U.S. patent application number 11/734662 was filed with the patent office on 2007-08-16 for quantified fluorescence microscopy.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Jean I. Montagu.
Application Number | 20070190566 11/734662 |
Document ID | / |
Family ID | 23990242 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190566 |
Kind Code |
A1 |
Montagu; Jean I. |
August 16, 2007 |
Quantified Fluorescence Microscopy
Abstract
The present invention relates to calibration apparatuses,
methods or tools used in microscopy. A calibration tool for
fluorescent microscopy includes a support, a solid surface layer
including a fluorescent material, and a thin opaque mask of
non-fluorescent material defining reference feature openings having
selected dimensions exposing portions of the surface layer. A first
type of the calibration tool may include an adhesion promoter
facilitating contact between the surface of the support and the
solid surface layer including the fluorescent material, which is in
contact with the opaque mask. A second type of the calibration tool
may include the thin opaque mask fabricated (with or without an
adhesion promoter) onto the support, and the solid surface layer
including the fluorescent material located on the thin opaque
mask.
Inventors: |
Montagu; Jean I.;
(Brookline, MA) |
Correspondence
Address: |
AFFYMETRIX, INC;ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3420 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
|
Family ID: |
23990242 |
Appl. No.: |
11/734662 |
Filed: |
April 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11510631 |
Aug 28, 2006 |
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11734662 |
Apr 12, 2007 |
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11139375 |
May 26, 2005 |
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11510631 |
Aug 28, 2006 |
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10214221 |
Aug 7, 2002 |
6984828 |
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11139375 |
May 26, 2005 |
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PCT/US01/04336 |
Feb 9, 2001 |
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10214221 |
Aug 7, 2002 |
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09500626 |
Feb 9, 2000 |
6472671 |
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10214221 |
Aug 7, 2002 |
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Current U.S.
Class: |
435/6.19 ;
435/287.2; 438/1 |
Current CPC
Class: |
G02B 21/34 20130101;
G01N 21/64 20130101; G01N 21/6452 20130101; B01J 2219/00585
20130101; G01N 2035/1037 20130101; G02B 21/16 20130101; G01N 21/278
20130101; B01J 2219/00527 20130101; G01N 35/1065 20130101; B01J
2219/00387 20130101; B01L 3/0244 20130101; B01L 3/0251 20130101;
B01J 2219/00605 20130101; B01J 2219/00659 20130101; G01N 21/274
20130101; B01J 2219/00677 20130101; B01J 2219/00596 20130101; B01J
2219/00691 20130101; B01J 2219/0059 20130101; B01J 2219/00612
20130101; C40B 60/14 20130101; G01N 21/6458 20130101; B82Y 30/00
20130101; B01J 2219/00364 20130101; Y10T 428/24355 20150115; G01N
2035/1034 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 438/001 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 3/00 20060101 C12M003/00; H01L 21/00 20060101
H01L021/00 |
Claims
1. A calibration device for confirming or calibrating a
biopolymeric array optical scanner, said device comprising: a
polymer layer comprising at least one fluorescent agent, wherein
said device has minimal local and global nonuniformities and is
dimensioned for placement in an optical scanner.
2. The device according to claim 1, wherein said at least one
fluorescent agent is distributed substantially uniformly throughout
said polymer.
3. The calibration device according to claim 1, wherein said
polymer is a polyimide.
4. The calibration device according to claim 1, wherein the
thickness of said polymer layer ranges from about 2 microns to
about 250 microns.
5. The calibration device according to claim 1, wherein the
thickness of said polymer layer ranges from about 1 micron to about
10 microns.
6. The calibration device according to claim 1, wherein said at
least one fluorescent agent absorbs and emits light in the portion
of the electromagnetic spectrum to which a photomultiplyer tube of
said optical scanner is sensitive.
7. The calibration device according to claim 1, wherein said at
least one fluorescent agent absorbs and emits light in the
wavelength range selected from the group consisting of ultraviolet,
visible and near infrared.
8. The calibration device according to claim 1, wherein said
polymer layer is a spin-coated polymer layer.
