20 of
the substrate 10 along the axial length of the grating 12 as
indicated by a curve 90 on a graph 91. Referring to FIG. 27,
illustration (b), a sample composite grating 12 comprises three
individual gratings that are co-located on the substrate 10, each
individual grating having slightly different pitches, .LAMBDA.1,
.LAMBDA.2, .LAMBDA.3, respectively, and the difference (or spacing)
.DELTA..LAMBDA. between each pitch .LAMBDA. being about 3.0% of the
period of an adjacent pitch .LAMBDA. as indicated by a series of
curves 92 on a graph 94. Referring to FIG. 27, illustration (c),
three individual gratings, each having slightly different pitches,
.LAMBDA.1, .LAMBDA.2, .LAMBDA.3, respectively, are shown, the
difference AA between each pitch .LAMBDA. being about 0.3% of the
pitch .LAMBDA. of the adjacent pitch as shown by a series of curves
95 on a graph 97. The individual gratings in FIG. 27, illustrations
(b) and (c) are shown to all start at 0 for illustration purposes;
however, it should be understood that, the separate gratings need
not all start in phase with each other. Referring to FIG. 27,
illustration (d), the overlapping of the individual sinusoidal
refractive index variation pitches .LAMBDA.1-.LAMBDA.n in the
grating region 20 of the substrate 10, produces a combined
resultant refractive index variation in the composite grating 12
shown as a curve 96 on a graph 98 representing the combination of
the three pitches shown in FIG. 27, illustration (b). Accordingly,
the resultant refractive index variation in the grating region 20
of the substrate 10 may not be sinusoidal and is a combination of
the individual pitches .LAMBDA. (or index variation).
[0175] The maximum number of resolvable bits N, which is equal to
the number of different grating pitches .LAMBDA. (and hence the
number of codes), that can be accurately read (or resolved) using
side-illumination and side-reading of the grating 12 in the
substrate 10, is determined by numerous factors, including: the
beam width w incident on the substrate (and the corresponding
substrate length L and grating length Lg), the thickness or
diameter D of the grating 12, the wavelength .lamda. of incident
light, the beam divergence angle .theta..sub.R, and the width of
the Bragg envelope .theta..sub.B (discussed more in the
aforementioned patent application), and may be determined by the
equation: N .apprxeq. .eta. .times. .times. .beta. .times. .times.
L 2 .times. .times. D .times. .times. sin .function. ( .theta. i )
Eq . .times. 6 ##EQU4##
[0176] Referring to FIG. 28, instead of having the input light 24
at a single wavelength .lamda. (monochromatic) and reading the bits
by the angle .theta.o of the output light, the bits (or grating
pitches .LAMBDA.) may be read/detected by providing a plurality of
wavelengths and reading the wavelength spectrum of the reflected
output light signal. In this case, there would be one bit per
wavelength, and thus, the code is contained in the wavelength
information of the reflected output signal.
[0177] In this case, each bit (or .LAMBDA.) is defined by whether
its corresponding wavelength falls within the Bragg envelope, not
by its angular position within the Bragg envelope 200. As a result,
it is not limited by the number of angles that can fit in the Bragg
envelope 200 for a given composite grating 12, as in the embodiment
discussed hereinbefore. Thus, using multiple wavelengths, the only
limitation in the number of bits N is the maximum number of grating
pitches .LAMBDA. that can be superimposed and optically
distinguished in wavelength space for the output beam.
[0178] Referring to FIGS. 28 and 29, illustration (a), the
reflection wavelength spectrum (.lamda.1-.lamda.n) of the reflected
output beam 310 will exhibit a series of reflection peaks 695, each
appearing at the same output Bragg angle .theta.o. Each wavelength
peak 695 (.lamda.1-.lamda.n) corresponds to an associated spatial
period (.LAMBDA.1-.LAMBDA.n), which make up the grating 12.
[0179] One way to measure the bits in wavelength space is to have
the input light angle .theta.i equal to the output light angle
.theta.o, which is kept at a constant value, and to provide an
input wavelength .lamda. that satisfies the diffraction condition
(Eq. 1) for each grating pitch .LAMBDA.. This will maximize the
optical power of the output signal for each pitch .LAMBDA. detected
in the grating 12.
[0180] Referring to 29, illustration (b), the transmission
wavelength spectrum of the transmitted output beam 330 (which is
transmitted straight through the grating 12) will exhibit a series
of notches (or dark spots) 696. Alternatively, instead of detecting
the reflected output light 310, the transmitted light 330 may be
detected at the detector/reader 308. It should be understood that
the optical signal levels for the reflection peaks 695 and
transmission notches 696 will depend on the "strength" of the
grating 12, i.e., the magnitude of the index variation n in the
grating 12.
[0181] In FIG. 28, the bits may be detected by continuously
scanning the input wavelength. A known optical source 300 provides
the input light signal 24 of a coherent scanned wavelength input
light shown as a graph 304. The source 300 provides a sync signal
on a line 306 to a known reader 308. The sync signal may be a timed
pulse or a voltage ramped signal, which is indicative of the
wavelength being provided as the input light 24 to the substrate 10
at any given time. The reader 308 may be a photodiode, CCD camera,
or other optical detection device that detects when an optical
signal is present and provides an output signal on a line 309
indicative of the code in the substrate 10 or of the wavelengths
present in the output light, which is directly related to the code,
as discussed herein. The grating 12 reflects the input light 24 and
provides an output light signal 310 to the reader 308. The
wavelength of the input signal is set such that the reflected
output light 310 will be substantially in the center 314 of the
Bragg envelope 200 for the individual grating pitch (or bit) being
read.
[0182] Alternatively, the source 300 may provide a continuous
broadband wavelength input signal such as that shown as a graph
316. In that case, the reflected output beam 310 signal is provided
to a narrow band scanning filter 318 which scans across the desired
range of wavelengths and provides a filtered output optical signal
320 to the reader 308. The filter 318 provides a sync signal on a
line 322 to the reader, which is indicative of which wavelengths
are being provided on the output signal 320 to the reader and may
be similar to the sync signal discussed hereinbefore on the line
306 from the source 300. In this case, the source 300 does not need
to provide a sync signal because the input optical signal 24 is
continuous. Alternatively, instead of having the scanning filter
being located in the path of the output beam 310, the scanning
filter may be located in the path of the input beam 24 as indicated
by the dashed box 324, which provides the sync signal on a line
323.
[0183] Alternatively, instead of the scanning filters 318,324, the
reader 308 may be a known optical spectrometer (such as a known
spectrum analyzer), capable of measuring the wavelength of the
output light.
[0184] The desired values for the input wavelengths .lamda. (or
wavelength range) for the input signal 24 from the source 300 may
be determined from the Bragg condition of Eq. 1, for a given
grating spacing .LAMBDA. and equal angles for the input light
.theta.i and the angle light .theta.o. Solving Eq. 1 for .lamda.
and plugging in m=1, gives:
.lamda.=.LAMBDA.[sin(.theta.o)+sin(.theta.i)] Eq. 7
[0185] It is also possible to combine the angular-based code
detection with the wavelength-based code detection, both discussed
hereinbefore. In this case, each readout wavelength is associated
with a predetermined number of bits within the Bragg envelope. Bits
(or grating pitches .LAMBDA.) written for different wavelengths do
not show up unless the correct wavelength is used.
[0186] Accordingly, the bits (or grating pitches .LAMBDA.) can be
read using one wavelength and many angles, many wavelengths and one
angle, or many wavelengths and many angles.
[0187] Referring to FIG. 30, the grating 12 may have a thickness or
depth D which is comparable or smaller than the incident beam
wavelength .lamda.. This is known as a "thin" diffraction grating
(or the full angle Bragg envelope is 180 degrees). In that case,
the half-angle Bragg envelope .theta.B is substantially 90 degrees;
however, .delta.n must be made large enough to provide sufficient
reflection efficiency, per Eqs. 3 and 4. In particular, for a
"thin" grating, D*.delta.n.apprxeq..lamda./2, which corresponds to
a .pi. phase shift between adjacent minimum and maximum refractive
index values of the grating 12.
[0188] It should be understood that there is still a trade-off
discussed hereinbefore with beam divergence angle .theta..sub.R and
the incident beam width (or length L of the substrate), but the
accessible angular space is theoretically now 90 degrees. Also, for
maximum efficiency, the phase shift between adjacent minimum and
maximum refractive index values of the grating 12 should approach a
.pi. phase shift; however, other phase shifts may be used.
[0189] In this case, rather than having the input light 24 coming
in at the conventional Bragg input angle .theta.i, as discussed
hereinbefore and indicated by a dashed line 701, the grating 12 is
illuminated with the input light 24 oriented on a line 705
orthogonal to the longitudinal grating vector 705. The input beam
24 will split into two (or more) beams of equal amplitude, where
the exit angle .theta..sub.o can be determined from Eq. 1 with the
input angle .theta..sub.i=0 (normal to the longitudinal axis of the
grating 12).
[0190] In particular, from Eq. 1, for a given grating pitch
.LAMBDA.1, the +/-1.sup.st order beams (m=++1 and m=-1),
corresponds to output beams 700,702, respectively. For the
+/-2.sup.nd order beams (m=+2 and m=-2), corresponds to output
beams 704,706, respectively. The 0.sup.th order (undefracted) beam
(m=0), corresponds to beam 708 and passes straight through the
substrate. The output beams 700-708 project spectral spots or peaks
710-718, respectively, along a common plane, shown from the side by
a line 709, which is parallel to the upper surface of the substrate
10.
[0191] For example, for a grating pitch .LAMBDA.=1.0 um, and an
input wavelength .lamda.=400 nm, the exit angles .theta..sub.o are
.about.+/-23.6 degrees (for m=+/-1), and +/-53.1 degrees (from
m=+/-2), from Eq. 1. It should be understood that for certain
wavelengths, certain orders (e.g., m=+/-2) may be reflected back
toward the input side or otherwise not detectable at the output
side of the grating 12.
[0192] Alternatively, one can use only the +/-1.sup.st order
(m=+/-1) output beams for the code, in which case there would be
only 2 peaks to detect, 712, 714. Alternatively, one can also use
any one or more pairs from any order output beam that is capable of
being detected. Alternatively, instead of using a pair of output
peaks for a given order, an individual peak may be used.
[0193] Referring to FIG. 31, if two pitches .LAMBDA.1,.LAMBDA.2
exist in the grating 12, two sets of peaks will exist. In
particular, for a second grating pitch .LAMBDA.2, the +/-1.sup.st
order beams (m=+1 and m=-1), corresponds to output beams 720,722,
respectively. For the +/-2.sup.nd order beams (m=+2 and m=-2),
corresponds to output beams 724,726, respectively. The 0.sup.th
order (un-defracted) beam (m=0), corresponds to beam 718 and passes
straight through the substrate. The output beams 720-726
corresponding to the second pitch .LAMBDA.2 project spectral spots
or peaks 730-736, respectively, which are at a different location
than the point 710-716, but along the same common plane, shown from
the side by the line 709.
