U.S. patent application number 09/122544 was filed with the patent office on 2002-01-24 for detector and screening device for ion channels.
Invention is credited to COASSIN, PETER J., HAROOTUNIAN, ALEC TATE, PHAM, ANDREW A., TSIEN, ROGER Y., VUONG, MINH.
Application Number | 20020008867 09/122544 |
Document ID | / |
Family ID | 22403320 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020008867 |
Kind Code |
A1 |
TSIEN, ROGER Y. ; et
al. |
January 24, 2002 |
DETECTOR AND SCREENING DEVICE FOR ION CHANNELS
Abstract
The invention provides for a detector assembly. fiber assembly
and screening system for optical measurements.
Inventors: |
TSIEN, ROGER Y.; (LA JOLLA,
CA) ; COASSIN, PETER J.; (ENCINITAS, CA) ;
PHAM, ANDREW A.; (DEL MAR, CA) ; HAROOTUNIAN, ALEC
TATE; (DEL MAR, CA) ; VUONG, MINH; (SAN DIEGO,
CA) |
Correspondence
Address: |
AURORA BIOSCIENCE CORPORATION
C/O GRAY CARY WARE FREIDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121
US
|
Family ID: |
22403320 |
Appl. No.: |
09/122544 |
Filed: |
July 24, 1998 |
Current U.S.
Class: |
356/35 |
Current CPC
Class: |
G01N 21/253 20130101;
G01N 2201/0639 20130101; G01N 2021/6484 20130101 |
Class at
Publication: |
356/35 |
International
Class: |
G01N 021/00 |
Claims
We claim:
1. A method of simultaneously measuring at least two optical
properties of emitted light from at least one sample in a plurality
of addressable wells of a multiwell plate comprising the steps of,
iv) aligning a plurality of addressable wells of a multiwell plate
to a plurality of ball lenses; v) directing electromagnetic
radiation substantially coaxially through the symmetry axis of each
of said plurality of ball lenses, vi) detecting the emitted light
focused by said plurality of ball lenses from said at least one
sample.
2. The method of claim 1, wherein said electromagnetic radiation is
directed to said plurality of ball lenses by at least one
laser.
3. The method of claim 1, wherein said electromagnetic radiation is
directed to said plurality of ball lenses by at least one fiber
optic bundle.
4. The method of claim 1, wherein said emitted light focused by
said plurality of ball lenses is directed to at least one detector
through at least one fiber optic bundle.
5. A device, comprising: i) a liquid handler comprising at least
one jetting tip, said at least one pipetting tip comprising
programmable control of aspiration from a first plurality of
addressable wells of a first multiwell plate and programmable
control of dispensation into a second plurality of addressable
wells of a second multiwell plate, ii) an optical detector module
comprising at least one detector, said at least one detector being
in optical connection to said second plurality of addressable wells
of said second multiwell plate and, said detector module
simultaneously detecting at least two optical properties from each
well of said second plurality of addressable wells of said second
multiwell plate, iii) a programmable moving conveying surface to
align said first multiwell plate and said second multiwell plate to
said liquid handler, and said detector module, and to move said
first multiwell plate and said second multiwell plate into and out
of said device. iv) a data processing and control module for
coordinating the operation of said automatic measuring device,
wherein said data processing and control module coordinates said
programmable moving conveying surface to move said second plurality
of addressable wells of said second multiwell plate to said liquid
handler, wherein said liquid handler simultaneously dispenses into
said second addressable wells of said second multiwell plate and
said detector module simultaneously measures at least two optical
properties from each well of said second plurality of addressable
wells of said second multiwell plate, and wherein, said data
processing and control module collects data from said detector
module.
6. The device of claim 5. wherein said data processing and control
module intermittently collects data from said detector module.
7. The device of claim 5, wherein said liquid handler dispenses
into said second addressable wells of said second multiwell plate
and there is a predefined delay before said detector module
simultaneously detects at least two optical properties from each
well of a plurality of said second plurality of addressable wells
of said second multiwell plate.
8. The device of claim 5 wherein the optical property is light
emission at a particular wavelength.
9. The device of claim 5, further comprising a optical irradiation
module comprising at least one light source, wherein said at least
one light source irradiates said second plurality of addressable
wells of said multiwell plate.
10. The device of claim 5, wherein said at least one light source
irradiates said second plurality of addressable wells of said
multiwell plate intermittently.
11. The device of claim 5, wherein, said at least one light source
is programably controlled to irradiate said second plurality of
addressable wells of said multiwell plate at predefined times.
12. The device of claim 5, wherein said detector module further
comprises at least one fiber optic bundle.
13. The device of claim 8, wherein said at least one fiber optic
bundle comprises at least one trifurcated fiber optic bundle.
14. The device of claim 5, wherein said detector module further
comprises at least one ball lens.
15. The device of claim 14, wherein said at least one ball lens is
formed of a material selected from the group consisting of glass,
fused silica, quartz and sapphire.
16. The device of claim 15, wherein said at least one ball lens
further comprises an anti-reflective coating.
17. The device of claim 14, wherein said detector module further
comprises at least one trifurcated fiber optic bundle, said at
least one trifurcated fiber optic bundle having a diameter that is
one third the diameter of said at least one ball lens.
18. The device of claim 14, wherein said detector module further
comprises at least one trifurcated fiber optic bundle, said at
least one trifurcated fiber optic bundle comprising at least one
central optical fiber that is in direct optical communication with
said at least one light source, and wherein said at least one
central fiber optic bundle is coaxially aligned to said at least
one ball lens.
19. A device, comprising: i) an optical detector module comprising
at least one detector, said at least one detector being in optical
connection to a plurality of addressable wells of a multiwell plate
and, said detector module simultaneously detecting at any instant,
at least two optical properties from each well of said plurality of
addressable wells of said multiwell plate, ii) a optical
irradiation module comprising at least one light source, wherein
said at least one light source irradiates said plurality of
addressable wells of said multiwell plate, iii) a programmable
moving conveying surface to align said plurality of addressable
wells of said multiwell plate to said detector module, and to move
said plurality of addressable wells of said multiwell plate into
and out of said device, iv) an integration and control module for
coordinating the operation of said automatic measuring device,
wherein said data processing and control module coordinates said
programmable moving conveying surface to move said plurality of
addressable wells of said multiwell plate to said detector module,
wherein said detector module simultaneously detects at lest two
optical properties from each well of said second plurality of
addressable wells of said second multiwell plate and. wherein said
data processing and control module collects data from said detector
module.
20. The device of claim 19, wherein said data processing and
control module intermittently collects data from said detector
module.
21. The device of claim 19, wherein the optical property is light
emission at a particular wavelength.
22. The device of claim 19, wherein said at least one light source
irradiates said plurality of addressable wells of said multiwell
plate intermittently.
23. The device of claim 19, wherein, said at least one light source
is programably controlled to irradiate said plurality of
addressable wells of said multiwell plate at predefined times.
24. The device of claim 19, wherein said detector module further
comprises at least one fiber optic bundle.
25. The device of claim 24, wherein said at least fiber optic
bundle comprises a trifurcated fiber optic bundle.
26. The device of claim 19, wherein said detector nodule further
comprises at least one ball lens.
27. The device of claim 26, wherein said at least one ball lens is
formed of a material selected from the group consisting of glass,
fused silica quartz and sapphire.
28. The device of claim 27, wherein said at least one ball lens
further comprises an anti-reflective coating.
29. The device of claim 26, wherein said detector module further
comprises at least one trifurcated fiber optic bundle, said at
least one trifurcated fiber optic bundle having a diameter that is
one third the diameter of said at least one ball lens.
30. The device of claim 26, herein said detector module further
comprises at least one trifurcated fiber optic bundle, said at
least one trifurcated fiber optic bundle comprising a central
optical fiber bundle that is in direct optical communication with
said at least one light source, and wherein said central fiber
optic bundle is coaxially aligned to said at least one all
lens.
31. The device of claim 26 wherein said at least one ball lens is
about three times larger in diameter than said addressable wells of
said multiwell plate.
32. The device of claim 26 wherein said at least one ball lens is
about the same diameter as said addressable wells of said multiwell
plate.
33. An optical assembly, comprising a ball lens and a trifurcated
fiber adapted for dual optical interrogation and in optical
communication with said ball lens.
34. The optical assembly of claim 33, wherein said trifurcated
fiber comprises a first optically isolated emission bundle to
collect light, second optically isolated emission bundle to collect
light, and an excitation bundle.
35. The optical assembly of claim 34, wherein said ball lens is
separated from said trifurcated fiber by a transmission space.
36. The optical assembly of claim 35, wherein said ball lens
comprises a sapphire material.
37. The optical assembly of claim 36, wherein said ball lens
comprises an anti-reflective coating.
38. The optical assembly of claim 33, wherein said trifurcated
fiber comprises a first plurality of emission bundles for receiving
light of a first wavelength and second plurality of emission
bundles for receiving light of a second wavelength and said first
plurality of emission bundles and said second plurality of emission
bundles are randomly distributed in plurality of excitation
bundles.
39. The optical assembly of claim 33, wherein said trifurcated
fiber comprises a first set of bundles for transmitting light of a
first wavelength and second set of bundles for transmitting light
of a second wavelength and third set of bundles for transmitting
light of a third wavelength.