9. A method for calibrating a biopolymeric array optical scanning
system, said method comprising: (a) illuminating a surface of a
calibration device with at least one light source, wherein said
calibration device is a calibration device according to claim 1;
(b) obtaining fluorescence data from said surface of said
calibration device; and (c) calibrating said optical scanning
system based upon said fluorescence data.
10. The method according to claim 9, wherein said step of
illuminating comprises illuminating said surface of said
calibration device in the portion of the electromagnetic spectrum
to which a photomultiplyer tube of said optical scanner is
sensitive.
11. The method according to claim 9, wherein said step of
illuminating comprises illuminating said surface of said
calibration device in the wavelength range selected from the group
consisting of ultraviolet, visible and near infrared.
12. The method according to claim 9, wherein said step of obtaining
fluorescence data comprises detecting a signal related to the
intensity of emitted light from said fluorescent agent.
13. The method according to claim 9, wherein said fluorescent
agent(s) is distributed substantially uniformly throughout said
surface.
14. A method for performing a hybridization assay, said method
comprising: (a) calibrating an optical scanner with a calibration
device, wherein said calibration device is calibration device
according to claim 1, (b) performing a hybridization assay with at
least one array, and (c) scanning said array with said calibrated
optical scanner.
15. A method for manufacturing a calibration device, said method
comprising spin-coating a composition onto a substrate to produce a
calibration device according to claim 1.
16. A calibration device for confirming or calibrating a
biopolymeric array optical scanner, said device comprising: a
polymer layer comprising at least one fluorescent agent, wherein
said device has minimal local and global nonuniformities; and a
transparent substrate; wherein said device is dimensioned for
placement in an optical scanner.
17. The calibration device according to claim 16, wherein said
transparent substrate is glass.
18. The calibration device according to claim 17, wherein said at
least one fluorescent agent is distributed substantially uniformly
throughout said polymer.
19. The calibration device according to claim 16, wherein the
thickness of said polymer layer ranges from about 1 micron to about
10 microns.
20. A calibration device for confirming or calibrating a
biopolymeric array optical scanner, said device comprising: a
polymer layer comprising at least one fluorescent agent, wherein
said device has minimal local and global nonuniformities; and a
substrate; wherein said device is dimensioned for placement in an
optical scanner.
21. The calibration device according to claim 20, wherein said at
least one fluorescent agent is distributed substantially uniformly
throughout said polymer.
22. The calibration device according to claim 20, wherein the
thickness of said polymer layer ranges from about 1 micron to about
10 microns.
23. An optical scanner comprising a calibration device according to
claim 1.
24. A calibration device for confirming or calibrating a
biopolymeric array optical scanner, said device comprising: a
polymer layer comprising at least one fluorescent agent, wherein
said device is dimensioned for placement in an optical scanner, and
wherein said at least one fluorescent agent is distributed
substantially uniformly throughout said polymer.
25. The device according to claim 24, wherein said polymer layer is
of uniform thickness.
26. A method for manufacturing a calibration tool for a fluorescent
microscope that is useful to image an array, comprising providing a
support, providing a solid surface layer including a fluorescent
material, and fabricating a thin opaque mask of non-fluorescent
material defining reference feature openings having selected
dimensions exposing portions of the surface layer; forming the
solid surface layer by depositing it onto the support, comprising
depositing an adhesion promoting layer onto the support for forming
the solid surface layer including the fluorescent material, the
deposition includes evaporating or sputtering, the depositing
includes spin coating to form the solid surface layer including the
fluorescent material.
27. A method for using a fluorescent microscope to image a
bioploymer array, comprising exciting the array at 473 nanometers,
producing 1/2 volt at a photomultiplyer tube in the microscope,
using a fluorophore having a fluorescent efficiency that is
approximately 1.times.10.sup.-6 or higher, and using a calibration
tool.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/139,375, filed May 26, 2005 which is a
continuation of U.S. patent application Ser. No. 10/214,221, filed
on Aug. 7, 2002, now U.S. Pat. No. 6,984,828, which is a
continuation of PCT Application PCT/US01/04336, filed on Feb. 9,
2001, and U.S. patent application Ser. No. 10/214,221 is also a
continuation-in-part of U.S. patent qpplication Ser. No.
09/500,626, filed on Feb. 9, 2000, now U.S. Pat. No. 6,472,671. The
disclosure of the above-mentioned applications is considered part
of, and is incorporated by reference in the disclosure of this
application.