[0194] Thus, for a given pitch .LAMBDA. (or bit) in a grating, a
set of spectral peaks will appear at a specific location in space.
Thus, each different pitch corresponds to a different elevation or
output angle which corresponds to a predetermined set of spectral
peaks. Accordingly, the presence or absence of a particular peak or
set of spectral peaks defines the code.
[0195] In general, if the angle of the grating 12 is not properly
aligned with respect to the mechanical longitudinal axis of the
substrate 10, the readout angles may no longer be symmetric,
leading to possible difficulties in readout. With a thin grating,
the angular sensitivity to the alignment of the longitudinal axis
of the substrate 10 to the input angle .theta.i of incident
radiation is reduced or eliminated. In particular, the input light
can be oriented along substantially any angle .theta.i with respect
to the grating 12 without causing output signal degradation, due
the large Bragg angle envelope. Also, if the incident beam 24 is
normal to the substrate 10, the grating 12 can be oriented at any
rotational (or azimuthal) angle without causing output signal
degradation. However, in each of these cases, changing the incident
angle .theta.i will affect the output angle .theta.o of the
reflected light in a predetermined predictable way, thereby
allowing for accurate output code signal detection or
compensation.
[0196] Referring to FIG. 32, for a thin grating, in addition to
multiplexing in the elevation or output angle based on grating
pitch .LAMBDA., the bits can also be multiplexed in an azimuthal
(or rotational) angle .theta.a of the substrate. In particular, a
plurality of gratings 750,752,754,756 each having the same pitch
.LAMBDA. are disposed in a surface 701 of the substrate 10 and
located in the plane of the substrate surface 701. The input light
24 is incident on all the gratings 750,752,754,756 simultaneously.
Each of the gratings provides output beams oriented based on the
grating orientation. For example, the grating 750 provides the
output beams 764,762, the grating 752 provides the output beams
766,768, the grating 754 provides the output beams 770,772, and the
grating 756 provides the output beams 774,776. Each of the output
beams provides spectral peaks or spots (similar to that discussed
hereinbefore), which are located in a plane 760 that is parallel to
the substrate surface plane 701. In this case, a single grating
pitch .LAMBDA. can produce many bits depending on the number of
gratings that can be placed at different azimuthal (rotational)
angles on the surface of the substrate 10 and the number of output
beam spectral peaks that can be spatially and optically
resolved/detected. Each bit may be viewed as the presence or
absence of a pair of peaks located at a predetermined location in
space in the plane 760. Note that this example uses only the
m=+/-1.sup.st order for each reflected output beam. Alternatively,
the detection may also use the m=+/-2.sup.nd order. In that case,
there would be two additional output beams and peaks (not shown)
for each grating (as discussed hereinbefore) that may lie in the
same plane as the plane 760 and may be on a concentric circle
outside the circle 760.
[0197] In addition, the azimuthal multiplexing can be combined with
the elevation or output angle multiplexing discussed hereinbefore
to provide two levels of multiplexing. Accordingly, for a thin
grating, the number of bits can be multiplexed based on the number
of grating pitches .LAMBDA. and/or geometrically by the orientation
of the grating pitches.
[0198] Furthermore, if the input light angle .theta.i is normal to
the substrate 10, the edges of the substrate 10 no longer scatter
light from the incident angle into the "code angular space", as
discussed herein and/or in the aforementioned patent
application.
[0199] Also, in the thin grating geometry, a continuous broadband
wavelength source may be used as the optical source if desired.
[0200] Referring to FIG. 33, instead of or in addition to the
pitches .LAMBDA. in the grating 12 being oriented normal to the
longitudinal axis, the pitches may be created at a angle .theta.g.
In that case, when the input light 24 is incident normal to the
surface 792, will produce a reflected output beam 790 having an
angle .theta.o determined by Eq. 1 as adjusted for the blaze angle
.theta.g. This can provide another level of multiplexing bits in
the code.
[0201] Referring to FIG. 34, instead of using an optical binary
(0-1) code, an additional level of multiplexing may be provided by
having the optical code use other numerical bases, if intensity
levels of each bit are used to indicate code information. This
could be achieved by having a corresponding magnitude (or strength)
of the refractive index change (.delta.n) for each grating pitch
.LAMBDA.. Four intensity ranges are shown for each bit number or
pitch .LAMBDA., providing for a Base-4 code (where each bit
corresponds to 0,1,2, or 3). The lowest intensity level,
corresponding to a 0, would exist when this pitch .LAMBDA. is not
present in the grating 12. The next intensity level 450 would occur
when a first low level .delta.n1 exists in the grating that
provides an output signal within the intensity range corresponding
to a 1. The next intensity level 452 would occur when a second
higher level .delta.n2 exists in the grating 12 that provides an
output signal within the intensity range corresponding to a 2. The
next intensity level 452, would occur when a third higher level
.delta.n3 exists in the grating 12 that provides an output signal
within the intensity range corresponding to a 3.
[0202] Referring to FIG. 35, the input light 24 may be incident on
the substrate 10 on an end face 600 of the substrate 10. In that
case, the input light 24 will be incident on the grating 12 having
a more significant component of the light (as compared to side
illumination discussed hereinbefore) along the longitudinal grating
axis 207 of the grating (along the grating vector k.sub.B), as
shown by a line 602. The light 602 reflects off the grating 12 as
indicated by a line 604 and exits the substrate as output light
608. Accordingly, it should be understood by one skilled in the art
that the diffraction equations discussed hereinbefore regarding
output diffraction angle .theta.o also apply in this case except
that the reference axis would now be the grating axis 207. Thus, in
this case, the input and output light angles .theta.i,.theta.o,
would be measured from the grating axis 207 and length Lg of the
grating 12 would become the thickness or depth D of the grating 12.
As a result, a grating 12 that is 400 microns long, would result in
the Bragg envelope 200 being narrow. It should be understood that
because the values of n1 and n2 are close to the same value, the
slight angle changes of the light between the regions 18,20 are not
shown herein.
[0203] In the case where incident light 610 is incident along the
same direction as the grating vector (Kb) 207, i.e., .theta.i=0
degrees, the incident light sees the whole length Lg of the grating
12 and the grating provides a reflected output light angle
.theta.o=0 degrees, and the Bragg envelope 612 becomes extremely
narrow, as the narrowing effect discussed above reaches a limit. In
that case, the relationship between a given pitch .LAMBDA. in the
grating 12 and the wavelength of reflection .lamda. is governed by
a known "Bragg grating" relation: .lamda.=2 n.sub.eff .LAMBDA. Eq.
8 where n.sub.eff is the effective index of refraction of the
substrate, .lamda. is the input (and output wavelength) and
.LAMBDA. is the pitch. This relation, as is known, may be derived
from Eq. 1 where .theta.i=.theta.o=90 degrees.
[0204] In that case, the code information is readable only in the
spectral wavelength of the reflected beam, similar to that
discussed hereinbefore for wavelength based code reading.
Accordingly, the input signal in this case may be a scanned
wavelength source or a broadband wavelength source. In addition, as
discussed hereinbefore for wavelength based code reading, the code
information may be obtained in reflection from the reflected beam
614 or in transmission by the transmitted beam 616 that passes
through the grating 12.
[0205] It should be understood that for shapes of the substrate 10
or element 8 other than a cylinder, the effect of various different
shapes on the propagation of input light through the element 8,
substrate 10, and/or grating 12, and the associated reflection
angles, can be determined using known optical physics including
Snell's Law, shown below: n.sub.in sin .theta.in=n.sub.out sin
.theta.out Eq. 9
[0206] where n.sub.in is the refractive index of the first (input)
medium, and n.sub.out is the refractive index of the second
(output) medium, and .theta.in and .theta.out are measured from a
line 620 normal to an incident surface 622.
[0207] Referring to FIG. 36, if the value of n1 in the grating
region 20 is greater than the value of n2 in the non-grating region
18, the grating region 20 of the substrate 10 will act as a known
optical waveguide for certain wavelengths. In that case, the
grating region 20 acts as a "core" along which light is guided and
the outer region 18 acts as a "cladding" which helps confine or
guide the light. Also, such a waveguide will have a known
"numerical aperture" (.theta.na) that will allow light that is
within the aperture .theta.na to be directed or guided along the
grating axis 207 and reflected axially off the grating 12 and
returned and guided along the waveguide. In that case, the grating
12 will reflect light having the appropriate wavelengths equal to
the pitches .LAMBDA. present in the grating 12 back along the
region 20 (or core) of the waveguide, and pass the remaining
wavelengths of light as the light 632. Thus, having the grating
region 20 act as an optical waveguide for wavelengths reflected by
the grating 12 allows incident light that is not aligned exactly
with the grating axis 207 to be guided along and aligned with the
grating 12 axis 207 for optimal grating reflection.
[0208] If an optical waveguide is used any standard waveguide may
be used, e.g., a standard telecommunication single mode optical
fiber (125 micron diameter or 80 micron diameter fiber with about a
8-10 micron diameter), or a larger diameter waveguide (greater than
0.5 mm diameter), such as is describe in U.S. patent application
Ser. No. 09/455,868, filed Dec. 6, 1999, entitled "Large Diameter
Waveguide, Grating". Further, any type of optical waveguide may be
used for the optical substrate 10, such as, a multi-mode,
birefringent, polarization maintaining, polarizing, multi-core,
multi-cladding, or microsturctured optical waveguide, or a flat or
planar waveguide (where the waveguide is rectangular shaped), or
other waveguides. Any other dimensions may be used for the
waveguide if desired, provided they meet the functional and
performance requirements of the application taking into account the
teachings herein.
[0209] Referring to FIG. 37, if the grating 12 extends across the
entire dimension D of the substrate, the substrate 10 does not
behave as a waveguide for the incident or reflected light and the
incident light 24 will be diffracted (or reflected) as indicated by
lines 642, and the codes detected as discussed hereinbefore for the
end-incidence condition discussed hereinbefore with FIG. 45, and
the remaining light 640 passes straight through.
[0210] Referring to FIG. 38, illustrations (a)-(c), in illustration
(a), for the end illumination condition, if a blazed or angled
grating is used, as discussed hereinbefore, the input light 24 is
coupled out of the substrate 10 at a known angle as shown by a line
650. Referring to FIG. 38, illustration (b), alternatively, the
input light 24 may be incident from the side and, if the grating 12
has the appropriate blaze angle, the reflected light will exit from
the end face 652 as indicated by a line 654. Referring to FIG. 38,
illustration (c), the grating 12 may have a plurality of different
pitch angles 660,662, which reflect the input light 24 to different
output angles as indicated by lines 664, 666. This provides another
level of multiplexing (spatially) additional codes, if desired.