40. The optical assembly of claim 39, wherein said trifurcated
fiber is separated from said ball lens by a transmission space of
about . 1 mm to 1 mm.
41. The optical assembly of claim 35, wherein said ball lens
comprises either sapphire material or a silica material.
42. The optical assembly of claim 39, wherein said first set of
bundles and said second set of bundles are coaxially arranged with
respect to said third set of bundles.
43. An optical detection system comprising: a) a light source that
launches at least one predetermined wavelength of light. b) sample
holder, c) a ball lens at a predetermined interrogation distance
from said sample holder, d) a trifurcated fiber adapted for dual
optical interrogation and in optical communication with said ball
lens, and e) a detector that detects light of at least one desired
wavelength and in optical communication with said ball lens.
44. The optical detection system of claim 43, wherein said
trifurcated fiber comprises a first plurality of emission bundles
for receiving light of a first wavelength and second plurality of
emission bundles for receiving light of a second wavelength and
said first plurality of emission bundles and said light source
launches at least one predetermined wavelength of excitation light
at said sample holder.
45. The optical detection system of claim 43, wherein said ball
lens is at a predetermined transmission distance from said
trifurcated fiber and further comprising at least one positioner to
controllably change said predetermined transmission distance.
46. The optical detection system of claim 43, further comprising at
least one positioner to controllably change said predetermined
interrogation distance.
47. The optical detection system of claim 43, further comprising a
liquid handling unit to dispense liquids into addressable
wells.
48. The optical detection system of claim 43, further comprising a
light activation system for triggering liquid handling unit to
dispense liquids into addressable wells.
49. The optical detection system of claim 43, wherein said light
source launches light through said trifurcated fiber to the
location at least one addressable well in a sample in said sample
holder to monitor epifluorescence.
50. The optical detection system of claim 49, further comprising a
computer control system to manage interrogation of a sample.
51. The optical detection system of claim 49, further comprising a
sample transfer system to transfer at least one sample platform to
said sample holder.
52. The optical detection system of claim 49, wherein said sample
holder further comprises a positioning system.
53. The optical detection system of claim 43, wherein said ball
lens is at a predetermined transmission distance from said
trifurcated fiber that approximately corresponds to a focal
length.
54. The optical detection system of claim 43, wherein said
trifurcated fiber comprises an end and said end is generally at a
focal plane of said ball lens.
55. The optical detection system of claim 43, wherein an object to
be interrogated is generally at a focal plane of said ball
lens.
56. An optical fiber assembly, comprising a trifurcated fiber
comprising a first plurality of emission bundles for receiving
light of a first wavelength and second plurality of emission
bundles for receiving light of a second wavelength and said first
plurality of emission bundles and said second plurality of emission
bundles are non-randomly distributed in plurality of excitation
bundles.
57. The optical fiber assembly of claim 56, wherein said first set
of bundles and said second set of bundles are coaxially arranged
with respect to said third set of bundles.
58. The optical fiber assembly of claim 56. wherein said first set
of bundles is coaxially arranged with respect to said second set of
bundles.
59. A method of identifying a useful chemical, comprising
interrogating a sample comprising a chemical of interest with one
of the above claimed devices, and detecting the activity of said
chemical from optical signals from said device.
60. A method of development for a therapeutic, comprising
interrogating a sample comprising a chemical with of interest on of
the above claimed devices, detecting the activity of said chemical
from optical signals from said device, administering said chemical
or a chemical derived from the structure activity of said chemical
to a cell, invertebrate or mammal, assessing a therapeutic effect
of said administering and optionally adding a suitable
pharmaceutical carrier to said chemical for administration into a
vertebrate or human.
61. A chemical identified by a process of detecting optical signals
from one of the above claimed devices, wherein said chemical is in
a sample interrogated by said device.
62. A pharmaceutical composition, comprising a pharmaceutical
acceptable carrier and a chemical identified by detecting optical
signals from one of the above claimed devices, wherein said
chemical is in a sample interrogated by said device.
Description
[0001] This application claims the benefit of an earlier filing
date under 35 U.S.C. .sctn.120 to patent application entitled
"Detector and Screening Device for Ion Channels" Tsien et al.,
filed Jul. 17, 1998, docket number 08366/006001, of which the
present application is a continuation in part of application, which
is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention generally relates to devices and
methods for rapidly identifying chemicals with biological activity
in liquid samples, particularly automated screening of low volume
samples for new medicines, agrochemicals, or cosmetics.
BACKGROUND
[0003] Drug discovery is a highly time dependent and critical
process in which significant improvements in methodology can
dramatically improve the pace at which a useful chemical becomes a
validated lead, and ultimately forms the basis for the development
of a drug. In many cases the eventual value of a useful drug is set
by the timing of its arrival into the market place, and the length
of time the drug enjoys as an exclusive treatment for a specific
ailment.
[0004] A major challenge for major pharmaceutical companies is to
improve the speed and efficiency of this process while at the same
time maintaining costs to an absolute minimum. One solution to this
problem has been to develop high throughput screening systems that
enable the rapid analysis of many thousands of chemical compounds
per 24 hours. To reduce the otherwise prohibitive costs of
screening such large numbers of compounds, typically these systems
use miniaturized assay systems that dramatically reduce reagent
costs, and improve productivity. To efficiently handle large
numbers of miniaturized assays it is necessary to implement
automatic robotically controlled analysis systems that can provide
reliable reagent addition and manipulations. Preferably these
systems and the invention herein are capable of interacting in a
coordinated fashion with other systems sub-components, such as a
central compound store to enable rapid and efficient processing of
samples.
[0005] Miniaturized high throughput screening systems require
robust, reliable and reproducible methods of analysis that are
sensitive enough to work with small sample sizes. While there are a
large number of potential analysis methods that can successfully
used in macroscopic analysis, many of these procedures are not
easily miniaturizable, or lack sufficient sensitivity when
miniaturized. This is typically true because absolute signal
intensity from a given sample decreases as a function of the size
of the sample, whereas background optical or detector noise remains
more or less constant for large or small samples. Preferred assays
for miniaturized high throughput screening assays have a high
signal to noise ratios at very low sample sizes.
[0006] Fluorescence based measurements have high sensitivity and
perform well with small samples, where factors such as inner
filtering of excitation and emission light are reduced.
Fluorescence therefore exhibit good signal to noise ratios even
with small sample sizes. A particularly preferred method of using
fluorescence based signal detection is to generate a fluorescent
(emission) signal that simultaneously changes at two or more
wavelengths. A ratio can be calculated based on the emission light
intensity at the first wavelength divided by the emitted light
intensity at a second wavelength. This use of this ratio to measure
a fluorescent assay has several important advantages over other
non-ratiometric types of analysis. Firstly. the ratio is largely
independent on the actual concentration of the fluorescent dye that
is emitting fluorescence. Secondly, the ratio is largely
independent on the intensity of light with which the fluorescent
compound is being excited. Thirdly, the ratio is largely
independent of changes in the sensitivity of the detector, provided
that is that these changes are the same for the detection
efficiency at both wavelengths. This combination of advantages make
fluorescence based ratiometric assays highly attractive for high
throughput screening systems, where day to day, and, assay to assay
reproducibility are important.
[0007] Fluorescence assays that produce ratiometric emission
readouts have gained in popularity as the advantages of the method
have grown in acceptance. Changes in emission ratios at two more
wavelengths can be created through a number of distinct mechanisms
including electronic and conformational changes in a fluorescence
compound. Typically, these changes can occur in response to a
chemical reaction or binding of the fluorescent compound to a
particular ion such as a metal ion like calcium or magnesium, or
through a change in pH that influences the protonation state of the
fluorescent compound.
[0008] Alternatively ratiometric changes in emission can be
conveniently be obtained by exploiting the use of fluorescence
resonance energy transfer (FRET) from one fluorescent species to
another fluorescent species. This approach is predictable,
sensitive and can give rise to large ratio changes at two
well-defined and well spectrally resolved wavelengths. Furthermore
FRET can be generally applied to create ratiometric assays for a
range of activities. For example patent WO 96/30540 (Tsien)
describes a FRET based system to measure gene expression using a
fluorogenic substrate of beta lactamnase. Patent WO 96/41166
(Tsien) describes the use of a FRET based system to measure voltage
across the plasma membrane of a cell. Patent WO 97/20261 (Tsien)
describes the use of FRET between two fluorescent proteins to
measure intracellular protein. Such assays can be used with the
inventions described herein.
[0009] The present invention is directed towards the development of
improved optical systems and methods for simultaneously measuring
emission ratios from a plurality of samples with high sensitivity,
speed, reproducibility and accuracy. The present invention has
several important advantages over prior methods and devices adapted
to measure fluorescence emission sequentially from samples.
Firstly, the simultaneous measurement of emission ratios enables
rapid fluctuations in lamp intensity, bleaching of the fluorescent
dye, or cycle to cycle errors in the alignment of multiwell plates
to be corrected for, thereby enabling much smaller changes in ratio
to be reliably observed. Secondly, no mechanical movements are
necessary during ratio measurement, eliminating mechanical design
challenges. Thirdly ratios can be acquired very rapidly, as
required for dynamic measurements of membrane potential or calcium,
and are not limited by the speed of filter changing. Fourthly the
overall throughput and duty cycle are improved by eliminating dead
times for filter changeover. Finally, residual ratio
non-uniformities between addressable wells should be constant and
easily correctable by using emission ratios previously measured on
reference samples to normalize sample ratios in software.