TECHNICAL FIELD
[0002] This invention relates to microscopy and more particularly
to calibrating a position of a sample with respect to optical
elements of a microscope and/or to calibrating fluorescence
detection in microscopes.
BACKGROUND
[0003] Testing or calibration targets are employed to evaluate
system performance of conventional microscopes. These are used to
establish a baseline between different microscope systems and to
characterize image quality in terms of its conventional components
resolution, contrast, depth of field and distortion. Common
offerings for conventional microscopy are the USAF Field Resolution
target, the USAF Contrast Resolution Target, the Star Target, the
Ronchi Ruling Targets, Modulation Transfer Function Targets, Depth
of Field Targets, and Distortion and Aberration Targets. There are
others.
[0004] The targets are typically printed or vapor deposited
patterns on plastic or glass substrates. The optical features on
the target are preferably finer than the resolution of the optical
system being tested. While it is desirable that the reference
features have dimensions or parameters an order of magnitude
smaller than those of the specimens to be examined by the
microscope, practitioners have had to accept reference dimensions
or parameters only 4 or 5 times smaller than those of the specimens
to be examined.
[0005] Fluorescent microscopy of specimens is different from and
more demanding than conventional microscopy because it is based on
relatively low-level fluorescent emissions excited by illumination
of the specimen, typically employing confocal arrangements for
detecting the relatively weak signal through a pin hole or the
like. An example is the detection of fluorescence from dried liquid
spots containing possibly fluorescing biological material, the
dried spots being essentially at the focal plane of the instrument
(dried spot thickness less than a few microns). Another example is
fluorescence from a biological microarray such as from a
GeneChip.RTM. biological array product, as produced by Affymetrix,
Inc., in which the fluorescing material is of relatively
insignificant thickness.
[0006] For testing or calibration targets for fluorescence
microscopy, besides the numerous conventional components of image
quality, there is the requirement of testing the optical efficiency
of the system in respect of fluorescence emission.
[0007] This introduces significant complications, as fluorescence
involves an excited photochemical effect, to produce a voltage or
signal level in the detector, that introduces signal to noise ratio
considerations that interact with measurements of the various
optical components involved in the calibration. In general the
signal to noise ratio must be at least 3 to 1 to obtain
satisfactory operation.
[0008] It has been an unsolved problem, to find a calibration
target that adequately simulates the fluorescent activity which it
is desired to quantify over a broad range of instruments and
conditions of use. It is wished to simulate fluorescing specimens
that generally lie within the depth of field of the microscope, and
in the case of micro dots of biological material, lie essentially
at a plane, e.g., in a depth of only a few microns or even
substantially less. As the dimensions of individual specimens to be
imaged become increasingly smaller as microarray technology
advances, the significance of not having a suitable calibration
tool has become increasingly severe.
[0009] The difficulties for fluorescent microscopy is that, without
the desired degree of calibration, it becomes difficult to compare
the results obtained in biological or other research performed with
different instruments, thus creating serious difficulties in
comparing and coordinating the results of different laboratories,
whether the laboratories be at different institutions, or separate
laboratory facilities within the same institution. Likewise, even
with a given instrument, the uncertainties of calibration can
introduce errors in the measurement of important actions such as
proportional expression, etc. In particular the lack of good
reference and calibration is felt at the forefront of research
where results are so new and there has been standards. The
development of true quantified fluorescence microscopy can fulfill
this need.
[0010] On the other hand, the availability of a strong calibration
tool will likely to open the possibilities of inexpensive and
reliable fluoresence instrumentation and procedures for the
clinical setting for diagnosis and treatment.
[0011] Existing calibration tools for conventional microscopy do
not satisfactorily fill the needs of fluorescence microscopy. A
number of special techniques have been offered
[0012] One technique, offered by Max Levy Reprographics, uses a
layer of organic fluorescent material e.g. of 3 micron thickness,
having fluorescence emission across a broad wavelength spectrum,
deposited on a non-fluorescent glass substrate such as synthetic
quartz. A suitable pattern is then etched away into the fluorescent
material, so that the critical edges of the reference are defined
by the exposed edges of the fluorescing material. One shortcoming
of this technology is that the minimum thickness of the fluorescent
material that can be deposited is of the order of 3 micron and,
with such thickness, the edges of the pattern do not etch squarely.