[0211] The grating 12 may be impressed in the substrate 10 by any
technique for writing, impressed, embedded, imprinted, or otherwise
forming a diffraction grating in the volume of or on a surface of a
substrate 10. Examples of some known techniques are described in
U.S. Pat. Nos. 4,725,110 and 4,807,950, entitled "Method for
Impressing Gratings Within Fiber Optics", to Glenn et al; and U.S.
Pat. No. 5,388,173, entitled "Method and Apparatus for Forming
Aperiodic Gratings in Optical Fibers", to Glenn, respectively, and
U.S. Pat. No. 5,367,588, entitled "Method of Fabricating Bragg
Gratings Using a Silica Glass Phase Grating Mask and Mask Used by
Same", to Hill, and U.S. Pat. No. 3,916,182, entitled "Periodic
Dielectric Waveguide Filter", Dabby et al, and U.S. Pat. No.
3,891,302, entitled "Method of Filtering Modes in Optical
Waveguides", to Dabby et al, which are all incorporated herein by
reference to the extent necessary to understand the present
invention.
[0212] Alternatively, instead of the grating 12 being impressed
within the substrate material, the grating 12 may be partially or
totally created by etching or otherwise altering the outer surface
geometry of the substrate to create a corrugated or varying surface
geometry of the substrate, such as is described in U.S. Pat. No.
3,891,302, entitled "Method of Filtering Modes in Optical
Waveguides", to Dabby et al, which is incorporated herein by
reference to the extent necessary to understand the present
invention, provided the resultant optical refractive profile for
the desired code is created.
[0213] Further, alternatively, the grating 12 may be made by
depositing dielectric layers onto the substrate, similar to the way
a known thin film filter is created, so as to create the desired
resultant optical refractive profile for the desired code.
FIGS. 39-50: Alternative Microbead Geometries
[0214] The substrate 10 (and/or the element 8) may have end-view
cross-sectional shapes other than circular, such as square,
rectangular, elliptical, clam-shell, D-shaped, or other shapes, and
may have side-view sectional shapes other than rectangular, such as
circular, square, elliptical, clam-shell, D-shaped, or other
shapes. Also, 3D geometries other than a cylinder may be used, such
as a sphere, a cube, a pyramid or any other 3D shape.
Alternatively, the substrate 10 may have a geometry that is a
combination of one or more of the foregoing shapes.
[0215] The shape of the element 8 and the size of the incident beam
may be made to minimize any end scatter off the end face(s) of the
element 8, as is discussed herein and/or in the aforementioned
patent application. Accordingly, to minimize such scatter, the
incident beam 24 may be oval shaped where the narrow portion of the
oval is smaller than the diameter D1, and the long portion of the
oval is smaller than the length L of the element 8. Alternatively,
the shape of the end faces may be rounded or other shapes or may be
coated with an antireflective coating.
[0216] It should be understood that the size of any given dimension
for the region 20 of the grating 12 may be less than any
corresponding dimension of the substrate 10. For example, if the
grating 12 has dimensions of length Lg, depth Dg, and width Wg, and
the substrate 12 has different dimensions of length L, depth D, and
width W, the dimensions of the grating 12 may be less than that of
the substrate 12. Thus, the grating 12, may be embedded within or
part of a much larger substrate 12. Also, the element 8 may be
embedded or formed in or on a larger object for identification of
the object.
[0217] The dimensions, geometries, materials, and material
properties of the substrate 10 are selected such that the desired
optical and material properties are met for a given application.
The resolution and range for the optical codes are scalable by
controlling these parameters as discussed herein and/or in the
aforementioned patent application.
[0218] Referring to FIG. 39, the substrate 10 may have an outer
coating 799, such as a polymer or other material that may be
dissimilar to the material of the substrate 10, provided that the
coating 799 on at least a portion of the substrate, allows
sufficient light to pass through the substrate for adequate optical
detection of the code. The coating 799 may be on any one or more
sides of the substrate 10. Also, the coating 799 may be a material
that causes the element 8 to float or sink in certain fluids
(liquid and/or gas) solutions.
[0219] Also, the substrate 10 may be made of a material that is
less dense than certain fluid (liquids and/or gas) solutions,
thereby allowing the elements 8 to float or be buoyant or partially
buoyant. Also, the substrate may be made of a porous material, such
as controlled pore glass (CPG) or other porous material, which may
also reduce the density of the element 8 and may make the element 8
buoyant or partially-buoyant in certain fluids.
[0220] Referring to FIG. 40, the grating 12 is axially spatially
invariant. As a result, the substrate 10 with the grating 12 (shown
as a long substrate 21) may be axially subdivided or cut into many
separate smaller substrates 30-36 and each substrate 30-36 will
contain the same code as the longer substrate 21 had before it was
cut. The limit on the size of the smaller substrates 30-36 is based
on design and performance factors discussed herein and/or in the
aforementioned patent application.
[0221] Referring to FIG. 41, one purpose of the outer region 18 (or
region without the grating 12) of the substrate 10 is to provide
mechanical or structural support for the inner grating region 20.
Accordingly, the entire substrate 10 may comprise the grating 12,
if desired. Alternatively, the support portion may be completely or
partially beneath, above, or along one or more sides of the grating
region 20, such as in a planar geometry, or a D-shaped geometry, or
other geometries, as described herein and/or in the aforementioned
patent application. The non-grating portion 18 of the substrate 10
may be used for other purposes as well, such as optical lensing
effects or other effects (discussed herein or in the aforementioned
patent application). Also, the end faces of the substrate 10 need
not be perpendicular to the sides or parallel to each other.
However, for applications where the elements 8 are stacked
end-to-end, the packing density may be optimized if the end faces
are perpendicular to the sides.
[0222] Referring to FIG. 42, illustrations (a)-(c), two or more
substrates 10,250, each having at least one grating therein, may be
attached together to form the element 8, e.g., by an adhesive,
fusing or other attachment techniques. In that case, the gratings
12,252 may have the same or different codes.
[0223] Referring to FIG. 43, illustrations (a) and (b), the
substrate 10 may have multiple regions 80,90 and one or more of
these regions may have gratings in them. For example, there may be
gratings 12,252 side-by-side (illustration (a)), or there may be
gratings 82-88, spaced end-to-end (illustration (b)) in the
substrate 10.
[0224] Referring to FIG. 44, the length L of the element 8 may be
shorter than its diameter D, thus, having a geometry such as a
plug, puck, wafer, disc or plate.
[0225] Referring to FIG. 45 to facilitate proper alignment of the
grating axis with the angle .theta.i of the input beam 24, the
substrate 10 may have a plurality of the gratings 12 having the
same codes written therein at numerous different angular or
rotational (or azimuthal) positions of the substrate 10. In
particular, two gratings 550, 552, having axial grating axes 551,
553, respectively may have a common central (or pivot or
rotational) point where the two axes 551,553 intersect. The angle
.theta.i of the incident light 24 is aligned properly with the
grating 550 and is not aligned with the grating 552, such that
output light 555 is reflected off the grating 550 and light 557
passes through the grating 550 as discussed herein. If the element
8 is rotated as shown by the arrows 559, the angle .theta.i of
incident light 24 will become aligned properly with the grating 552
and not aligned with the grating 550 such that output light 555 is
reflected off the grating 552 and light 557 passes through the
grating 552. When multiple gratings are located in this rotational
orientation, the bead may be rotated as indicated by a line 559 and
there may be many angular positions that will provide correct (or
optimal) incident input angles .theta.i to the grating. While this
example shows a circular cross-section, this technique may be used
with any shape cross-section.
[0226] Referring to FIG. 46, illustrations (a), (b), (c), (d), and
(e) the substrate 10 may have one or more holes located within the
substrate 10. In illustration (a), holes 560 may be located at
various points along all or a portion of the length of the
substrate 10. The holes need not pass all the way through the
substrate 10. Any number, size and spacing for the holes 560 may be
used if desired. In illustration (b), holes 572 may be located very
close together to form a honeycomb-like area of all or a portion of
the cross-section. In illustration (c), one (or more) inner hole
566 may be located in the center of the substrate 10 or anywhere
inside of where the grating region(s) 20 are located. The inner
hole 566 may be coated with a reflective coating 573 to reflect
light to facilitate reading of one or more of the gratings 12
and/or to reflect light diffracted off one or more of the gratings
12. The incident light 24 may reflect off the grating 12 in the
region 20 and then reflect off the surface 573 to provide output
light 577. Alternatively, the incident light 24 may reflect off the
surface 573, then reflect off the grating 12 and provide the output
light 575. In that case the grating region 20 may run axially or
circumferentially 571 around the substrate 10. In illustration (d),
the holes 579 may be located circumferentially around the grating
region 20 or transversely across the substrate 10. In illustration
(e), the grating 12 may be located circumferentially around the
outside of the substrate 10, and there may be holes 574 inside the
substrate 10.
[0227] Referring to FIG. 47, illustrations (a), (b), and (c), the
substrate 10 may have one or more protruding portions or teeth 570,
578,580 extending radially and/or circumferentially from the
substrate 10. Alternatively, the teeth 570, 578,580 may have any
other desired shape.
[0228] Referring to FIG. 48, illustrations (a), (b), (c) a D-shaped
substrate, a flat-sided substrate and an eye-shaped (or clam-shell
or teardrop shaped) substrate 10, respectively, are shown. Also,
the grating region 20 may have end cross-sectional shapes other
than circular and may have side cross-sectional shapes other than
rectangular, such as any of the geometries described herein for the
substrate 10. For example, the grating region 20 may have a oval
cross-sectional shape as shown by dashed lines 581, which may be
oriented in a desired direction, consistent with the teachings
herein. Any other geometries for the substrate 10 or the grating
region 20 may be used if desired, as described herein.
[0229] Referring to FIG. 49, at least a portion of a side of the
substrate 10 may be coated with a reflective coating to allow
incident light 510 to be reflected back to the same side from which
the incident light came, as indicated by reflected light 512.
[0230] Referring to FIG. 50, illustrations (a) and (b),
alternatively, the substrate 10 can be electrically and/or
magnetically polarized, by a dopant or coating, which may be used
to ease handling and/or alignment or orientation of the substrate
10 and/or the grating 12, or used for other purposes.
Alternatively, the bead may be coated with conductive material,
e.g., metal coating on the inside of a holy substrate, or metallic
dopant inside the substrate. In these cases, such materials can
cause the substrate 10 to align in an electric or magnetic field.