SUMMARY OF THE INVENTION
[0010] The invention includes a method of simultaneously measuring
at least two optical properties of emitted light from at least one
sample in a plurality of addressable wells of a multiwell plate,
comprising the steps of,
[0011] i) aligning a plurality of addressable wells of a multiwell
plate to a plurality of ball lenses;
[0012] ii) directing electromagnetic radiation substantially
coaxially through the symmetry axis of each of said plurality of
ball lenses,
[0013] iii) detecting the emitted light focused by said plurality
of ball lenses from said at least one sample.
[0014] The invention includes an optical detection system,
comprising a light source that launches at least one predetermined
wavelength of light, sample holder, a ball lens at a predetermined
interrogation distance from said sample holder, a trifurcated fiber
adapted for dual optical interrogation and in optical communication
with said ball lens, and a detector that detects light of at least
one desired wavelength and in optical communication with said ball
lens. Typically, the optical detection system includes a
trifurcated fiber comprising a first plurality of emission bundles
for receiving light of a first wavelength and second plurality of
emission bundles for receiving light of a second wavelength and
said first plurality of emission bundles and said light source
launches at least one predetermined wavelength of excitation light
at said sample holder. The optical detection system may further
comprise at least one positioner to controllably change the spatial
relationship between the ball lens and the fiber or sample or
multiwell plate or a combination thereof. Typically, the light
source launches light through said trifurcated fiber to the
location at least one addressable well in a sample in said sample
holder to monitor epifluorescence. Preferably, the trifurcated
fiber comprises an end that is generally at a focal plane of the
ball lens.
[0015] The invention also includes an optical fiber assembly,
comprising a trifurcated fiber comprising a first plurality of
emission bundles for receiving light of a first wavelength and
second plurality of emission bundles for receiving light of a
second wavelength and said first plurality of emission bundles and
said second plurality of emission bundles are non-randomly
distributed in plurality of excitation bundles.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows one embodiment of a fluorescence measuring
device utilizing the detection system of the invention.
[0017] FIG. 2 shows one embodiment of a detection arrangement
according to the invention.
[0018] FIG. 3 shows a cross sectional view of the trifurcated fiber
optic bundles showing potential arrangements of the individual
fiber optic fibers. Excitation fibers being represented by X or
cross hatching, and emission fibers being represented by the
letters (A) for the first emission leg of a trifurcated fiber optic
bundle, and (B), for the second emission leg of a trifurcated fiber
optic bundle.
[0019] FIG. 4 shows several embodiments of a ball lens of the
invention in a cross sectional view depicting the light directing
ability of the lens to focus light from the sample, 400 and 401 to
the fiber optic bundle face plate 402. FIG. 4A. depicts a 5 mm
sapphire ball lens spaced 1 mm from the sample, FIG. 4B, depicts a
10 mm glass ball lens spaced 1 mm from the sample and FIG. 4C,
depicts a 20 mm glass ball lens spaced 1 mm from the sample.
[0020] FIG. 5 shows a perspective view of one embodiment of the
ball lens assemblies of the present invention. The ball lens 500,
is engaged by a ball lens holding assembly. 501 & 502, and
spring 503 to maintain accurate alignment of the ball lens and
fiber optic bundle 504. The mounting assembly for the assembly 505
mounts the assembly to the z-axis mover (see FIG.6)
[0021] FIG. 6 shows a perspective view of one embodiment of a ball
lens assembly Z-axis mover according to the invention. The stepper
motor 600, z-axis mounting assembly 601, cam 602 and 603, ball lens
assemblies 604, platform for ball lens assemblies, 605 guiding
pillar 606, switch 607 and trifurcated fiber optic bundle 608.
[0022] FIG. 7 shows a perspective view of one embodiment of a
filter changer of the invention. The filter holder enclosure 700
& 701, filter holder support 702 & 703, trifurcated fiber
optic assembly 704, photomultiplier (PMT) 705, support 706, holding
platform 707, and light tight O-ring 708.
[0023] FIG. 8A shows the rapid detection and continuous analysis of
voltage changes induced within a cell measured using one preferred
embodiment of the invention.
[0024] FIG. 8B shows a dose response curve of voltage changes
induced within a cell measured in response to the addition of an
ion channel blocker, using one preferred embodiment of the
invention.
[0025] FIG. 9 shows the use of one embodiment of a device
comprising the trifurcated ball lens assemblies of the invention to
screen for ligand gated ion channel receptor antagonists.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Definitions
[0027] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs.
Generally, the nomenclature used herein and many of the automation.
computer, detection, chemistry and laboratory procedures described
below are those well known and commonly employed in the art.
Standard techniques are usually used for engineering, robotics,
optics, molecular biology, computer software and integration.
Generally, chemical reactions, cell assays and enzymatic reactions
are performed according to the manufacture's specifications where
appropriate. The techniques and procedures are generally performed
according to conventional methods in the art and various general
references (see generally Lakowicz, J. R. Principles of
fluorescence spectroscopy, New York: Plenum press (1983), and
Lakowicz, J. R. Emerging applications of fluorescence spectroscopy
to cellular imaging: lifetime imaging, metal-ligand probes,
multi-photon excitation and light quenching. Scanning Microsc Suppl
VOL. 10 (1996) pages. 213-24, for fluorescent techniques, Sambrook
et al Molecular Cloning: A laboratory manual, 2.sup.nd ed. (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. for
molecular biology methods, Optics Guide 5 Melles Griot.RTM. Irvine
Calif. for general optical methods, Optical Waveguide Theory,
Snyder & Love published by Chapman & Hall, and Fiber Optics
Devices and Systems by Peter Cheo, published by Prentice-Hall for
fiber optic theory and materials.
[0028] As employed throughout the disclosure, the following terms,
unless otherwise indicated, shall be understood to have the
following meanings:
[0029] "Multiwell plate" refers to a two dimensional array of
addressable wells located on a substantially flat surface.
Multiwell plates may comprise any number of discrete addressable
wells, and comprise addressable wells of any width or depth. Common
examples of multiwell plates include 96 well plates, 384 well
plates and 3456 well nanoplates.
[0030] "Addressable well" refers to spatially distinct location on
a multiwell plate that may or may not have a physical
representation outside of the computer representation of the
plate.
[0031] "Chemical plate" refers to a multiwell plate containing
chemicals. such as stock solutions or dilutions thereof.
[0032] "Pharmaceutical agent or drug" refers to a chemical compound
or composition capable of inducing a desired therapeutic effect
when properly administered to a patient.
[0033] As used herein, "Optical property" refers a measurable
property of light, such as the intensity of emission light at a
particular wavelength, the intensity or degree of light
polarization, the transmittance of a compound or composition, or
the reflectance of a compound or composition.
[0034] "Ball lens" refers to a sphere, truncated sphere, cylinder,
or truncated cylinder of suitable transparent refractive material
and is usually a sphere.
[0035] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner.
[0036] "Optical Interrogation" means the process of detecting, or
measuring at least one optical property of a sample by at least one
detection device. A detection device typically would comprise a
photon detection device such as a photon multiplier tube (PMT).
DESCRIPTION OF ONE EMBODIMENT OF THE INVENTION
[0037] FIG. 1 shows one device of the invention. In one embodiment
of the invention, a device integrates a liquid handler 115, a
multiwell positioning stage 112 and a detection device comprising
the ball lens trifurcated fiber optic bundle ball lens assembly of
the invention. The vertical position of the optical assembly can be
adjusted by a stepper motor driven cam system (further described in
FIG. 6). The assemblies are lowered when the plate is moved in or
out of the system to allow the skirt of the microplate to pass over
the trifurcated fiber optic bundle ball lens assembly. The
assemblies (further described in FIG. 5) are raised once the plate
is in the system to maximize fluorescence detection efficiency.
Plates containing cells and compounds are loaded into the device
either manually or by a computer-controlled arm. The device then
takes the plate(s) into the light-tight reading area 116. A
multiwell plate positioning stage 112, such as a 500000 series,
Parker Hannifin Corp, Harrison City, Pa. may also be used to
control movement of the multiwell plate. In one embodiment, the
liquid handler 115 may be a modified Hamilton Micro Lab 2000 MPH,
Hamilton Co, Reno, Nev.), with at least 1 dispensing tip 114 and
associated pump 107, waste container 108 and diluent container 109.
The detector module 111, comprises 16 photomultipliers, (Hamamatsu
HC 124 -01) that are used to detect fluorescence emission at a rate
of 1 Hz or 10 Hz. Two photomultiplier tubes are used to detect
fluorescence from each well in a column of 8 wells allowing for
continuous emission ration detection. The blue-sensitive bi-alkali
photomultiplier tube is typically used to detect the shorter
wavelength emission (300 to 650 nm) while the multi-alkali
photomultiplier tube is used to detect longer wavelength emission
(300 to 850 nm). A computer 105, and graphical user interface 101
coordinates the functions of the liquid handler 115, multiwell
plate positioning stage 112, detector module 111 and data
collection 106. Data collection can be viewed through a monitor in
real-time via a computer monitor 103. A central power switch can be
used to switch the device on and off 104.