The finest reference details that can be formed in this material
are believed to be approximately 4 micron width lines, spaced apart
8 microns on center. This is unsatisfactory for calibration with
respect to instruments employing conventional 5 micron spot size
and is an order of magnitude greater than required to evaluate
optical spots that are 1/2 micron in diameter, achievable with a
microscope having an 07 NA objective in air, or 1/4 micron diameter
achievable with a 14 NA, oil immersion objective. The relatively
large thickness of the fluorescent layer poses problems of edge
definition, particularly because the fluorescent rays emit at acute
angles to the surface and can be blocked by the edges of the
material, or on the other hand, the edges themselves fluoresce, to
produce confusion.
[0013] Another technique for testing a fluorescent microscope uses
as a substrate a fluorescent glass on which is deposited a very
thin metal layer e.g. a few hundred Angstrom thick. Preferably a
nickel layer is employed. A suitable pattern is subsequently etched
in the metal to create fine features, as small as 1/2 micron
dimension. Whereas this technique does not have the foregoing edge
problem, I have realized that there are shortcomings to this
approach, owing to the fact that the glass constitutes a
significant fluorescing volume, i.e., a substantial thickness, 1
millimeter, far exceeding the depth of field. (Notably, for a spot
size of 5 or 11/2 micron, the depth of field is typically about 50
micron and 45 micron, respectively, and progressively less for
smaller spot sizes). The fluorescent radiation emitted from this
volume causes focus to be difficult to define accurately and hence
is an unsatisfactory standard for many purposes.
[0014] Accordingly, there is a need for calibration tools,
calibration apparatuses, methods and tools used in microscopy
SUMMARY OF THE INVENTION
[0015] The present invention relates to calibration apparatuses,
methods and tools used in microscopy. The present invention may
separately be used for calibrating a location of a sample with
respect to optical elements and for calibrating fluorescence
detection in microscopes.
[0016] According to one aspect, a calibration tool for fluorescent
microscopy includes a support, a solid surface layer including a
fluorescent material, and a thin opaque mask of non-fluorescent
material defining reference feature openings having selected
dimensions exposing portions of the surface layer.
[0017] Preferably, a first type of the calibration tool may surface
of the support and the solid surface layer including the
fluorescent material, which is in contact with the thin opaque
mask. Alternatively, a second type of the calibration tool may
include the thin opaque mask fabricated (with or without an
adhesion promoter) onto the support, and the solid surface layer
including the fluorescent material located on the thin opaque
mask.
[0018] Preferred embodiments of this aspect may include one or more
of the following. The thin opaque mask is fabricated onto the
support using an adhesion promoter. The support is flat and rigid.
The support includes fused quartz. The surface layer is opaque. The
mask comprises a thin metal film. The support forms an optical
window of a cassette. Several suitable types of cassettes are
described in U.S. Pat. No. 6,140,044, which is incorporated by
reference.
[0019] The fluorophores are excitable by optical radiation passing
through the support and/or through the openings in the mask. The
fluorophores are excited by optical radiation mask and the support
absorbs excited fluorescent radiation. The solid surface layer
provides a broadband fluorescence emitter. The solid surface layer
provides a fluorescence emitter active at least two wavelengths and
having emission characteristics similar to Cy3 and Cy5 fluorescent
dies. The solid surface layer provides a fluorescence emitter
having effective fluorescent emittance that can produce a full
scale response for microscope calibration. The solid surface layer
including the fluorescent material has a thickness in the range of
about 2 micron to about 250 micron. The solid surface layer is
polyimide.
[0020] The thin opaque mask has a thickness in the range of about
10 nm to about 10 micron The thin opaque mask has a thickness in
the range of about 10 nm to about 100 nm.
[0021] According to another aspect, a process for producing a
calibration tool for fluorescent microscopy includes providing a
support, providing a solid surface layer including a fluorescent
material, and fabricating a thin opaque mask of non-fluorescent
material defining reference feature openings having selected
dimensions exposing portions of the surface layer.