Alternatively, the substrate can be doped with an element or
compound that fluoresces or glows under appropriate illumination,
e.g., a rare earth dopant, such as Erbium, or other rare earth
dopant or fluorescent or luminescent molecule. In that case, such
fluorescence or luminescence may aid in locating and/or aligning
substrates.
Further Alternative Embodiments for Groove Plates and
Loading/Unloading Beads
[0231] Referring to FIGS. 55 and 56, the bead cell, chamber, or
cuvettes 900, 920, respectively, may be segmented into regions each
associated with a different reaction or used for a different
identification process/application. In particular, referring to
FIG. 55, for a cell having circular grooves 1258, the cell may have
a plurality of separate sections 902 which are physically separated
from each other by barriers, 904. In that case, the beads may be
loaded through separate holes or ports 906, which communicate only
with an associated section 902. The sections 902 may be
mechanically isolated, so that the beads 8 placed in a given
section 902 all remain in that section, and/or fluidically
isolated, so that any fluid with the beads 8 placed in a given
section 902 remains in that section with no cross-over into any
other section 902.
[0232] Further, referring to FIG. 56, for a cell having straight
grooves 205, the cell 940 may have a plurality of separate sections
942 which are physically separated from each other by barriers,
944. In that case, the beads may be loaded through separate holes
or ports 946, which communicate only with an associated section
942. The sections 942 may be mechanically isolated, so that the
beads 8 placed in a given section 942 all remain in that section,
and/or fluidically isolated, so that any fluid with the beads 8
placed in a given section 942 remains in that section with no
cross-over into any other section 942.
[0233] Referring to FIG. 57, one example of a sectored cell 920
with straight grooves 205 has a base groove plate 930, a spacer
932, and a cover 934. The groove plate may be made of fused silica,
borosilicate glass, or plastic, acrylic, Zeonex made by Zeon Corp.
or any other support material that is transparent or substantially
transparent to desired incident wavelength light or can be made of
reflective by coating a transparent material or using a reflective
material, such as silicon or other support material that reflects
the desired wavelengths of incident light. Also, the groove plate
930 may be made of a material that has minimal fluorescence to
minimize background fluorescence in the desired fluorescence
wavelength range, for applications where fluorescence of the beads
8 is measured.
[0234] The base plate 930 has a substantially circular shape having
a diameter of about 100 mm, with a mechanical alignment key or
notch 952 about 32.5 mm long, which may be used for mechanical
alignment during wafer fabrication of the groove plate 930. The
thickness 948 is about 1 mm. The base plate 930 has the grooves 205
therein, which may be formed by direct reactive ion etching (REI)
of the glass base plate 930, photo-patterning with photoresist,
photoresist and plating process, or any other process that provides
the grooves 205 that meet the requirements for the application. The
sectors 944 have a length 950 of about 50 mm. Also, one or more
reference lines 948 (or fiducials) may be provided for reader head
alignment with the grooves 205. The length 940 of each grooved
section or sector 944 is about 7 mm and the space 946 between each
section 944 is about 2 mm. The grooves 205 are about 34 microns by
24 microns deep and have about a 55 micron pitch spacing. For a 7
mm long groove, each groove 205 would hold about 28 cylindrically
shaped beads 8 each bead 8 having a dimension of about 30 microns
in diameter and 250 microns in length. The sectors 944 having a
length of about 50 mm, may have about 900 grooves and hold a total
capacity about 25,200 beads 8. While the number of physically
separated sectors 944 in the cell 938 shown is eight, any number of
sectors may be used if desired.
[0235] Referring to FIG. 58, the grooves 205 have a depth of about
22 to 24 microns, and have a top width of about 34 microns, and a
base width 953 of about 30 microns for .theta.g=5 deg., and a
spacing pitch of about 55 microns, for a bead 8 having a diameter
D1 of about 28 micons. The side walls 958 may have an angle
.theta.g of about 0 to 10 degrees. Other angles may be used,
depending on the application, e.g., whether the beads will be
removed from the plate and how they will be removed.
[0236] For example, referring to FIG. 58, with the angle .theta.g
is between 0 and about 10 degrees the beads may be flushed or
washed out of the grooves 205 with fluid flow transversely across
the top of the grooves 205, using a fluid flow rate of about 3 to 6
ml/second cleans out the beads. The flush may be done with dionized
water, regular water, saline, detergent with water, or other
liquid. Using a detergent reduces the viscosity and surface tension
so beads do not stick to the surface of the cell. The angle
.theta.g may be greater than 10 degrees if desired, depending on
certain design parameters, including, flush flow rate,
groove-to-groove separation, and groove depth. Alternatively, if
the angle .theta.g is less than 0 deg., the beads will be more
likely to stay in the grooves 205.
[0237] Other dimensions and geometries for the groove plate 930,
grooves 205, spacer 932, and cover 934 and/or for any features or
characteristics thereof may be used if desired.
Loading/Moving Beads Using Pressure Wave/Vibrations
[0238] The present invention, which is predicated on two
observations, eliminates the need for mechanically distributing
beads. The first observation is that small particles are easily
moved by a fluid stream, and the orientation of cylindrical
particles is generally with the long axis of the particle
perpendicular to the direction of the flow. And the second is that
particles in a liquid can be moved in a particular direction by a
temporally asymmetric oscillatory flow. Regarding the later, it was
observed that when an oscillatory flow was used in a closed fluidic
cell containing cylindrical glass particles, whereby the rate of
the outgoing wave was higher than the return wave, the particles
would acquire a net displacement in the direction of the outgoing
wave. When the flow rates were reversed, i.e. when the outgoing
wave was slower than the return wave, the particles moved inward.
Again it was observed that the particles would tend to orient
perpendicular to the direction of the pressure wave.
[0239] This behavior was first observed using a closed fluidic cell
in the shape of a round disk with a floor and ceiling spaced by
approximately 500 micron. The cell was entirely closed except for a
hole in the center of the top, which allowed the particles
(400.times.40 um cylindrical glass "beads") to be inserted into the
center of the cell. An asymmetric flow was established by tapping
the bottom of the cell with a blunt object. A time sequence is
shown FIG. 1(a-f), illustrating how the particles form a ring
shaped pattern and how the size of the ring increased, indicating
that the particles were moving outward, after a series of pulses
were applied in one direction. FIG. 1 (g-1), illustrate how the
size of the ring decreased after the direction of the pulses was
reversed. In subsequent experiments, oscillatory flow was
established by coupling fluid through the open port in the top of
the cell. The general behavior of the cell was the same in either
case. By applying rapid pressure pulses, coupled through a flexible
tube inserted into the center hole, and allowing the waves to
slowly return, beads were made to move outward, thereby forming the
familiar circular shape. The radius of the circle depended on such
things as: the number of pulses, the amplitude of the pulses, the
separation between the floor and the ceiling, the size of the beads
and the geometry of the cell. An important feature of the cell was
an air buffer around the perimeter of the cell to allow the fluid a
place to move, since the fluid itself is non-compressible, the air
gap acted as a pneumatic spring. Another important feature was the
space between the floor and ceiling. It was important to maintain a
small gap (<500 um) between the floor and the ceiling to keep
the velocity of the fluid in the cell high enough to move the
particles.
[0240] Other experiments relating to the general behavior of
fluidic-induced particle movement include placing cylindrical beads
on the bottom of a an open vessel such as a beaker, then moving the
beads by introducing the tip of a syringe into the pile of beads
and blowing the liquid out through the tip. In this experiment, the
beads all moved radially away from the tip, leaving behind a region
void of all beads. Again, it was observed that the beads tended to
generally align parallel to the wave front. FIG. 11 shows a
schematic of a concept that uses two such flow-generating tips. The
flow from the tips can be operated such that they oppose each
other, thus acting to push the beads into the region half way
between the tips. Or they can be operated in a push-pull fashion
whereby the beads tend to move toward one tip or the other. A
synthetic circular force field can be generated by rotating the
plate while operating the tips in either of the previously
mentioned methods.
[0241] An of the invention involves combining the ability to
transport beads across the floor of a substrate using either
continuous fluid flow or a type of asymmetric oscillatory flow,
with the technology for trapping beads, such as the previously
described groove plate. This would enable a highly efficient
assembly of beads with precise orientations in the smallest
possible area. With respect to reduced operating cost and high
throughput, all three of these attributes are important elements of
a commercial encoded particle reader. FIG. 70 shows a schematic of
a concept that incorporates a closed liquid cell and the elements
required to load the cell efficiently. Key elements of the method
include: a closed cell including a top and a bottom, the bottom
contains a plate with grooves for aligning beads, both the top and
the bottom are transparent, the top has an opening in the center
for loading beads and for coupling a pressure generating device
such as a bellows or a tube, and finally a region of trapped air
around the perimeter adjacent to and in contact with the fluid in
the cell. The loading operation consists of: filling the cell with
a liquid such as water, spinning the cell to remove the air
bubbles, dispensing beads through the center hole in the lid,
applying a pulsating flow such that the rate of the outward going
pulse is higher the return pulse. This will tend to move the pile
of beads away from the center of the cell. As the beads move
outward they populate the grooves. The direction of the pulsation
can be reversed to move the pile of beads back toward the center to
enhance the probability that the grooves are fully populated before
allowing beads to move out to a larger radius. By moving the beads
in and out it should be possible to fully populate the inner most
grooves, thus maximizing the overall loading density. It may
further be desirable to include an azimuthal (or circumferential)
agitation or vibration to stimulate the beads to move along the
channels of the grooves once they have fallen in, thereby enhancing
the probability that an open space is created to allow room for
additional beads to fall into the groove.
[0242] Also see FIGS. 77-79 for "puffing" (pressure pulses) done
with straight grooves with the actuator on one end.
Unloading Beads
[0243] Referring to FIG. 80-81, the beads may be unloaded by
flushing with fluid as shown and discussed herein before.
Cylindrical Groove Platform
[0244] Alternatively, referring to FIGS. 62-65, the groove plate
may be cylindrical shaped. In that case, the grooves 205 may run
along the longitudinal axis as shown in FIGS. 62,63 or
circumferentially as shown in FIGS. 64,65. The grooves 205 may be
oriented in any other direction along the cylindrical groove if
desired. Also, the grooves 205 may be located on the outside of the
cylinder as shown in FIGS. 62,64. Alternatively, the grooves 205
may be located on the inside of the cylinder as shown in FIGS.
63,65. When the grooves 205 are located on the inside, the cylinder
may be spun about the longitudinal axis to locate the beads 8
within the grooves 205. The orientation of the longitudinal axis of
the cylinder may be such that the longitudinal axis is vertical or
horizontal or at any other desired angle.