[0038] Referring to FIG. 2, monolayers of cells can be detected on
the bottom of microplate wells 206 by the common end of a
trifurcated optical fiber bundle 203. One leg of the each
trifurcated fiber bundle is used as an excitation source 201; each
of the eight excitation legs is fused into a single bundle 204 to
provide uniform light intensity to each of the eight trifurcated
bundles. The other two legs of the trifurcated fiber are used for
to detect fluorescence emission 214 and 213. The common end of the
trifurcated bundle is used to both excite and collect fluorescence
emission. Eight trifurcated fibers are used to detect two emission
channels from each well in a column of eight wells. A ball lens
205, (RB-707004, Bird Precision, Waltham, Mass.) may be included at
the top of the common end of the trifurcated fiber bundle to
increase the efficiency of fluorescence detection.
[0039] A 300 watt xenon arc lamp 201, e.g. CXP300, ILC Technology,
Sunnyvale, Calif.) with a parabolic reflector can be used as the
fluorescence excitation source. The excitation light is filtered by
two 2" diameter interference filters (e.g. 400RDF 15 or 480RDF20,
Omega Optical, Brattleboro, Vt.) and then focused by a lens 202 on
to the excitation leg of the trifurcated bundle. Both a IR heat
absorbing water filter 208 and shutter system 207 are also included
in the optical path to protect the interference filters from heat
damage. A 1" diameter "head-on" photomultiplier (e.g. HC124 series,
Hamamatsu Corp, Bridgewater, N.J.) tubes are used to detect the
fluorescence emission. Fluorescence emission from one leg of the
fiber bundle is detected by a blue-sensitive bi-alkalai
photomultiplier tube 209; emission from detected by a red-sensitive
multi-alkalai photomultiplier tube 210. Data is collected by the
A/D portion of a multifunction board (e.g. PCI-MIO-E-1. National
Instruments, Dallas, Tex.) in a Pentium.TM. based personal computer
212. The computer controls data acquisition, plate and fiber
movement, and shutter opening and closing 215.
[0040] Components of the Detection System
[0041] Typically, the greatest issue in fluorescent detection is
the reduction of background signal in the detection system. In this
case the detection system might comprise the excitation source and
associated optics (dichroic filters, interference filters,
focussing lenses, collimators, etc), the fiber optic assembly
(excitation and emission pathways and patterns), the substrate
containing sample to be analyzed, and the emission filters and
associated optics that direct the emission radiation to the
detection element. A key challenge in epifluorescent detection
(where the excitation light and emission light are directed and
collected from the same plane) is to maximize the excitation light
energy and the area (the field of view created by the excitation
light) this energy is delivered to the sample, without comprising
the efficient collection of the fluorescent emission or generating
a high background from the reflection of excitation radiation.
Typically, a tradeoff exists between optimal radiant energy, the
field of view illuminated by the excitation energy and the
fluorescent emission collection efficiency. For example, the
wavelength to be utilized for excitation may preclude the use of
certain materials (which might have other desirable features like
high numerical aperture (NA)) due to the incompatibility of the
material (high autofluorescence) with the excitation wavelength
that is required. The ultimate sensitivity of fluorescent detectors
is thus often limited by the amount, and drift in background noise
sources that are mainly generated at the various optical
interfaces, where reflection and refraction takes place.
[0042] Typically, a detection system of the invention includes a
ball lens in optical communication with a tetra, tri- or
bi-furcated fiber optic bundle that is in optical communication
with a photon detector. A liquid handling system is included to
provide predetermined dispensing at a designated time and volume.
Preferably dispensation and optical interrogation are coordinated
by computer control. Preferably, a first positioner controls the
interrogation distances between a ball lens and sample or sample
holder. A second positioner may be included (either with or without
the first positioner to control the transmission distance between a
ball lens and the fiber bundle.
[0043] Ball lenses and trifuarcated fiber optic assemblies
[0044] Ball lenses provide a compact, wide field of view lens, that
when coupled with a suitable fiber optic bundle arrangement
significantly reduces background noise. The ball lens trifurcated
fiber optic assemblies of the invention are effective in directing
light from the light source into the sample in the addressable
well, and of efficiently focusing emitted light from the sample to
the emission legs of the trifurcated fiber optic bundle. The light
focusing ability of ball lens of various sizes and compositions are
shown in FIG. 4. Such ball lens, comprising of glass, sapphire or
fused silica can collect emission light from up to about 65.degree.
from the optical axis, and maintain a high numerical aperture (NA)
even with an air gap of 50-100 .mu.m between the apex of the ball
lens and bottom of the multiwell plate. Furthermore, vignetting,
the variation in lens image intensity between the center to the
edge of the image, is minimal across the fiber optic face plate.
These advantages are further enhanced when a trifurcated fiber
optic bundle is used in conjunction with a ball lens. In one
embodiment, a plurality of excitation fibers are coaxially arranged
within the trifurcated fiber optic bundle and direct the light
substantially through the axis of symmetry of the ball lens. Under
these conditions, the excitation light is confined to
<11.degree. of the optical axis, therefore the side walls of the
addressable wells of the multiwell plate are not illuminated,
reducing background scattering and fluorescence. Additionally all
reflected light that returns from the multiwell plate with an angle
of <11.degree. of the optical axis enters the excitation fibers
and not the emission fibers. This sacrifices a small amount of
fluorescence but rejects light specularly reflected from both
surfaces of the multiwell plate bottom.
[0045] To select the preferred optical components for a specific
application it is often preferred to determine the signal to noise
or signal to background level for particular combinations of ball
lens and fiber optic assemblies. Signal to noise ratios can be
determined by comparing the magnitude of a defined amount of
fluorescent material measured in the optical system, compared to
the noise obtained by measuring an empty well under exactly the
same conditions. S/N ratios can be calculated at a range of
concentrations of the calibration material (for example,
fluorescein) to determine overall detector sensitivity and
linearity. Additionally. variability of measurements can be
expressed in terms of standard deviation (S.D.) and Coefficient of
Variance (C.V.) to establish reproducibility and alignment
sensitivity of each of the systems.
[0046] Additionally, it is preferred to select the spacing of the
fiber optic bundle to the ball lens and the ball lens to the
surface of the object to be optically interrogated. This can be
quickly accomplished by generating a graph of S/N ratio versus
interrogation or transmission distance for each of the optical
arrangements desired. In the same way, similar S/N ratio graphs can
be created for each of the combinations in response to different
illumination intensities and wavelengths of excitation light (in
conjunction with appropriate fluorescent samples). This analysis
would create a matrix of performance characteristics as represented
by S/N ratios that are used to select the optimal fiber optic
assemblies, ball lens size. composition, antireflective coating,
and spatial alignments of the components for specific
applications.
[0047] For example, fiber optic bundles may be created with varying
packing patterns of excitation and emission bundle numbers and
arrangements, and with different numbers of fibers in the
excitation legs and emission legs. In one embodiment, the packing
of the fibers of both the excitation and emission legs in the
bundle is randomly packed. In another embodiment the fibers are
arranged in specific and defined patterns, that confers a preferred
optical characteristic to the system. For example, in one preferred
embodiment discussed above, the excitation fibers could be bundled
to together centrally in the fiber optic bundle and the emission
filters arranged around the outside to create a coaxial fiber optic
bundle. Both bifurcated and trifurcated fiber bundles can be
produced in this preferred configuration. Alternatively, the
emission bundles could be arranged in small groups to create an
array, or radially around the axis of the bundle, or any other
symmetrical or non-symmetrical pattern.
[0048] Fiber optic assembles may also vary in total number of
fibers of both the excitation and emission legs and overall size.
The number of excitation fibers and the number of emission fibers
and the relative ratios of excitation fibers to emission fibers may
be widely varied depending upon the other components in the system,
as well as the type of light source, sensitivity of the detector
and size of the addressable well in which the sample is located.
The optimization of these factors is discussed herein. In one
embodiment a fiber optic bundle may contain a total of 341 fibers
of which 55 will be excitation fibers arranged randomly within the
fiber. In another embodiment the fiber may have 341 fibers of which
85 fibers are excitation fibers arranged in preferentially within
the center of the bundle, but also distributed randomly through the
remainder of the emission bundles. In another embodiment the fiber
may contain 112 fibers of which 7 fibers are excitation fibers
arranged in the center of the bundle, and the remaining emission
fibers are located around the excitation fibers. In another
embodiment the fiber may contain 1417 fibers of which 163 fibers
are excitation fibers arranged in the center of the bundle, and the
remaining emission fibers are located around the excitation fibers.
In another embodiment of the fiber optic bundle, the excitation
fibers are centrally located within the bundle and extend beyond
the point where the emission fibers terminate. In a preferred
version of this embodiment the emission filters terminate into a
liquid light guide that is in contact with the ball lens.
Typically, the percent of excitation to emission fibers in the
trifurcated fiber optic bundle ranges from about 5 to 10 percent
excitation fibers, or about 10-20 percent excitation fibers or
about 20-40 percent excitation fibers.