[0022] Preferred embodiments of this aspect may include one or more
of the following. The solid surface layer may be deposited onto the
support the solid surface layer having the fluorescent material.
This may include depositing an adhesion promoting layer onto the
support for forming the solid surface layer including the
fluorescent material. The deposition includes delivering vapor
forming the solid surface layer including the fluorescent material,
such as evaporating or sputtering. The depositing includes spin
coating to form the solid surface layer including the fluorescent
material.
[0023] The fabrication of the thin opaque mask includes depositing
onto the support the non-fluorescent material and patterning the
non-fluorescent material to form the reference feature openings.
Then, the solid surface layer including the fluorescent material is
deposited onto the thin opaque mask.
[0024] The support may be fused quartz. The thin opaque mask may
include metal. The support may be used as an optical window for a
cassette used in examination of biological material.
[0025] The solid surface layer provides a broadband fluorescence
emitter.
[0026] The solid surface layer provides a fluorescence emitter
active at least two wavelengths and having emission characteristics
similar to Cy3 and Cy5 fluorescent dyes.
[0027] According to another aspect, a method of calibrating a
microscope includes providing a microscope, employing the
above-described calibration tool, bringing in focus an excitation
beam emitted from an objective of the microscope by detection
intensity of the microscope.
[0028] The bringing in focus may include adjusting a position of a
sample table wherein the calibration tool is located. The
microscope may be an on-axis flying objective microscope. The
microscope has a micro-lens objective carried upon an oscillating
rotary arm.
[0029] The calibration tool is suitable for use with a confocal
microscope having a restricted depth of field and the solid surface
layer that comprises fluorophores has an effective depth of less
than the depth of field of the confocal microscope, preferably the
surface layer having an effective fluorescent emittance that can
produce a full scale response of the microscope.
[0030] According to yet another aspect, a method of quantified
fluorescence microscopy includes providing a fluorescence detecting
microscope, employing a calibration tool as described above to
calibrate the microscope, and performing fluorescence microscopy of
specimens employing the calibrated microscope Preferably the
microscope is an on-axis flying objective microscope, and most
preferably, the microscope has a micro objective lens carried upon
a rapidly oscillating rotary arm.
[0031] In a preferred embodiment, a fluorescent calibration tool is
built with a suitably fluorescent solid surface layer of constant
thickness that is opaque, made of organic material or inorganic
material, carried on a suitable support. For materials that are not
naturally opaque, dyes or pigments are added. A very thin metal
layer is subsequently deposited on the opaque fluorescent material
and covered with a layer of photo-resist. An appropriate pattern is
then imaged on the photo-resist and chemically etched. The
resulting fluorescent pattern showing through the etched openings
has extremely fine features because the metal layer is as little as
a fraction of a micron thick, preferably about 100 to 300 Angstrom
thick. The pattern-creating process can be identical to the process
used to create integrated circuits. Presently that technology
enables the formation of features as narrow as 02 micron width
lines separated by spaces of the same dimension.
[0032] In the calibration tool, the fluorescence is caused to be a
surface emission phenomenon, which permits reliable focusing and
fluorescence calibration, that can be used as a standard, and
enable all instruments to be set to the same standard. Preferably,
the calibration tool uses a very stable fluorescing material, that
is insensitive to photobleaching.
[0033] An important fluorescing material with a broad band of
fluorescent response, is a selected polyimide such as Kapton.TM.
available in liquid form and used for spin coating substrates and
creating sheets with 1 to 10 micron thickness. A suitable product
is available under the trade designation WE-IIII or PI-IIII from H
D MicroSystems, Wilmington, Del. This material is a polyimide which
has as a backbone a high molecular weight polyimic acid precursor
comprised of specific aromatic decanydride and an aromatic
diamine.
[0034] The fluorescing material may preferably be another polyimide
product, Probonide 116A, available from Arch Chemicals of
Portsmouth, N.H. This material exhibits broad band fluorescence of
approximately 1/4 the intensity of the H D Microsystems product,
that can be satisfactorily used. Alternatively, fluorescing
polyimide material is one that is provided to the semiconductor
industry as a self-priming, non-photosensitive polyimic acid
formulation which becomes a fully stable polyimide coating after
thermal curing.