[0245] Referring to FIG. 89, an apparatus for transporting
microbeads for the present invention includes a container or well
400 with a sealed lid 410 having microbeads 8 and liquid 412
therein and a second container 402 having a sealed lid 411 with
liquid 412 therein. Two tubes 406,408 penetrate the lid 410, the
first tube 406 connects the first container 400 to a pump 416 and
the second tube 408 connects the first container to the second
container 402 for receiving the beads from the first container 200.
The first container 400 is filled with a liquid 412, and one end or
tip 414 of the first tube 406 is in the liquid 412 a predetermined
distance into the container 400, e.g., 20% to 50% of the full depth
of the container. A tip 409 of the tube 408 is mounted
substantially flush with the bottom surface of the lid 410. The
pump 416 pumps liquid 412 from a reservoir 405 into the tube 406 to
the first container 400. When the pump 416 pumps the liquid 412
into the container 400, the beads 8 are agitated as indicated by
the lines 418. The liquid 412 and the beads 8 exit the container
400 through the tube 408 as indicated by a line 420 and are emptied
into the container 402. The liquid 412 enters the container 402 and
exits the container 402 through a filter 424 then through a tube
428, as indicated by a line 426. The filter 424 prevents the beads
8 from exiting the container 402. The liquid 412 that exits the
container 402 is dispensed via the tube 428 into a waste pan or
container 430.
[0246] Instead of the pump 416 being connected to the tube 406, a
vacuum pump 432 may draw a vacuum on the tube 428. In that case the
tube 406 would be open ended. We have found that this technique
transports all the beads 8 from the container 400 to the second
container 402.
[0247] The tip 409 of the tube 408 may be placed further into the
container (i.e., not flush with inner surface of the lid 410), if
desired. In that case, some air may be pumped along the tube 408
with the liquid and the beads 8. If air exists at the top of the
container 400, the beads 8 may stick to the wall or inner surface
of the lid 410 and not be transported to the other container
402.
[0248] Referring to FIG. 90, instead of the second container 402
being a sealed container, it may be an open container. In that
case, the container 402 should have sufficient volume to receive
the fluid from the pump 416.
[0249] Referring to FIGS. 90 and 91, instead of having the filter
424 on the exit port of the sealed container 402, the container may
have a volume that is large enough such that when the beads 8 enter
from the tube 408, they stay substantially near the bottom of the
container 402 and do not get sucked out of the tube 428 to the
waste container 430. This technique for transporting the beads 8
may be referred to as the "telegraph" technique.
[0250] The seal between the lids 410,411 and the containers 400,
402, may be any type of seal that retains the liquid inside the
container, e.g., a radial seal/inner surface seal on the inside
wall of the container, a top surface/axial seal to the top surface
of the container, or any other seal that will perform the function
required.
[0251] We have found that a flow rate of 1.0 to 2.0 ml/sec., with a
tube inner diameter of 0.031 to 0.063 inches, and a total transport
time of about 0.73 seconds will transport all the beads from a well
of a 96 well plate to a bead reader cell, such as that described in
copending U.S. patent application Ser. No. (CyVera Docket No.
CV-0082 PR). In that case, the first container 400 would be an
individual well in the well plate, and the second container 402
would be the reader cell.
[0252] Also, this can be automated such that the lid 410 is a probe
head which comes down on top of the well to create a seal on the
well. The probe would contain the two tubes 406, 406, the tube 406
would be an aspirate tube and the tube 408 would be a dispense tube
for dispensing or transporting the beads 8 from the first container
400 to the second container 402. As discussed herein, the system
can operate under pressure or a vacuum. For a system operating
under pressure, the liquid 412 is driven into the dispense line,
pressurizing the well and sending the fluid out of the aspirate
tube 408. This permits use of drive pressure greater than 1 atm.
However, there is a risk that fluid (and possibly beads) will leak
out of the well if the lid seal fails. In a vacuum configuration,
the aspirate tube 408 is connected to negative pressure, and drive
pressure is limited to 1 atmosphere. However, in that case, if a
seal fails, air leaks into the system instead of liquid (and
possibly beads) leaking out.
[0253] Referring to FIG. 92, a similar configuration to that shown
in FIG. 91, using a syringe pump.
[0254] Referring to FIGS. 93-97, various alternative configurations
for pulling or pushing the beads 8 out of a well 440 having a
sealed lid 446, through a tube 442, to a larger diameter holding
area 440, using a syringe pump 448. In each case, once the beads 8
are in the holding area 440, the lid 446 is removed from the
container 440 and placed in the target or destination well or
container (not shown).
[0255] In particular, two tips penetrate the upper seal on the
container as discussed hereinbefore, with one tip connected to a
syringe pump and the other connected to a reservoir. When the
syringe pump is aspirated, fluid will be pulled from the reservoir
through the second tip. The fluid is thus dispensed from the second
tip, agitating the slurry, and aspirated by the first tip. In this
way, an arbitrary volume of fluid can be dispensed and aspirated
using only a single pump, without overflowing the well or
prematurely emptying the well of fluid. The dispensing head is then
moved to the new location desired. To dispense the beads, flow is
reversed. The flow rate is set lower to avoid re-aspirating the
beads into the reservoir. Also, in general, the volume can be set
much lower to simply dispense the beads into a new well. The volume
can be set the same, however, to refill the reservoir to the
original volume. Alternatively, the actuation direction can be
reversed. The second tip can be connected to a pump, while the
first tip is connected to the reservoir. When fluid is dispensed
under pressure through the second tip, fluid will flow up through
the first tip, providing an effective aspiration. Re-dispense then
involves aspiration through the second tip.
[0256] Referring to FIG. 98, a fluidic circuit for loading beads 8
from wells 400 in a known multi-well plate (e.g., a 96 well plate
having 8 rows and 12 columns) to a multi-segmented bead
cell/chamber, such as is discussed in copending U.S. patent
applications Ser. No. (CyVera Docket No. CV-0082 PR) and copending
U.S. patent applications Ser. No. (CyVera Docket No. CV-0086 PR) is
shown. This system uses an air pump to create a vacuum to pull the
beads into the cell 402 (second container) from the wells 400
(first container).
[0257] In particular, source fluid is contained in reagent bottles.
A bottle is selected by opening the valve which leads by a tubing
connection through the bottle cap to the desired bottle. Three
bottles are shown, actuated by valves V1, V2 and V3. Additional
bottles could be added, each with a companion valve. All valves are
electrically operated solenoid valves, such as clean valves sold by
Takasago Corp. Valve V6 is ideally a tubing pinch type valve for
reliability as beads may damage a conventional solenoid valve.
[0258] The prime mover in this embodiment is an air pump, such as
that made by Boxer Corp., which creates a vacuum condition in a
pressure vessel that acts as a vacuum trap. Fluid is then pulled
into this container when valve V6 is open.
[0259] Alternatively, a liquid pump can serve as the prime mover.
In this case, a filter should be placed in front of the pump to
block beads from entering the pump. If a liquid pump is used, the
pressure vessel is unnecessary as an unsealed waste container can
be used. Alternatively, a syringe pump, such as that sold by Kloehn
Inc., can serve as the prime mover (as shown in FIG. 94). In this
case, a filter should be placed in front of the syringe pump to
block beads from entering the syringe. A three way valve must be
used with the syringe pump so that after filling the syringe, fluid
the valve can be switched to then dump fluid to waste. If a syringe
pump is used, the pressure vessel is unnecessary as an unsealed
waste container can be used.
[0260] Referring to the valve state table shown as Table 1 below,
to describe the process of filling the cell, begin with a null
state of all valves closed and the pump off. The pump is turned on
to stabilize a vacuum condition in the pressure vessel. One of
valves V1, V2 or V3 is opened. Valve V4 is opened to direct fluid
into the cell. Valve V6 is opened, thereby pulling fluid from the
reagent bottles, through the cell and into the pressure vessel.
Valve V6 controls the fill cycle time and is held open for a
specified length of time, e.g., 1 second, calibrated to pull the
desired volume of fluid through the cell.
[0261] A bubble sensor, such as that made by Introtek, may be used
to aid in filling the cell with fluid, by ensuring that the fluid
line is free of air before ending the fill cycle. The bubble sensor
may also be used to detect if air is being pulled into the cell or
system by an improperly seated lid 410 or other air leak. An
optional bubble sensor may also be used near the reagent bottles to
detect when one of the reagent bottles are empty. Alternatively, a
level sensor, such as that made by The Madison Company, in each
reagent bottle may be used instead of the bubble sensor to detect
empty bottles. Also, another level sensor may be used to in the
waste container to detect a full containter.
[0262] Continuing the cycle, with the pump on, valve V6 closed and
V4 open, to move beads from the well plate into the cell, valve V4
is closed and either valve V5 or valve V7 is opened. Valve V7 is
only used if the single well to 8 output divider is intended to be
used. Valve V6 is opened, thereby pulling fluid from the reagent
bottles, into the sealed well plate, out the well plate into the
cell, pushing fluid out of the cell into the pressure vessel. Beads
are pulled along with the fluid from the well plate into the cell.
While excess fluid exits the cell, the beads remain due to the
manifold design within the cell.
[0263] To flush beads from the cell, the process of filling the
cell is repeated. Cycle time is set longer, e.g., 2 to 5 seconds,
for flush than for filling the cell, as several volume changes are
desired to clean fluid and beads from the cell. As the flush volume
is several times greater than the volume held within the cell, and
the fluid velocity is high, the beads are propelled out of the
cell, pass through valve V6 and enter the pressure vessel. A filter
in the pressure vessel can be used to capture the beads. Standard
household or industrial water filter housing makes an excellent
pressure vessel, as does bag filter housing, such as the Giant Bag
Housings by MetPro Corporation, Keystone Filter Division.
TABLE-US-00001 TABLE 1 Valve Number V1 V2 V3 V4 V5 V6 V7 Fill Cell
With Fluid OPEN OFF OFF OPEN OFF OPEN OFF Transfer Beads from 1 OFF
OPEN OFF OFF OPEN OPEN OFF Well to 1 Sector in Cell Transfer Beads
from 1 OFF OPEN OFF OFF OFF OPEN OPEN Well to 8 Sectors in Cell
Load Beads OFF OFF OFF OFF OPEN OFF OFF Flush Reagent #1 OPEN OFF
OFF OPEN OFF OPEN OFF Flush Reagent #2 OFF OPEN OFF OPEN OFF OPEN
OFF Flush Reagent #3 OFF OFF OPEN OPEN OFF OPEN OFF In Table 1, Off
= fluid flow is blocked; Open = valve passes fluid flow.