[0049] Additional optimization of the composition and size of the
ball lens is desirable for each fiber optic bundle arrangement and
application. Ball lens compositions of materials of different
refractive index and of different sizes can be easily evaluated
with each fiber optic arrangement to establish a preferred optical
arrangement. Ball lens of about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm,
8 mm, 10 mm and 20 mm diameter may be evaluated depending on the
size of the instrument and spatial requirements of the imaging
system desired. Suitable compositions of the ball lens include
fused silica, sapphire, optical glass (such as BK7, SF11 or LaSF9),
borosilicate glass or zinc selenide (for infrared applications).
Preferred compositions of the ball lens for use within the
wavelength range 300-750 nm include fused silica and sapphire. For
low light applications it is often necessary to include a suitable
anti-reflective coating such as single or multi-layer MgF.sub.2,
V-coatings, HEBBAR.TM. (High Efficiency BroadBand AntiReflection)
and Extended range AntiReflective coatings for a ball lens. To
determine the optimum composition, size and AntiReflective coating
(AR) of the lens different coatings, each size of ball lens above
made of each of the materials above would be prepared with each of
the AR coatings above, and in the absence of an AR coating and
evaluated as described herein.
[0050] Detectors
[0051] The detector can include at least one photon sensitive
surface or material for measuring photon emission, such as a
charged coupled device (CCD), photodiode, or a photomultiplier tube
(PMT). The detector can intensify the signal, and gate if desired,
using a photon intensifier. Preferably, the detector can utilize a
high quantum efficiency CCD without an intensifier for long
detection integration. Alternatively, the detector can utilize a
plurality of PMT's or multi-site PMTF's for simultaneous photon
detection and quantitation at two wavelengths from a plurality of
addressable wells.
[0052] The detector preferably functions in the epi-fluorescence
mode where the preferred illumination is from the bottom of the
multiwell plate and the preferred collection is also from the
bottom of the multiwell plate. The detector usually is capable of
three to four orders of magnitude of dynamic range in signal
response from a single reading. The detector, in one embodiment,
utilizes a CCD chip for imaging and detecting photons emitted from
the assay wells.
[0053] Light Source
[0054] In the preferred embodiment, the detector comprises a light
source assembly (e.g., Xenon lamp) that can be switched (either
manually or through computer control) between continuous and pulsed
(1 kHz) output depending upon power supply. Suitable light sources,
for example lasers, light emitting diodes (LEDs) or mercury arc
lamps are also suitable as are other light sources that are
described herein and other suitable sources can be developed in the
future.
[0055] Liquid Handlers
[0056] In one embodiment, the liquid handler can comprise a
plurality of nanoliter pipetting tips that can individually
dispense a predetermined volume. Typically, pipetting tips are
arranged in two-dimension array to handle plates of different well
densities (e.g., 96, 384, 864 and 3,456).
[0057] Usually, the dispensed volume will be less than
approximately 2,000 microliters of liquid that has been aspirated
from a predetermined selection of addressable wells and dispensed
into a predetermined selection of addressable wells. Preferably,
nanoliter pipetting tips can dispense less than approximately 500
nanoliters, more preferably less than approximately 100 nanoliters,
and most preferably less than approximately 25 nanoliters.
Dispensing below 25 nanoliters can be accomplished by pipetting
tips described herein. Preferred, minimal volumes dispensed are 5
nanoliters, 500 picoliters, 100 picoliters, 10 picoliters. It is
understood that pipetting tips capable of dispensing such minimal
volumes are also capable of dispensing greater volumes. The maximal
volume dispensed will be largely dependent on the dispense time,
reservoir size, tip diameter and pipetting tip type. Maximum
volumes dispensed are about 10.0 microliters, 1.0 microliters, and
200 nanoliters. Preferably, such liquid handlers will be capable of
both dispensing and aspirating. Usually, a nanoliter pipetting tip
(or smaller volume dispenser) comprises a fluid channel to aspirate
liquid from a predetermined selection of addressable wells (e.g.,
chemical wells containing drug candidates). Liquid handlers are
further described herein, and for some volumes, typically in the
microliter range, suitable liquid pipetting tips known in the art
or developed in the future can be used. It will be particularly
useful to use liquid handlers capable of handling about 1 to 20
microliter volumes when it is desired to make daughter plates from
master plates. Preferably, in such instances a liquid handler has a
dispensing nozzle that is adapted for dispensing small volumes and
can secure a tip having a fluid reservoir.
[0058] In one embodiment nanoliter pipetting tips comprise solenoid
valves fluidly connected to a reservoir for liquid from an
addressable chemical well. The fluid reservoir can be a region of a
dispenser that can hold fluid aspirated by the nanoliter pipetting
tip. Usually,. a tip reservoir will hold at least about 100 times
the minimal dispensation volume to about 10,000 times the
dispensation volume and more preferably about 250,000 times the
dispensation volume. The solenoid valves control a positive
hydraulic pressure in the reservoir and allow the release of liquid
when actuated. A positive pressure for dispensation can be
generated by a hydraulic or pneumatic means, e.g., a piston driven
by a motor or gas bottle. A negative pressure for aspiration can be
created by a vacuum means (e.g., withdrawal of a piston by a
motor). For greater dispensing control, two solenoid valves or more
can be used where the valves are in series and fluid
communication.
[0059] In another embodiment, nanoliter pipetting tips comprise an
electrically sensitive volume displacement unit in fluid
communication to a fluid reservoir. Typically, the fluid reservoir
holds liquid aspirated from an addressable chemical well.
Electrically sensitive volume displacement units are comprised of
materials that respond to an electrical current by changing volume.
Typically, such materials can be piezo materials suitably
configured to respond to an electric current. The electrically
sensitive volume displacement unit is in vibrational communication
with a dispensing nozzle so that vibration ejects a predetermined
volume from the nozzle. Preferably, piezo materials are used in
dispensers for volumes less than about 10 to 1 nanoliter, and are
capable of dispensing minimal volumes of 500 to 1 picoliter. Piezo
pipetting tips can be obtained from Packard Instrument Company,
Connecticut, USA (e.g., an accessory for the MultiProbe 104). Such
small dispensation volumes permit greater dilution, conserve and
reduce liquid handling times.
[0060] In some embodiments, the liquid handler can accommodate bulk
dispensation (e.g., for washing). By connecting a bulk dispensation
means to the liquid handler, a large volume of a particular
solution to be dispensed many times. Such bulk dispensation means,
for example a modified Hamilton Micro Lab 2200, (MPH, Hamilton Co,
Reno, Nev.) are known in the art and can be developed in the
future.
[0061] Positioners, Transitional Stages
[0062] Interrogation, aspiration or dispensation into multiwell
plates of different densities can be accomplished by automated
positioning (e.g. orthogonal) of a multiwell plate. Typically, the
multiwell plates are securely disposed on an orthogonal positioner
that moves the wells of a multiwell plate with a first density in
an X,Y position with respect to the X,Y position of the liquid
handler. Usually, the liquid handler will have an array of
aspiration and/or dispensation heads, or both. Many
aspiration/dispensation heads can operate simultaneously. The
orthogonal positioner will align each addressable well with the
appropriate dispensing head. Preferably, a predetermined location
(e.g., center) of a pre-selected addressable well will be aligned
with the center of a dispensing head's fluid trajectory. Other
alignments can be used, such as those described in the examples.
With a head substantially smaller than a well diameter, orthogonal
positioning permits aspiration or dispensation into plates of
different densities and well diameters.
[0063] An orthogonal positioner can typically match an array of
dispensing heads with an array of addressable wells in X,Y using a
mechanical means to move the addressable wells into position or the
liquid handler (e.g., dispensing heads) into position. Preferably,
arrays of addressable wells on a plate are moved rather than the
liquid handler. This design often improves reliability, since
multiwell plates are usually not as heavy or cumbersome as liquid
handlers, which results in less mechanical stress on the orthogonal
positioner and greater movement precision. It also promotes faster
liquid processing times because the relatively lighter and smaller
multiwell plates can be moved more quickly and precisely than a
large component. The mechanical means can be a first
computer-controlled servo motor that drives a base disposed on a X
track and a second computer-controlled servo motor that drives a Y
track disposed on the X track. The base can securely dispose a
multiwell plate and either a feedback mechanism or an accurate
Cartesian mapping system, or both that can be used to properly
align addressable wells with heads. Other such devices, as
described herein, known in the art or developed in the future to
accomplish such tasks can be used. Usually, such devices will have
an X,Y location accuracy and precision of at least .+-.0.3 mm in X
and .+-.0.3 mm in Y, preferably of at least .+-.0.09 mm in X and
.+-.0.09 mm in Y, and more preferably of at least .+-.0.01 mm in X
and .+-.0.01 mm in Y. It is desirable that such devices comprise
detectors to identify the addressable wells or multiwell plates
being orthogonally positioned. Such positioners for predetermined
X, Y coordinates can be made using lead screws having an accurate
and fine pitch with stepper motors (e.g., Compumotor Stages from
Parker, Rohnert Park, Calif. USA). Positioners (e.g. X, Y or Z) can
be used to move the detector assembly. the sample, liquid handler
or a combination there of.