[0035] Another material for the surface layer, suitable for a
specific wavelength of interest, is an extremely thin layer of
fluorescent glass deposited e.g. by evaporation or by a sol-gel
process on a non-fluorescent support. In the case of a sol gel,
large molecules of a glassy type of material are suspended, with
selected fluorophores in a water or alcohol carrier, and applied as
a film coating to a support. It is baked at a relatively low
temperature to form a thin glassy fluorescent film. In these and
other cases, fluorescing dyes for specific wavelengths are
incorporated in a suitable non-fluorescing and preferably opaque
binder, applied as a thin, uniform thickness coating. Examples of
fluorescing dyes are Cy3, Cy5, and fluorescence.
[0036] In all events, the substance of the surface layer must be
selected to produce sufficient fluorescence to be detected in the
way that is normal to use in operating the instruments for
examining fluorescent specimens. The specific selection of a
fluorescing reference material is dependent upon numerous
parameters such as the response of the instrument, the selected
wavelength, the size of the features to be examined and the spot
size of the excitation beam. In the case of the commercial
instrument as described as an example in the accompanying appendix,
the fluorescent material must produce of the order of one million
times the radiation detected at the detector. The polyimide
materials described above provide a great benefit over others in
being broad band and hence suitable as a single reference that is
useful over a range of selected wavelengths at which important
experiments are performed.
[0037] Some fluorescent microscopy applications demand that the
material under inspection be located behind a transparent
protective window, typically made of non-fluorescent optical glass
such as synthetic quartz. In such cases the alignment tool
preferably duplicates the application and the metalized target is
first created on the glass and the fluorescent media is applied as
a coating covering the metalization as well as the glass, or is
provided as a planar coating on a second optically flat member
which is then mounted face-to-face with the metal layer on the
first optically flat member.
[0038] Importantly, in many cases, the effective fluorophores for
producing photons that reach the detector lie substantially only in
a surface layer. (As used herein, the term, "effective
fluorophores" is meant to include substantially all of those
fluorophores which are effective to produce meaningful fluorescent
radiation from the face of the surface layer that can reach the
detector of a microscope, and does not refer to fluorophores which
are either out of the range of excitation radiation of the
microscope due to the opacity of the matrix, or, though within the
range of effective excitation radiation, do not produce fluorescent
radiation that reaches the detector of the microscope, due, e.g.,
to absorption by the opaque matrix material). The surface
fluorophores approximate what occurs when dots of biological
specimen material only a fraction of a micron thick produce
fluorescence in response to an incident excitation beam.
[0039] In the present calibration tool, limitation of fluorescence
to the surface layer suitable for a given application may
accomplished by one or a combination of techniques. In one case,
the binder material for forming the solid matrix in which the
calibration fluorophores are contained, is made essentially opaque
at the excitation or detection wavelength or both, such that a
large fraction, e.g. 80% or more, preferably as much as 99% of the
detected fluorescing radiation, emanates from a surface layer of
depth, .DELTA..sub.t, that is only of the order of the thickness of
the specimen to be inspected, and within the depth of field of the
instrument. In another case, the micro thickness of a layer in
which the fluorophores are confined is controlled to a high degree
of uniformity, the layer sitting on an opaque support devoid of
fluorophores, such that even if some fluorescence occurs from a
depth beyond the preferred bound, the resulting fraction of
luminescence outside of the bound is uniform across the tool
because of the uniformity of coating thickness, and hence is not
effective to significantly disturb the calibration.
[0040] In another case, the fluorophores are introduced to a
surface layer after performing the surface layer, e.g., by
diffusion, spray or implantation techniques that confine the
fluorophores essentially to the surface that is to be exposed by
openings in the pattern. Thus, in the calibration tool, an
effective solid fluorescent surface layer is provided that can
serve as a proxy for the specimen to be examined.
[0041] The thickness of the thin metal layer or other material
forming the reference pattern also matters, because many rays of
the detected fluorescence form an angle as great as 45 degrees with
the surface being examined, and can be blocked by the edge walls of
the pattern elements, if the elements are too thick, to impair the
resolution of detection of the pattern edge. The finer the features
to be inspected, the finer must be the calibration of the
instrument, hence the more critical becomes the thickness of the
pattern elements, i.e., the reference lines, circles, etc.,
included in the thin opaque mask.