[0264] Referring to FIG. 99, an alternative fluidic circuit for
loading beads 8 from wells 400 in a known multi-well plate (e.g., a
96 well plate having 8 rows and 12 columns) to a multi-segmented
bead cell/chamber is shown. The bead cell is similar to that
described in copending U.S. patent applications Ser. No. (CyVera
Docket No. CV-0082 PR) and/or copending U.S. patent applications
Ser. No. (CyVera Docket No. CV-0086 PR). This system uses an air
pump to create a vacuum to pull the beads into the cell 402 (second
container) from the wells 400 (first container). This system also
uses a 3-way valve to route the various fluids into the cell 402 or
into the wells 400.
[0265] Referring to FIG. 100, an alternative fluidic circuit for
loading beads 8 from wells 400 in a known multi-well plate (e.g., a
96 well plate having 8 rows and 12 columns) to a multi-segmented
bead cell/chamber 402 is shown. The bead cell is similar to that
described in copending U.S. patent applications Ser. No. (CyVera
Docket No. CV-0082 PR) and/or copending U.S. patent applications
Ser. No. (CyVera Docket No. CV-0086 PR). This system uses an air
pump to create a vacuum to pull the beads into the cell 402 (second
container) from the wells 400 (first container). This system also
uses a cell having a filter or frit as described in the
aforementioned patent application to help collect the beads at the
entry of the cell prior to distributing the beads across the cell
for reading.
[0266] Referring to FIGS. 101-102, an example of an O-ring sealed
lid 410 that fits on top of the well (or first container) 400 that
would contain the beads 8. FIG. 102 shows a head having eight lids
410, one for each well of an eight row well plate and the tubes
406,408, as well as a housing to which they are all mounted.
[0267] Referring to FIG. 110, a cross-section of a head having 8
lids 410, engaged with eight wells, and also showing a housing and
springs and the tubes 406, 408.
[0268] Referring to FIG. 103, a fluidic circuit for loading beads 8
from wells 400 in a known multi-well plate (e.g., a 96 well plate
having 8 rows and 12 columns) to a multi-segmented bead
cell/chamber 402, similar to that described herein with FIGS. 98
and 99, except that this system uses a pipetting technique instead
of a "telegraph" technique for moving the beads. In that case, the
pipette is placed in a well 400 and beads are extracted into the
pipette tip. Then the pipette tip is moved and inserted into a
pipette port on the cell 402. Also, this embodiment uses a 3-way
valve for flow management.
[0269] Referring to FIGS. 108-109, instead of moving beads from one
well to one of the sectored cells in the cell 402, a 1 to 8 flow
manifold may be used to distribute beads from one well to eight
separated sectors in the cell. FIG. 109 (a) is a perspective view
and FIG. 109 (b) is a side cross-section view of the 1 to 8 fluid
manifold. This 1 to 8 manifold is also shown as one option in the
fluidics schematic if FIG. 98. The 1 to 8 manifold may be used to
move fluid (with or without beads) from one well or port to 8 wells
or port or used in as an 8 to 1 manifold to move fluid (with our
without beads) from 8 wells or ports to 1 well or port.
[0270] Referring to FIGS. 104-106, one technique for pipetting a
predetermined number of the beads 8 from a well 462 is as
follows:
[0271] 1. Start with a highly accurate estimate of the total number
of beads 8 in a large population in a separate container (not
shown). This can be done by aspirating a certain volume of beads 8
and knowing the packing density is around 40%. N=volume of beads
(ul).times.40%/volume per bead (ul/bead).
[0272] 2. Dispense the beads 8 into a known volume of buffer
solution, e.g., SSC, SDS, or any other buffer solution or desired
fluid in a vial or well 462.
[0273] 3. Calculate the concentration of the beads 8.
[0274] 4. Agitate the mixture of the beads 8 in the vial 462 by
repeatably and rapidly cycling the pipette 464 in the buffer
solution 462, thereby causing the beads 8 to mix and suspend
substantially "homogeneously" in the solution 462. The agitation
volume should be about 2-10% of the total volume and the rate of
agitation should be fast enough to suspend the beads in solution.
Also, it was found that in order to generate good fluid currents
and, thus, good bead mixing/suspension, the pipette tip should be
placed away from the center of the well 460, and near to the side
wall if possible. Note that the pipette tip should be inserted into
the liquid 462 such that the pipette tip is near the top of the
liquid when the fluid is fully aspirated. Therefore, as the liquid
level decreases from successive aspirations, the tip will need to
be placed deeper into the vial each time a new group of beads is
removed. Also, the size of the opening in the pipette tip
opening/orifice determines the velocity of the mixing currents for
a given agitation volume and rate. For example, a larger orifice
will result in lower velocities for the same rate and volume. We
have found that a tip with a small orifice (<about 600 um) works
well for the 28.times.250 micron beads, solution and volume used.
However, the tip orifice should not be so small (<about 300 um)
that the flow through the tip for the pressure generated and
decreased to the point where the velocities are too low to generate
good mixing and suspension of the beads.
[0275] 5. When the beads 8 are substantially "homogeneously" mixed
and suspended in the liquid, then the next (final) aspiration of
beads 8 should determine how many of the beads 8 are drawn from the
vial 460. The number of beads 8 drawn=concentration
(beads/ul).times.aspiration volume (ul).
[0276] 6. Dispensing the beads 8 into the target well 468 should
include a brief time delay of about 1-3 sec to allow the beads 8 to
fall to the bottom of the pipette tip before they are completely
dispensed into the target well 468.
[0277] In particular, referring to FIG. 104, for example, starting
with the vial 460 with about 1000 beads having a diameter of about
28 microns and a length of about 250 microns, in about 1000
microliters of known buffer solution 462, e.g., SCC,SDS. First
insert a pipette tip 464 into the liquid 462 such that the pipette
tip is near the top of the liquid when the fluid is fully
aspirated. Then, holding the pipette tip substantially still,
aspirate/dispense about 4 times with about 150 microliters over a
period of about 4 seconds; however other times may be used provided
sufficient bead mixing and suspension is achieved. Then, draw a
final aspiration of about 50 microliters. Then, transfer the
pipette to a target well 468 and wait about 1-3 seconds to allow
the beads to settle to the end of the pipette tip, then dispense
the beads 8 into the well 468. The size of the opening in the
pipette tip was 400 microns (0.4 mm) and the size of the well 460
was about 1000 micron liters, and the pipette tip was placed about
mid way between the center of the well 460 and the side wall.
[0278] Referring to FIG. 105, a picture of a Hamilton Syringe Pump
syringe pump used to pipette beads with the present invention is
shown. The pump having a storage buffer of 1.times.SSC, 0.1% SDS,
and using 200 microliter ultrafine points (VWR) pipette tips.
[0279] Referring to FIG. 106, a graph of syringe pump bead
pipetting results is shown using the process described herein. For
36 tests, the average number of beads removed each time was 18,
with a bead diameter of about 28 microns and length of 250 microns,
agitation volume of 150 microliters, final aspiration volume of 27
microliters, and a starting bead concentration of 0.68 beads per
microliter.
[0280] Referring to FIG. 107, a diagram of how a kitting process
may be performed with the present invention. A plurality of
containers or wells 500-504 are provided, each well having beads
with a specific code. For example, the well 500 has beads 8 with a
code of 345, as shown by the digital representation image 506, the
well 502 has beads 8 having the code of 8197, as shown by the
digital representation image 508, and the well 504 has beads 8
having the code of 15606, as shown by the digital representation
image 510. The plurality of wells 500-504 having the beads 8 can
create Multiplex Bead Kits 1 through N, each Kit in a separate
container 516-520, and each Kit having a predetermined number of
any one or more of the codes in the wells 500-504. The beads 8 may
be transported from the wells 500-504 using the any embodiment of
the present invention or using any technique now known or later
developed to move a predetermined number the beads 8 into the
containers 516-520 for the Kits. The predetermined number of beads
8 of each code in each kit may have a tolerance, e.g., +/-10 beads.
Other bead kit tolerances may be used depending on the application.
Referring to FIGS. 111-114, a method for making a "kit" consisting
of N unique codes, where N may range from 1 to 5000, represented by
M replicates (beads), where M may range from 5-100, can be
accomplished by a two step process, consisting of transferring a
small number (M) of beads from a vial or well containing beads of
all the same code to a target well or vial, then, combining the
small number of beads representing each code in the kit to a single
vial or well, thus forming the "kit". The first step, transferring
M beads from the source, could be performed in a 48,96 or 384 well
format using the pipetting approach previously described, or from
an arbitrary configuration of individual vials. It is recognized
that this can be done in parallel with a conventional multi-head
pipetting machine such as those found in many laboratories. The
second step of combining individual sets of N codes together to
form the final "kit" may be accomplished by either dispensing the
individual sets into a funnel-like device where the beads are
flushed into a single well or vial containing a filter bottom such
that copious amounts of fluid may be used to sufficiently flush all
the beads through the funnel, leaving substantially no beads
behind. Another approach, which accomplishes the combining effect,
is to "telegraph" (previously described) the beads representing
individual codes into a single vial or well all at once. This
process is very fast and highly efficient in terms of transferring
all the beads from the source to the destination. This two-step
process would enable "kits" to be made with an arbitrary number of
codes and represented by an arbitrary number of beads per code, in
a rapid and efficient manner.
[0281] Referring to FIG. 115 shows a perspective view of the 8
sector bead cell having 8 input tubes 408 which transport beads and
fluid from 8 cells to 8 corresponding sectors of the bead cell. It
also shows a 1 to 8 flow manifold which takes in fluid and
distributes it to 8 sectors in the cell.
[0282] In particular, FIG. 111 shows a multi-well plate having
beads which are pipetted individually to another multi-well plate
which are then telegraphed as a group of wells (as described
herein) to a filter well Kit container. FIG. 112 shows a multi-well
plate having beads which are pipetted as a predetermined group or
individual pipette tips to another multi-well plate, which are then
telegraphed as a group of wells (as described herein) to a filter
well Kit container. FIG. 113 shows an 8 to 1 manifold for receiving
8 pipette tips which will simultaneously dispense fluid and beads
into the manifold and the manifold combines the received fluid and
beads to a single output port which dispenses the fluid and beads
into the Kit container having a filter on the bottom to catch the
beads. The beads are then transferred to a final kitting container.
FIG. 114 shows a pipetting machine having a flush port for flushing
fluid through the pipette tip, which can be used after the beads 8
are dispensed into the manifold shown in FIG. 113, or whenever
flushing with a fluid is needed. In each of the above cases, the
Kit container may have a filter on the bottom to catch the beads
and allow the fluid to exit. The beads are then transferred to a
final kitting container. Alternatively, the container may be large
enough to hold the fluid and the beads and then the beads and a
portion of the fluid may be transported (e.g., by the telegraph
method described herein) to a smaller kit container if needed.