[0064] Alternatively, the liquid handler can be disposed on a
Z-positioner, having an X,Y positioner for the liquid handler in
order to enable precise X,Y and Z positioning of the liquid handler
(e.g., Linear Drives of United Kingdom).
[0065] A reference point or points (e.g., fiducials) can be
included in the set up to ensure that a desired addressable well is
properly matched with a desired addressable head. For instance, the
multiwell plate, the orthogonal positioner or the liquid handler
can include a reference point(s) to guide the X,Y alignment of a
plate, and its addressable wells, with respect to the liquid
handler. For example, the liquid handler has a detector that
corresponds in X,Y to each comer of a plate. The plate has orifices
(or marks) that correspond in X,Y to the liquid handler's position
detectors. The plate's orifices allow light to pass or reflect from
a computer-controlled identification light source located on the
orthogonal positioner in the corresponding X,Y position. Optical
locators known in the art can also be used in some embodiments (PCT
patent application WO91/17445 (Kureshy)). Detection of light by the
liquid handler emitted by the orthogonal positioner verifies the
alignment of the plates. Once plate alignment is verified,
aspiration or dispensation can be triggered to begin. Stepper
motors can be controlled for some applications as described in U.S.
Pat. No. 5,206,568 (Bjornson).
[0066] The liquid handler will also typically be disposed on a
Z-dimensional positioner to permit adjustments in liquid transfer
height. This feature allows for a large range of plate heights and
aspirate and dispense tips, if desired, to be used in the sample
distribution module. It also permits the dispense distance between
a addressable well surface, or liquid surface in an addressable
well, and a liquid handler to be adjusted to minimize the affects
of static electricity, gravity, air currents and to improve the X,Y
precision of dispensation in applications where dispensation of a
liquid to a particular location in a addressable well is desired.
Alternatively, multiwell plates can be positioned on a
Z-dimensional positioner to permit adjustments in liquid transfer
height. Static neutralizing devices can also be used to minimize
static electricity. Generally, the liquid transfer height will be
less than about 2 cm. Preferably, small volumes will be dispensed
at a liquid transfer height of less than about 10 mm, and more
preferably less than about 2 mm. Occasionally, it may be desirable
to contact the tips with a solution in a controllable fashion, as
described herein or known in the art
[0067] Control of Z-axis Movement of Ball Lens Assembly
[0068] The ball lens assembly will also typically be disposed on a
Z-dimensional positioner to permit adjustments in interrogation
distance and transmission distance. For example through the use of
a stepper motor driven cam system or other positioners as described
herein. The assemblies are lowered when the plate is moved in or
out of the system to allow the skirt of the microplate to pass over
the trifurcated fiber optic bundle ball lens assembly. The
assemblies may be raised once the plate is in the system to control
the interrogation distance to improve fluorescence detection
efficiency. Alternatively, multiwell plates can be positioned on a
Z-dimensional positioner to permit adjustments in interrogation
distance. Typically the transmission distance between the ball lens
and fiber optic bundle would be fixed at a preferred distance, for
optimal fluorescence detection. In a preferred embodiment,
adjustments in transmission distance could be under programmable
control to optimize the sensitivity and reproducibility of
fluorescence measurements.
[0069] Control, Data Processing and/or Integration Modules
[0070] In one embodiment, a data processing and integration module
can integrate and programmably control a liquid handler module, and
a detector module to facilitate rapid processing of the multiwell
wells. In a preferred embodiment the data processing and
integration module can also control the distance of the ball lens
assembly to the sample (the interrogation distance), and the
distance of the ball lens to the trifurcated fiber optic bundle
(the transmission distance). To manage information in the system,
the data processing and integration module comprises elements to
store, manage and retrieve data, including a data storage device
and a processor. The data storage device can hold a relational
database, an array of physical disk drives (e.g., random access
disk drives), and a connection to other system components via a
network. A data storage device can, for instance, store a
relational database for environmental, diagnostic, and drug
discovery applications. For instance, one particularly useful
relational database can be provided by Oracle, and the network can
be a TCP/IP (transfer communication protocol) ethernet LAN (local
area network).
[0071] Interface Designs
[0072] In most embodiments, it will be advantageous to integrate
and operably link device of the invention with at least one other
workstation, usually a sample transporter. The integration can be
accomplished with a computer and associated control programs to
instruct the translational stage and sample processor to operate
coordinately. Alternatively, the device may be used without
directly integrating to another workstation by tracking addressable
wells in groups and either mechanically or manually transporting
multiwell plates to another workstation where the multiwell plates
are identified. For instance. the device of the invention may be
directly integrated and operably linked to a storage and retrieval
module and sample transporter, and indirectly linked to an
integration and control module. While this approach is feasible,
especially for lower throughputs, it is not desirable for higher
throughputs as it lacks direct integration that can lead to faster
throughput times. Manual operations also are more frequently
subject to error especially when processing large numbers of
samples. Preferably, the device of the invention can be integrated
with other workstations and operate in a mode with minimal or
substantially no manual intervention related to transferring
multiwell plates to other work stations.
[0073] Usage Modes
[0074] The detector module and its system are often capable of many
different operating modes that facilitate drug discovery assay
requirements. These operating modes can include: single excitation
wavelength with single emission wavelength detection, single
excitation wavelength, dual emission wavelength detection,
sequential or dual excitation wavelength with dual emission
wavelength detection and ratio measurement determination,
sequential dual excitation wavelength with four emission wavelength
detection and ratio measurement determination, homogeneous time
resolved fluorescence with single excitation wavelength and single
emission wavelength detection, homogeneous time resolved
fluorescence with single excitation wavelength and dual emission
wavelength detection and ratio determination measurement,
homogeneous time resolved fluorescence with sequential dual
excitation wavelength and dual emission wavelength detection and
ratio determination measurement, absorbance (e.g. dual),
transmittance (e.g. dual), reflectance, dual sequential excitation
wavelengths and single emission wavelength detection with ratio
determination measurement, luminescence measurement at a single
wavelength with luminescence measurement at dual wavelengths,
luminescence measurement at dual wavelengths with a ratio
determination, and time resolved fluorescence emission (intrinsic
dye properties with or without a binding event).
[0075] Fluorescence Measurements
[0076] It is recognized that different types of fluorescent
monitoring systems can be used to practice the invention with
fluorescent probes, such as fluorescent dyes or substrates.
Preferably, systems dedicated to high throughput screening, e.g.,
96-well or greater microtiter plates, are used. Methods of
performing assays on fluorescent materials are well known in the
art and are described in, e.g., Lakowicz, J. R., Principles of
Fluorescence Spectroscopy, New York: Plenum Press (1983). Herman,
B., Resonance Energy Transfer Microscopy, in: Fluorescence
Alicroscopy of Living Cells in Culture, Part B, Methods in Cell
Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego:
Academic Press (1989), pp. 219-243; Turro, N. J., Modern Molecular
Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.
(1978), pp. 296-361 and the Molecular Probes Catalog (1997), OR,
USA.
[0077] Preferably, FRET (fluorescence resonance energy transfer) is
used as a way of monitoring probes in a sample (cellular or
biochemical). The degree of FRET can be determined by any spectral
or fluorescence lifetime characteristic of the excited construct,
for example, by determining the intensity of the fluorescent signal
from the donor, the intensity of fluorescent signal from the
acceptor, the ratio of the fluorescence amplitudes near the
acceptor's emission maxima to the fluorescence amplitudes near the
donor's emission maximum, or the excited state lifetime of the
donor. For example, cleavage of the linker increases the intensity
of fluorescence from the donor, decreases the intensity of
fluorescence from the acceptor, decreases the ratio of fluorescence
amplitudes from the acceptor to that from the donor, and increases
the excited state lifetime of the donor. Preferably, changes in
signal are determined as the ratio of fluorescence at two different
emission wavelengths, a process referred to as "ratioing."
Differences in the absolute amount of probe (or substrate) cells,
excitation intensity, and turbidity or other background
absorbencies between addressable wells can affect the fluorescence
signal. Therefore, the ratio of the two emission intensities is a
more robust and preferred measure of activity than emission
intensity alone.
EXAMPLES
Example 1
Construction and Testing of a Ball Lens Trifurcated Fiber Optic
Assembly
[0078] Arrangements of ball lenses and trifurcated fibers can be
tailored to their intended application. To determine the
appropriate arrangement of fiber optic bundles and ball lens a
series of experiments can be conducted to determine the highest
signal to noise ratio, preferred sensitivity, lowest background,
preferred field of optical interrogation or excitation or a
combination thereof.