[0042] An important aspect is the fluorescent wavelength produced
by the fluorescing surface layer. Preferably, the calibration tool
is made employing a broad band fluorescent material and thus is
useful with various lasers and wavelengths used in a microscopes
and with different types of fluorophores used in various lines of
scientific or industrial inquiry. In one example, a polyimide
material is selected which has effective fluorescence for use as a
reference at wavelengths from 473 nm to 850 nm, or more preferably
from 450 nm to 800 nm, covering essentially the entire visible
spectrum. (The visible spectrum is important, since a great deal of
historical biological data has been generated in that region, and
is available for reference and comparison as research proceeds).
However, fluorophores in the near infrared and ultraviolet may be
employed, given suitable circumstances with respect to the biology
and the available sources of illumination and detection.
[0043] According to another aspect, the calibration tool is used in
combination with a flying objective, on-axis scanner, to achieve
highly reproducible quantified fluorescence microscopy. While
microscopes with any means of moving the lens preferably a micro
lens, is included, significant further advantages are obtainable by
employing an oscillating rotary arm to transport the micro lens
over the specimen or calibration tool. The calibration of
fluorescence detection microscopes, to the calibration of on-axis,
wide field of view scanning fluorescence microscopes, and
ultimately to quantified fluorescent microscopy having application
from the forefront of genomic research and drug discovery to
clinical use. The invention has particular application to the
accurate reading of biochips and micro arrays.
DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 is a plan view of an alignment tool used in
microscopy.
[0045] FIG. 2 is a cross-section of the tool taken through certain
alignment features along lines 2-2 of FIG. 1.
[0046] FIG. 3 is a diagram depicting the fabrication of the
alignment tool of FIG. 1.
[0047] FIG. 4 shows one embodiment of the alignment tool used with
a microscope.
[0048] FIG. 4A shows an alternative embodiment of the alignment
tool used with a microscope.
[0049] FIG. 5 illustrates diagramatically another method for
fabrication an alignment tool, while FIG. 5a shows the resulting
tool.
[0050] FIG. 5b illustrates diagramatically another method for
fabrication an alignment tool, while FIGS. 5c and 5dare schematic
cross-sectional views of the tool fabrication.
[0051] FIG. 6 is a view of an alternative to the construction
of
[0052] FIG. 7 illustrates the use of the tool shown in FIGS. 5B or
6.
[0053] FIGS. 8 and 8A illustrate diagrammatically a wide-angle
fluorescent scanning microscope employing a flying micro objective
lens on a rapidly rotating, oscillating arm.
[0054] FIG. 9 is a perspective representation of an oscillating arm
of the scanning microscope shown in FIG. 8 employing an alignment
tool.
[0055] FIG. 10 is a diagrammatic plan view of the oscillating arm
shown in FIG. 9 used for scanning a microarray of biological
material on a glass slide or biochip following the calibration of
the scanning microscope shown in FIG. 9.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0056] Referring to FIGS. 1 and 2, the alignment tool 2a comprises
a generally planar rigid member carrying on its face a detailed
pattern of optical features suitable for calibration of the
instrument. The rigid member is typically of the same dimensions as
the microscope slide, microarray chip or other object to be
examined, to fit in the same position on the instrument. The
optical features of the alignment tool include lines and circular
dots of various dimensions to emulate the various sizes of dots and
linear features of biological or other material to be examined. The
finest features have dimensions of the order of 1 micron or less to
suitably calibrate for detection of features of a few micron
dimensions or less.
[0057] The calibration tool shown in FIGS. 1 and 2 includes regions
T-1, T-2, T-3, T-4, and T-5. Region T-1 includes a set of "barcode"
lines having thickness 2 micron, 4 micron, 6 micron, 8 micron, 10
micron, and 12 micron, separated by twice their size. Region T-2
include a long 5 micron and 10 micron line across. Region T-3
includes a solid layer of the fluorescing material. Region T-4
includes three arrays of circular features having diameters from
300 micron to 25 micron, and squares having sizes from 300 micron
to 25 micron. Region T-5 is a solid metallic area.