An Alternative Embodiment of the Fluidic Subsystem
[0283] FIGS. 118-133 show an alternative embodiment of the present
invention. In summary, FIGS. 118-121 show the basic achitecture and
governing design principles; FIGS. 122a to 125 show steps of the
overall method and the sequencing thereof; FIG. 126 shows details
related to a groove plate design; FIGS. 127a-d show basic
experiments; and FIGS. 128-133 show more detailed diagrams of
components of the basic architecture.
[0284] Consistent with that discussed above, the microbead platform
will perform biological assays on beads by attaching a type of
biomolecule to the beads then placing the beads in a vessel
containing sample material, which will react in varying degrees to
the biomolecules. The extent to which the sample reacts is
determined by measuring the intensity of a fluorescent tag
molecule, and the indentity of the fluorescent beads is determined
by reading its holographic code. Both fluorescence and code
detection methods place requirements on how beads are oriented
relative to the interrogation lasers and collection optics. The
purpose of the fluidic sub-system is to manage all fluid and bead
manipulation activities entailed in the interrogation process.
These include movement of beads from the microtiter plate to the
cell, alignment of beads in the cell and finally removal of beads
from the cell so that the next batch can be interrogated. The
fluidic system must also provide a means by which it can be cleaned
of all biological and chemical contamination.
[0285] The following 6 steps describe the basic functions of the
reader from a fluidic point of view: [0286] 1) Prime Cell [0287] 2)
Transfer Beads [0288] 3) Load beads into grooves [0289] 4) Scan
beads [0290] 5) Flush [0291] 6) Clean cell (after N cycles)
[0292] Steps 2-5 are performed every cycle on 8 wells at a time on
an 8.times.12 well plate. Therefore a full 96 well plate requires
12 cycles to complete. Step 1 is performed on start up and whenever
the fluidic system either accumulates too much air or to remove
persistent beads from grooves. Likewise, step 6 is performed when
an unacceptable level of fluorescent contamination has
accumulated.
[0293] FIG. 119 shows the governing design principles.
[0294] FIG. 120 shows a diagram of the fluidic system process flow
including the 6 basic steps.
[0295] FIG. 121 shows the fluidic system including the architecture
generally indicated as 1000 and major subsystems, including a
transfer tube or probe assembly 1012, a well plate 1012, a cell or
cell assembly 1008, a puffer tube or puffer tube assembly 1006 and
syringe pump 1001, consistent with that shown in detail below in
FIG. 118.
Fluidics Architecture
[0296] FIG. 118 shows in detail the basic fluidics architecture
generally indicated as 1000 for performing the 6 steps of the
alternative embodiment of the present invention, and includes the
following elements and functionality as set forth below:
[0297] The Syringe pump 1001 aspirates or dispenses from selected
valve position.
[0298] The Rotary valve 1002 rotates to select from 5
positions--Output to cell, Reagent 1, Reagent 2, Reagent 3,
Dispense Excess/Aspirate Air.
[0299] The Tube Assembly, Output to Cell 1003 is fluoropolymer
tubing to allow fluid to flow from syringe 1001 to the manifold
leading to cell chimney 1007.
[0300] The Manifold (1.times.8) 1004 divides flow from 1 input line
to 8 output lines leading to cell.
[0301] Check valves 1005: Check valves are connected to each of the
8 lines in the manifold. The check valves prevent fluid from
siphoning between lanes of the cell. Without the check valves, a
small height imbalance of the fluid in one probe versus another,
could cause the siphoning of fluid out of the line carrying the
shorter fluid path. This could then lead to a chain reaction where
all the fluid siphons out of the cell.
[0302] Puffer tube assemblies 1006: The puffer tubes are
constructed of silicone tubing with inner diameter 3/16 inch and
outer diameter 5/16 inch. Compression of the tubing with the puffer
block displaces fluid. The check valves prevent fluid from flowing
back to the pump. Also, even without the check valves, the syringe
is a stiff system which would prevent flow in this direction.
Therefore all flow from compression moves out through the cell and
out the probes. Release of the tubing then aspirates fluid through
the probes back into the cell. The silicone tubes were selected for
their elasticity and low compression set.
[0303] The Chimney 1007--The chimney 1007 is a molded part that is
critical to the transfer of beads into the cell and loading of
beads into grooves. The chimney 1007 terminates in the cell in a
narrow line shaped nozzle which we call the line port. This shape
provides a relatively flat flow velocity profile across the width
of the alley. The narrowness of the port (generally less than 2
bead lengths at the narrowest portion), prevents large slow moving
eddy regions when beads turn the corner from chimney to cell. Also,
the spacer is aligned with the back of the nozzle to prevent
significant dead zones of flow. Bead loading into grooves takes
place at flow velocities that are in the laminar regime away from
the immediate vicinity of the grooves. The chimney 1007 expands
into a wider region. The height of the chimney and width of the
expanded region were designed to limit the height to which beads
rise in the chimney during transfer. By limiting this height, beads
are not aspirated out of the chimney, which would lead to
cross-contamination in later cycles.
[0304] Cell Assembly is labelled 1008.
[0305] Top Plate 1008a: The top plate is the top optical window of
the cell sandwich. It contains a row of 8 holes for attachment of
the chimney to the 8 lanes of the cell. At the opposite end of the
alleys, it contains a row of 8 holes for attachment of the bead
ports.
[0306] Spacer 1008b--The spacer maintains a gap between the groove
plate and top window. It also seals and separates the 8 alleys from
each other and from the outside world. The spacer is made of a
silicone gasket. The gasket is attached to the two glass plates
under compression and heat to create a seal. The spacer thickness
is 0.015 inch or 380 microns. This thickness appears to be near an
optimum value for balancing competing needs. On one hand, the
thinner the gap the higher the velocity near the grooves, this aids
in bead loading with the puffer and in bead removal. On the other
hand, if the gap is too small, bubbles are not effectively cleared
from the cell. Note that other materials could serve as a
gasket.
[0307] Groove Plate 1008c--the groove plate arranges the beads in
an orderly fashion to be read by the reader optics. The groove
plate is made of fused silica and is produced by an RIE (reactive
ion etch) process. Fused silica is used for its low fluorescence,
permitting better sensitivity to low fluorescence signals. Several
other processes have been explored for constructing a groove
plate.
[0308] Bead Entrance/Exit Ports 1009--The bead tubes from the
probes terminate in this port. The gasket taper to a rounded cone,
with the port at the apex. The goal is to minimize dead volume, so
that beads maintain momentum as they enter and exit the cell
through the port. Each alley has a fluidically isolated port.
[0309] Bead Tubes, fluoropolymer 1010--The bead tubes carry beads
into the cell through the transfer process, and carry them out of
the cell during the flush process. Fluoropolymer tubing is used for
inertness, to minimize friction and reduce bubble adhesion.
[0310] Probe Assemblies 1011--The probes are connected and integral
with the bead tubes. The probes enter the well plate for
transferring beads into the cell. The probes are designed to
withstand "bottoming out" in the well plate and are spring
loaded.
[0311] Well Plate 1012
[0312] Water Tube Assembly 1013 [0313] Tube Assembly, External,
Water 1013a
[0314] Buffer Tube Assembly 1014 [0315] Tube Assembly, External,
Buffer 1014a
[0316] Alcohol Tube Assembly 1015 [0317] Tube Assembly, External,
Alcohol 1015a
[0318] Air & Fluid Excess Tube Assembly 1016
[0319] Tee 1016a
[0320] Air Inlet to Valve Check Valve 1016b-On syringe aspirate
from this valve position, allows air to enter the syringe. The
check valve blocks flow in the dispense direction
[0321] Air Inlet to Valve Check Valve 1016c--This check valve
allows flow in the dispense direction to waste, but blocks return
flow, to prevent aspiration from waste.
[0322] Waste Drain Tube Assembly 1017 [0323] Tube Assembly,
External, Waste Drain 1017a
[0324] Panel Connections 1018--Field connections for the customer.
Luer locks are preferred.
[0325] Reagent Bottles 1019
[0326] Bottle Caps 1019a
[0327] Tube Connections 1019b
[0328] Straws 1019c
[0329] Filters 1019d
[0330] Level Switches 1020--Sense reagent bottles empty below a set
point or waste bottle full over a maximum level.
[0331] Waste Bottle 1021
[0332] Waste/Wash Tray 1022--Divided into two sections--one for
dumping waste fluid and beads, and a section for washing the probe
tips. Spillage from the wash overflows into waste. Fluid may be
pumped using the auxiliary pump into the wash section to augment
cleaning and to add bleach.
[0333] Wash subsystem 1023
[0334] Auxiliary pump 1023a
[0335] Check Valve 1023b-Prevents back pressure on the auxiliary
pump.
[0336] A rotary valve selects among reagent and waste bottles, or
output to the cell. The syringe pump aspirates or dispenses to the
selected valve.
[0337] Either by hand or using laboratory automation, the user
places a 96 well micro-titre plate on the platform.
[0338] An actuator moves the platform with the plate into
position.
[0339] Transfer beads into cell [0340] a) Probe assemblies descend
to near top of well [0341] b) Slow compression of the puffer tubing
to displace fluid into the wells [0342] c) Probe assemblies move to
bottom of well [0343] d) Rapid release of puffer tubing to draw
fluid and beads into the bead tubes
[0344] FIG. 119 sets forth the the governing design principles.
Description of the 6 Steps
[0345] The 6 steps are described in detail, as follows:
1) The Prime Cell Step
[0346] FIGS. 122a-e show the basic sequence of the prime cell
step.
[0347] The purpose of the Prime Cell step is to configure the cell
and its associated fluidic components in a state that allows
effective transfer of beads from the well plate 1012 to the cell.
Such a state is characterized by having the entire fluidic system,
from the syringe pump to the probe tips filled with a buffer
solution and having substantially all the air removed from the
fluidic system, including both small air bubbles and larger
cavities. Once in this state, the fluidic system is considered
"stiff" from a fluidic point of view, and is capable of supporting
bead transfer, bead loading and bead flushing operations.
[0348] The following sequence, which relies on a syringe pump as
the motive for fluids through the system, is designed to prime the
cell:
[0349] Displace fluid in system with air,
[0350] Displace Air with Ethanol,
[0351] Displace Ethanol with Water, and
[0352] Displace Water with Buffer.