[0079] For example, one embodiment of a trifurcated fiber optic
assembly adapted for miniaturized sample analysis of a 1 mm well
diameter with a variable interrogation layer of approximately 0.1
mm to 2.0 mm could comprise the following arrangement. A ball lens
made of fused silica material coated with an antireflective coating
such as HEBBAR, with a diameter of about 3 mm. A trifurcated fiber
optic assembly, optically coupled to the ball lens comprising 91
fibers, of which 7 fibers in the center are for excitation, and the
remaining fibers are for emission collection. The fiber assembly
being about 3 mm in diameter and packed into a hexagonal ferrule to
maximize packing efficiency and ease of assembly. The emission
fibers (42 for each optical property to be measured) are selected
so as to maximize collection efficiency, signal intensities and
signal to noise or signal to background) properties of the
assembly. The following table (Table 1) illustrates the effect that
the spatial position of the ball lens to the fiber assembly has on
signal-to-background ratios. In this example, the interrogation
fluorescent sample was kept constant as the transmission distance
of the ball lens to the fiber optic assembly was varied. Different
signal (10 nM fluorescien-10 nM F) to background (BB) ratio's were
obtained from which an optimal transmission distance could be
selected, which under these conditions was 0.482 mm
1TABLE 1 Emission filter: 535RDF30 (no long pass) Sample volume: 2
microliters, hand loaded. Ball lenses: Fused silica. Distance from
lens to plate = 0. (Signal-background)/ Fiber-to-lens Empty Buffer
(BB) 10 nM F background (mm) (mV) Background Signal (10 nM F-BB)/BB
0 10.8 12.4 119 9 0.384 9 7.95 117.67 14 0.482 7.8 6.95 130.33 18
0.533 8 7.25 126 16 0.584 7.9 7.15 122 16 0.71 4.9 5.8 34 5
[0080] Further subsequent analysis (Table 2) of this particular
system demonstrates that the interrogation distance between the
test sample relative to the ball lens fiber optic assembly can be
relatively widely varied (within the range 0 and 0.152 mm) without
significantly impacting signal to background values. Thus,
demonstrating one useful aspect of the invention.
2TABLE 2 Distance from fiber to lens = 0.584 mm lens-to-plate Empty
Buffer (BB) 10 nM F (mm) (mV) (mV) (mV) (10 nM F-BB)/BB 0 7.9 7.15
122 16 0.152 7.8 7.2 126 17 0.203 8.4 8.15 119.5 14 0.305 10 8.9
118 12
[0081] In order to analyze the influence of the fiber optic
arrangement on Signal to Background measurements, a series of fiber
optic bundle assemblies were compared under identical conditions.
In this case, a 3 mm HEBBAR coated sapphire ball lens was utilized
with four different fiber optic assemblies (as shown in FIG. 3) .
These assemblies contained varying numbers and arrangements of both
excitation and emission fibers. Fiber optic bundle assembly
performance was assessed by measuring the minimum detectable level
(MDL) of a particular dye as described herein.
3TABLE 3 Number Number MDL of of Fiber Description in nM of Ex Em
Assembly of Assembly Fluorescein Fibers Fibers Diameter Assembly #1
0.50 7.00 84.00 2.6 mm Assembly #2 0.86 1.00 6.00 1.2 mm Assembly
#3 0.50 3.00 16.00 1.5 mm Assembly #4 0.50 7.00 30.00 1.6 mm
[0082] Surprisingly, as shown in Table 3, assemblies #3 and #4
perform (as shown in FIG. 3) as well as assembly #1. Even though
assembly one is significantly larger than the other assemblies, and
contains more fiber optic fibers (FIG. 3). This indicates that
there is a relationship between fiber optic bundle size and the
optimal ball lens size, and that this optimally about equal or 1 to
3 times greater in diameter than the fiber optic assembly. The ball
lens can thus aid in reducing the complexity or quantity of fibers
required in a fiber optic assembly for optimal detection
sensitivity particularly when the need to reduce the size of the
fiber optic assembly is important in a miniaturized system.
[0083] In a similar example to above, (Table 4) the fiber assembly
is kept constant but the size of the ball lens is varied. In this
example, a 3 mm diameter coaxial fiber optic--(3 mmCoAX) assembly
containing 112 fiber arranged with 7 XXF200/210/235T fused silica
excitation fibers in the center of the assembly surrounded by 105
XXF200/210/235T fused silica emission fibers is used with three
different size sapphire ball lenses. of external diameters of 3 mm,
5 mm and 10 mm. As Table 4 illustrates. sensitivity as measured by
MDL determinations, sensitivity improves as ball lens size
increases. Surprisingly, sensitivity increases by a factor of 15 in
moving from a 3 mm ball lens to a 10 mm ball lens.
4TABLE 4 Different Size Ball Lens Experiment Description Glass
Bottom Plate with Solution Standards Relative Cermax 300 w Xenon
Lamp, dual excitation and Sensitivity emission filtering Molar
equivalents of Sapphire Ball Lens with HEBBAR coating dye PMT-3
mmCoAX-10 mmHB-DF 1.125E-12 PMT-3 mmCoAX-5 mmHB-DF 1.108E-11 PMT-3
mmCoAX-3 mmHB-DF 1.727E-11
[0084] Protocols, Materials and Methods for the Experiments
Herein
[0085] The minimum detectable level (MDL) was calculated by
generating a fluorescein calibration curve that enabled the
concentration of fluorescein that was equivalent to 4 times the
standard deviation of the buffer blank to be calculated. Buffer
blank (BB) measurements are determined from the variance of
readings from many buffer blanks and would be affected by well to
well variability, positioning, artifacts and other errors.
[0086] MDL2 is determined from the variance of repeated
measurements of the same buffer blank and presumably would be
affected only by the noise of the detector.
[0087] The optical detectors utilized to evaluate fluorescent
intensity in the experiments were either a Hamamatsu PMT and
associated electronics as described in the Fluorocount instrument
or a Hamamatsu HC 135-01/100 Mhz PMT sensor module with embedded
micro controller and RS-232-C interface. This sensor operates in
the 360-650 nm range. A Labview.TM. software interface was Aritten
to control the PMT and acquire data. When needed. excitation
radiant power was measured using a Newport Corporation 1835-C power
meter equipped with a 818-UV NIST traceable silicon photodiode
detector. The filters used in these experiments were obtained from
Chroma Technology Corporation or Omega Optical Inc., with the
exception of neutral density filters that were obtained from Oriel
Corp. In general and except where noted, all experiments were
conducted with the Hamamatsu PMT were double filtered on the
excitation and emission ends with a 0.2 neutral density filter
sandwiched in between the interference filters. The excitation
filters were HQ475/40 +0.2 ND+D480/20x. The emission filters were
535DF35t +0.2ND+535DF.
[0088] Three different light supplies were utilized for the
experiments and are identified as appropriate in the experimental
results section. The first was a Quartz Tungsten Halogen (QTH)
light obtained from Cole-Palmer Model #H-41700-00. The second was a
Cermax LX-300W xenon Arc with integral parabolic reflector. The
third was a 175 watt Xenon Arc lamp with ultra stable power supply
from Hamamatsu.
[0089] All of the ball lenses were coated with HEBBAR. Experiments
with the Hamamatsu PMT were performed on a Newport Corporation
optical bench with Vibration dampening. Certain fixtures and mounts
were specially made through local machine shops and others were
obtained through Newport Corporation.
[0090] Three types of plates were utilized. The standard plate is a
96 well black top clear bottom polystyrene plate filled with
fluorescent standards. The glass bottom plates were specially
modified black polystyrene 96 well plates with 175 micron glass
bottoms. 384 well black polystyrene glass bottom plates were
utilized for the 384 well readings. These specially modified plates
were obtained from polyfiltronics/Whitman.
[0091] The fiber optic assemblies were composed of fused silica
coated with a black polyimide coating obtained from Fiberguide. The
individual fibers are 200/220/240 in microns in diameter for the
core/cladding/coating respectively unless otherwise specified in
particular experiments.
Example 2
Sensitivity, and Background Testing of Optical Assemblies of One
Embodiment of the Invention
[0092] This example demonstrates the ability of the optical
assemblies to achieve uniform illumination of the addressable wells
while at the same time avoiding illumination of the sides of the
well and the illumination of adjacent wells. This leads to reduced
background fluorescent signals caused by reflections from the plate
and wells and reduces punch through of excitation light through
emission filters into detection system, yet enables high
sensitivity detection at two wavelengths.
[0093] This is exemplified by the determinations of minimum
detectability of a number of fluorescent standards. For example,
the minimum detectable fluorescein level achieved using a detector
incorporating the optical system of the invention was better than
50 pM fluorescein in a standard 96 well plate Table 5. Emission was
collected at wavelengths centered at both 535 nm and 580 nm. Both a
blank solution and a solution containing 2 nM fluorescein were
measured. The minimum detectable level (MDL) was calculated by
generating a fluorescein calibration curve that enabled the
concentration of fluorescein that was equivalent to 4 times the
standard deviation of the buffer blank to be calculated. Because
the detector typically measures changes of brightness within a
single well, the standard deviations for readings within the same
well at 1 Hz for eight seconds are given. It was found that the
plate material also affected the MDL levels. Both buffer and
fluorescein statistics were determined from 100 .mu.L volumes in 40
wells (5 columns of 8 wells) of a 96 well plate. Fluorescein MDL
levels measured using 480.+-.10 nm excitation 535.+-.17.5 nm and
580.+-.30 nm emission filters.
5TABLE 5 Plate bottom material Glass Glass Polystyrene Polystyrene
Emission wavelength 535 nm 580 nm 535 nm 580 nm MDL (nM
fluorescein) 0.0017 0.0085 0.034 0.072
[0094] Because the fluorescent dyes typically used with the
detector are not excited at fluorescein wavelengths, more relevant
standards are the fluorophores 3-glycine chlorocoumarin (3GCC) and
rhodamine 101 Table 6. MDL measurements were determined for these
fluorescent dyes as described above except that a fluorescent dye
solution also containing 25 nM fluorophores 3-glycine
chloro-coumarin and 4.mu.M rhodamine 100 was used in place of the
fluorescein solution
6 TABLE 6 Fluorescent dye 3GCC rhodamine 101 Plate bottom material
Polystyrene polystyrene Emission wavelength 460 nm 580 nm
Excitation Wavelength 400 nm 400 nm MDL (nM fluorescent dye) 0.181
20.8
[0095] Two Dye MDIL levels measured both excited using a 400.+-.7.5
nm filter. The 3GCC fluorescence was collected using a 460.+-.22.5
nm filter; the rhodamine 101 fluorescence was collected using a
580.+-.30 nm filter. Because 400 nm excitation light is not optimal
for the efficient excitation of rhodamine 101, the MDL level for
this fluorophore is relatively high when compared to those for 3GCC
or fluorescein.
[0096] A desirable feature of the invention is that the fiber optic
bundle and ball lens assemblies enable efficient excitation of the
addressable wells, as well as the ability to simultaneously measure
at least two optical properties. The average measured excitation
intensity at 400 nm emerging through each of the fiber optic
bundles and ball lens of the invention is 529.+-.75 .mu.W when
using two 400.+-.7.5 nm excitation filters. The light source used
was an ILC CXP300 300 watt Xenon arc lamp, with 6.3 mm anti
reflection coated fused silica ball lenses at the common ends of
each of eight 5.18 mm diameter bundles containing 333 fibers, 111
fibers from each leg of the randomly packed trifurcated bundles.
Light power was measured using a calibrated Newport 1835-C
powermeter.
[0097] The use of the trifurcated fibers and ball lens system, and
the calculation of an emission ratio significantly reduces
experimental noise, eliminates relative excitation variability
between the 8 fiber optic assemblies in the detector and leads to
smaller C.V.s and improves the dynamic range of FRET based assays.
A major additional advantage is the removal of addition artifacts
to enable continuous measurements during reagent addition. In these
phenomena, intensities of cells loaded with fluorescent dye often
decline upon reagent addition. This decline in intensity may be due
to some cells being washed from the detection area during addition
and mixing of reagents. By taking the emission ratio at two
separate wavelengths these artifacts are eliminated. In the data
set below Table 7, a mammalian neuronal cell line was loaded using
a FRET based fluorescent dye system. In this example, the majority
of the emission change was in the 460 nm channel. For this
experiment, monolayers (e.g. about 5 to 50 micrometers) of
mammalian cells were plated into the first 6 columns of a 96 well
plate. The emission intensities measurements were made at two
wavelengths and the ratio determined for 35 seconds at 1 Hz for
each of the 8 wells in a column. Reagent solutions were added
following the 12.sup.th read of each column. In this example, test
cells stimulated by depolarization by addition of 100 uL high
potassium solution (90 mM K). Control cells received normal Hank's
buffered saline solution (HIBS) without high potassium to test for
addition artifacts. Both intensity data and emission data were
normalized versus basal levels to account for well to well
variations in cell number or loading brightness and normalized
basal levels prior to reagent addition. This enables direct
comparisons between intensity data and ratiometric data.
7TABLE 7 Comparison of ratio versus non-ratio measurements (Data
Normalized to Initial Values) Non-ratio measurements Ratio
measurements 460 alone Emission Ratio (460/580) HBS HBS AV 91.7% AV
99.2% SD 4.0% SD 1.4% CV 4.4% CV 1.4% HiK HiK AV 139.3% AV 155.6%
SD 6.3% SD 4.9% CV 4.5% CV 3.1% Difference 47.6% Difference
56.5%
[0098] As can be seen in Table 7 both the standard deviations (SD)
and coefficient of variation (CV) are about 30% lower for the
ratiometric data (1.4% compared to 4.4% for HBS controls). There is
also an addition artifact (91.7% of basal) in the intensity data
but not in the emission data (99.2% of basal) for the control HBS
additions. Because the emission ratio data factors both the
increase in intensity at 460 nm and the slight decrease in
intensity at 580 nm upon depolarization with HiK solution, the
dynamic range of the emission ratio data is larger than that of the
single intensity data. Statistics were determined from 24 wells (3
rows of 8 wells).
Example 3
Determination of Na+ Dependent Depolarization in Mammalian
Cells
[0099] An advantage of the use of the optical assemblies of the
invention is the ability to rapidly measure two wavelengths
simultaneously thereby enabling the rapid analysis of cellular
responses. In the field of voltage sensing, the use of rapid
depolarization measurements has several significant advantages over
earlier relatively slow depolarization approaches that are subject
to artifacts and reduce throughput of the assay. The use of the
device thus allows the development of sensitive and rapid assay
systems for membrane voltage measurements in whole cells. As shown
in Table 8, these assays are highly sensitive, reliable and able to
discriminate relatively small changes in membrane potential with
high precision.
[0100] Mammalian neuronal cells were grown in F12 complete medium
supplemented with 20% fetal bovine serum. Prior to experiments
cells were washed twice with sodium free buffer (140 mM
N-methyl-D-glucamine, 10 mM HEPES, pH 7.2, 0.34 mM
Na.sub.2HPO.sub.4, 0.4 mM MgCl.sub.2, 0.5 mM KH.sub.2PO.sub.4,
5.37mM KCl, 1.26 mM CaCl.sub.2, 2 g/L D-glucose). The cells were
then harvested using calcium and magnesium free buffer and washed
once. The cells were then loaded with the fluorescent dye CCl-DMPE
(4 .mu.M for 30 minutes at room temperature) and washed in sodium
free buffer. The fluorescent dye DiSBAC.sub.2 was then added to the
cells, after 30 minutes the plates were loaded onto the device of
the invention. All wells treated with a channel opener to open
Na.sup.+ channels and maintained in low Na.sup.+ solution. Each
well contained approximately 10.sup.5 cells. The average, standard
deviation, and standard error of the mean are given in the for
cells treated with three different experimental conditions, low
extracellular sodium (0 Na.sup.+, buffer with high sodium (HBS) and
buffer with high sodium in the presence of a sodium channel blocker
(HBS-TTX). The results (Table 8) show that the change in membrane
voltage exerted by the change in extracellular sodium ion
concentration can be accurately measured using a device comprising
the present invention.
8 TABLE 8 0 Na+ HBS HBS-TTX AV 99.5% 130.2% 98.9% SD 0.9% 4.3% 0.9%
C.V. 0.9% 3.3% 0.9% Difference N/A 30.7% -0.6%
Example 4
Determination of Dose Response Relationships
[0101] The large ratio changes observed with this method enable the
creation of highly reproducible assays and provide signals large
enough for dose response curves to be generated. Furthermore
because the device can acquire data continuously, the responses
from the individual wells can be viewed as a function of time. FIG.
8A shows the real time changes in voltage for individual wells.
[0102] The cells were stained and handled as described in Table 8.
All wells contained a sodium channel agonist . Traces show the
effect of different doses of an anesthetic RS-105914-197 on
blocking Na.sup.+ channel activity in the neuronal cells. FIG. 8B
shows the dose response of the anesthetic RS-105914-197 for
blocking sodium channel activity using the device of the invention.
The data represents the average of 4 wells and the error bars
represent the CV value. 1 mM of the drug completely blocks the
Na.sup.+ induced depolarization. These results with error analysis
are summarized in Table 9. The results show that a device of the
invention provides a sensitive, accurate and reproducible method of
measuring relatively small changes in fluorescence
measurements.
9 TABLE 9 Mean S.D. C.V. 0 mM RS-105914-197 186.4% 3.7% 2.0% 0.1 mM
RS-105914-197 172.2% 8.2% 4.7% 0.3 mM RS-105914-197 117.7% 5.6%
4.8% 1.0 mM RS-105914-197 100.5% 2.6% 2.6%
Example 5
Screening for Antagonists
[0103] To test whether it would be possible to identify antagonists
on a single plate assay in a screening format, a protocol was set
up. This protocol was designed such that compound additions were
made from a chemical multiwell plate to the test plate, and the
wells read continuously during compound addition FIG. 9
demonstrates the use of the device to identify antagonists in a
screening mode. The results show ratio vs well number for the assay
run in antagonist screening mode. End ratio values were averaged as
in FIG. 9. A test antagonist (100 .mu.M) was used to test screening
sensitivity. Vehicle control wells had an equivalent final
concentration of DMSO as the test antagonist treated wells.
Negative controls received an addition of buffer instead of
agonist. In this experiment, cells (HEK-293) were washed with assay
buffer (160 mM NaCl, 10 mM HEPES pH 7.4, 0.34 mM Na.sub.2HPO.sub.4,
0.4 mM MgCl.sub.2, 0.5 mM KH.sub.2PO.sub.4, 5.37 mM KCl, 1.26 mM
CaCl.sub.2, 2 g/L D-glucose) and loaded with the fluorescent dyes
CC2-DMPE and DiSBAC.sub.2 as described in Table 8.
[0104] Publications
[0105] All publications. including patent documents and scientific
articles, referred to in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication were individually incorporated by
reference.
[0106] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
* * * * *