[0353] Each of the four basic elements of the prime cycle has a
specific purpose, as does the order of operations. The importance
of the first step, pushing air through the system to displace any
fluid that may already be in the system, was found to help with the
second step, ethanol purge; though it is still unclear why it
helps. Ethanol is the first fluid pushed through the system after
purging with air. Ethanol has a very low surface tension and is a
good wetting agent; properties that make it ideal for removing
bubbles throughout the system, especially in the cell where bubbles
trapped in the small cross-section device are most difficult to
remove. Once the interior surfaces of the fluidic system are wetted
with ethanol and the air bubbles are removed, water is pushed
through. Although the ethanol is highly effective at removing air
bubbles, the one source of trapped air ethanol cannot remove
resides in the chimney. The pocket of air trapped in the chimney is
a consequence of pushing fluid down the chimney rather than up the
chimney. The natural tendency of the air in the chimney is to rise
since it is less dense than all the fluids. When ethanol is pushed
through, it simply spills around the air pocket as it travels from
the top of the chimney to the bottom on its way to the cell. The
spilling effect, a result of very low surface tension, prevents the
air from being displaced by the liquid. To overcome this, water is
pushed through the system next. Because the ethanol wets all
surfaces the water can come through next and wet the surfaces by
simply displacing the ethanol rather than trying the wet dry
surfaces directly. Once the water displaces the ethanol, its high
surface tension allows it to form a meniscus at the top of the
chimney, which follows the shape of the chimney as it travel from
the inlet at the top of the chimney to the cell at the bottom.
Provided the inside diameter of the chimney never exceeds a
critical diameter (approximately 9 mm for a round geometry and less
for shapes that deviate from round), it is possible to support a
column of water above the air without spilling around the air
pocket. As the water is introduced into the chimney by the syringe
pump the pocket of air is continuously pushed down toward the
bottom of the chimney and eventually out through the cell and
finally through the probe tips. In addition to the critical
diameter, it is also important that surfaces and transitions inside
the chimney be smooth and continuous; asperities will tend to break
the meniscus as it travels slowly down the chimney. Once the water
is pushed entirely through the system and the cell is free of all
air both in the form of bubbles and cavities the sequence proceeds
to the final step, which is to simply displace the water with a
buffer solution. The system is now considered primed.
2) The Transfer Beads to Cell Step
[0354] FIGS. 123a-e show the basic sequence of the step for
transferring of beads 8 to the cell or well-plate 1008.
[0355] The transfer process refers to the movement of beads from
the well-plate 1012 to the cell 1008 through a path, which includes
the transfer tube, the cell and finally the chimney. In most cases,
beads begin their journey at the bottom of a round bottom well;
since they are denser than the buffer fluid they sink to the bottom
of the well. The method of transferring beads from the well plate
involves vacuuming them off the bottom of the well with an
open-ended tube attached to the cell, called a transfer tube. At
the distal end of the transfer tube is the probe tip which has
attached to it a cone-shaped vestige designed to enhance the flow
rate around beads that are more than a few tube diameters away from
the center of the tube, thereby enhancing the efficiency of the
transfer process. However, unlike a typical vacuum whereby the flow
rate is always in the same direction (i.e. into the vacuum) the
method employed here involves alternating the direction of the flow
and varying the rate of flow.
[0356] Responsibility for this action is a device called a
"puffer," which consists of a flexible silicone tube approximately
2'' long by 1/4'' diameter. The tube is connected in-line between
the syringe pump and the cell and is placed between two metal
surfaces, on of which moves in order to squeezed the tube. Fluid
rushes out of the tube when it's squeezed and back in when it's
released. Since one end of the tube is dead-ended at the syringe
and the other end (the part that goes into the well with the beads)
is open, the net flow is always through the open end, both inward
and outward.
[0357] Beads are transported from the well-plate to the cell by
repeating a cycle of slow contractions and fast expansions of the
puffer tube. Slow contractions re-set the puffer tube to a state
whereby a vacuum can be applied to the transfer tube (expansion),
thereby pulling beads toward the cell. While the flow rate is slow
liquid moves past the beads without carrying them very far in the
direction of the flow. While the flow is rapid, beads are
effectively moved in the direction of the flow. Therefore,
repeating the cycle causes the beads to acquire a net motion in the
direction of the fast flow. This method is employed to lift beads
out of the well plate and transport them to the cell during the
transfer process and move beads out on the groove plate during the
load process and remove beads from the grooves and flush them out
of the cell during the flush process.
[0358] Another key element of the fluidic architecture with regard
to the transfer process is the chimney. The chimney is made to have
a rapidly increasing inner diameter starting from the point at
which it is attached to the cell. The purpose of the large inner
diameter is to decrease the flow rate to the extent that beads
cannot travel past the chimney and become lost in tubing. The inner
volume of the chimney is designed to be 2 to 5 times larger than
the volume of fluid displaced by the puffer during the transfer
process. It was found that beads entering the chimney at high rates
of speed travel about 3/4 the height of the chimney before the flow
is reversed (slow contraction) which then pushes the beads to the
bottom of the chimney and even out onto the groove plate. After a
certain number of puffer cycles (10-15), the puffer stops and beads
fall under their own weight to the bottom of the chimney and pile
up in small rectangular opening called the line port. The
distribution of beads in the port is uniform across the opening,
which is important for the next step; bead load.
[0359] Beads are considered transferred after a set number of
(empirically determined) puffer cycles. Once the beads are
transferred excess cycles cause them to harmlessly rise and fall in
the chimney. Therefore, without a means of feedback, the transfer
process is always run with an excess number of cycles to ensure
that a high percentage of beads are transferred. Efficiencies that
range between 95 and 99.9% are obtained after about 15 cycles,
approximately 40 seconds. The proc0ess concludes with beads
settling to the bottom of the chimney on the groove plate in a pile
substantially uniform in distribution within the port, a
consequence of randomization caused by turbulent flow in the
chimney.
[0360] The port, which is defined as the opening of the chimney to
the cell, is rectangular in shape, approximately 6 mm long and 250
.mu.m wide. It is surrounded on three edges by the gasket, which
forms the perimeter of each of the 8 independent lanes. The fourth
edge is open to the lane leading to the grooved region. The back
edge of the port is aligned as closely as possible to the edge of
the gasket so as to minimize dead zones in the flow field caused by
eddy currents. Gaps that range from 0 to 50 .mu.m were found to
eliminate such dead regions behind the opening of the port where
beads could potentially become stuck. Similarly, the width of the
port opening is large enough to ensure beads don't form a log jam
but small enough ensure the velocity of the flow through any
portion of the opening is sufficiently large to carry beads out of
the port region during the bead load process. The range of openings
found to be effective were 200 to 400 .mu.m. The length of the port
opening, which spans nearly the entire 7 mm width of the lane, was
found to produce the most uniform distribution of beads in the
grooved region of any combination of port and gasket shapes. Other
geometries tried involved ports of various sizes of circles with
gaskets cut into linear tapers, horn shaped tapers and parabolic
shaped taper, which depending on the flow rate, produced either
narrow beam-like distributions or lobed distributions characterized
by a low density region of beads in the middle of the lane and high
density regions near the edges of the lane. Both types of profiles
produced unacceptably low total packing densities, a feature that
plays heavily into the overall throughput of the instrument. Unlike
the circular port shapes that rely on the flow field to produce
distribution functions, the rectangular port shape allows the beads
to form a uniform distribution across the width of the lane by the
simple process of mixing in the chimney then settling to the
bottom.
[0361] Another feature of the cell that plays in important roll in
the dynamics of bead transport is the thickness of the gasket. The
gasket not only defines the perimeter of the lane around the
grooved region and the ports, it also defines the height of the
column of fluid in the cell. With a density of 2.2 glass beads sink
in aqueous solutions, which means when they are in the cell they
will lay on the surface of the groove plate (the bottom of the
cell), where the velocity of the laminar flow is close to zero.
When the height of the laminar flow field (thickness of the gasket)
becomes very large compared with the diameter of the bead the
velocity of the flow intercepting the bead approaches zero.
Therefore, to maximize the interaction of the bead with the flow
field the gasket thickness should be kept as thin as possible.
[0362] Countering this requirement are two issues that occur when
the gasket is too thin. The first pertains to the persistence of
small air bubbles. The smaller the gap between the groove plate and
the top plate the harder it becomes to flush small air bubbles
away. It was found that a gasket thickness of less than 300 .mu.m
resulted in such problems. The second relates to the pressure drop
across the cell during the transfer cycle. Because the entire
pressure generated by the puffer during transfer drops across both
the cell and the transfer tube, since they're in series with each
other, the impedance of the cell cannot be much larger than the
transfer line. Otherwise the flow rate at the distal end of the
transfer tube will be insufficient to cause bead transport out of
the well. Therefore it is important to balance the impedance of the
transfer tube with the cell. Again, the minimum gasket thickness
was found to be around 300 .mu.m.
3) Load the Beads into the Grooves
[0363] FIGS. 124a-b show the basic sequence of the step for loading
of the beads 8 into the grooves of the well-plate 1008.
4) Scan the Beads in the Well-Plate
[0364] The step for scanning the beads in the well-plate 1008 is
consistent with that described above.
5) Flushing the Beads from the Grooves
[0365] FIGS. 125a-b show the basic sequence of the step for
flushing the beads 8 from the grooves of the well-plate 1008.
FIG. 126: The Groove Plate Design
[0366] FIGS. 126 a and b show the groove plate design.
FIGS. 127a-d: Bead Alignment Feasibility Experiments
[0367] FIGS. 127 a-d relate to bead feasibility experiments,
including FIG. 127a that shows bead alignment feasibility
experiments with performance requirements and drivers and the basic
parameters of the experiment; FIG. 127b that shows multiplex
ranges; FIG. 127c that shows bead loss feasibility experiments; and
FIG. 127d that shows bead flush feasibility experiments.
FIGS. 128-133
[0368] FIGS. 128-133 show more detailed diagrams of components of
the basic architecture 1001.
The Scope of the Invention
[0369] Unless otherwise specifically stated herein, the term
"microbead" is used herein as a label and does not restrict any
embodiment or application of the present invention to certain
dimensions, materials and/or geometries.
[0370] The dimensions and/or geometries for any of the embodiments
described herein are merely for illustrative purposes and, as such,
any other dimensions and/or geometries may be used if desired,
depending on the application, size, performance, manufacturing
requirements, or other factors, in view of the teachings
herein.
[0371] It should be understood that, unless stated otherwise
herein, any of the features, characteristics, alternatives or
modifications described regarding a particular embodiment herein
may also be applied, used, or incorporated with any other
embodiment described herein. Also, the drawings herein are not
drawn to scale.
[0372] Although the invention has been described and illustrated
with respect to exemplary embodiments thereof, the foregoing and
various other additions and omissions may be made therein and
thereto without departing from the spirit and scope of the present
invention.
* * * * *