[0058] Referring to FIG. 3, in one preferred embodiment the first
step in manufacture of the calibration tool is to provide a rigid
support plate having effective fluorophores confined to a solid
surface layer 4 of only an incremental thickness, see FIG. 2.
Typically this depth, delta.sub.t, is negligible such that
fluorescent emission occurs essentially as a surface phenomenon.
Upon this layer, step two, a nickel or other suitable very thin and
opaque metal film as applied that is etchable to form a reference
pattern. Sputter coating, vacuum metal deposition or other known
techniques may be employed. A photo-resist is then applied in
general to the metal layer, the photoresist on the tool perform is
exposed through a precision mask defining the alignment features
and then the surface is chemically etched to form the resulting
reference pattern. The resultant tool is used to calibrate
fluorescnce measurements as well as the conventional image
components of fluorescent microscopes.
[0059] Referring to FIG. 4, an oscillating arm 19, rotating about
axis A, carries an on-axis micro objective lens 18 for on-axis
scanning over the alignment tool 2A which is positioned in the
place ordinarily occupied by specimens to be examined. Mirrors 15
and 17 are effective to Introduce excitation light from a
stationary laser source, along axis of rotation A, thence out along
the arm to lens 18, thence to the specimen (or in this case, to the
calibration tool). Light reaching the surface layer 4 of the tool
excites effective fluorophores, which emanate in all directions at
a different wavelength. A significant feature is that this
radiation is captured by micro lens 18 (whose axis is always
perpendicular to the object plane), and directed back through the
optical path and through a restriction such as a pin hole 103, to
detector D, 95, typically a photo multiplier tube (PMT).
[0060] In the case of FIG. 4, the surface layer 4 is a separately
applied layer of uniform minimum thickness applied to a solid,
optically flat, opaque support plate. Preferably surface layer 4 is
also opaque such that excited light does not substantially
penetrate even the surface layer, but even if it does penetrate to
a degree, because of the great uniformity of the layer, and the
non-emitting character of its support, any incidental fluorescence
from below the surface layer.sub..DELTA.t is uniform throughout,
hence its disturbing effect can in many instances be tolerated.
Depending upon the particular instrument and application, in some
cases, in which the solid surface layer is extremely thin and
sufficiently uniform in thickness and distribution of fluorophore,
the surface layer need not be opaque and will still function
appropriately to produce essentially only surface emissions.
[0061] The alternative processes of FIGS. 5 and 5b are self
explanatory, both producing calibration tools which, in use, are
illuminated by light passing through the transparent pattern
support. The tool of FIG. 5A is produced by the steps of FIG. 5
while the tools of FIGS. 5D and 6 are produced employing the steps
of FIG. 5B. The tools of FIGS. 5D and 6 differ from each other in
the same respect that tools of FIGS. 4 and 4A differ, described
above.
[0062] The resultant tools are effective to enable standardization
of wide field of view fluorescent scanning microscopes such as the
microscope depicted in FIGS. 8 and 9.
[0063] This microscope is described in detail in PCT Application
PCT/US99/06097, published as WO99/47964, entitled "Wide Field of
View and High Speed Scanning Microscopy," which is hereby
incorporated by reference as if fully set forth herein. It is
sufficient to say that the micro objective lens 18, mounted on a
rotary arm 19 for on-axis scanning, is driven in rapid rotary
oscillation movement by galvanometer or oscillating motor 3, whose
position is detected by position sensor 43 for the purpose of
relating data acquisition to position on the specimen. By employing
a pin hole or other restriction 103 in front of the light sensor
95, the resulting confocal microscope has a significantly limited
depth of field, which could not be calibrated well by prior
techniques but which can be readily calibrated to high accuracy
using calibration tools featuring broad band surface emission by
fluorescence as has been described here.
[0064] FIG. 9 depicts employing a calibration tool as described
above with a flying objective microscope, whereas FIG. 10 depicts
the subsequent scanning of a microarray using the same instrument,
now calibrated, to achieve quantified fluorescence microscopy that
can readily be compared to the results produced by other
microscopes that have been calibrated in the same way.
[0065] A rudimentary analysis of the amount of fluorescence
required to stimulate an actual specimen is presented in the
following appendix with respect to a commercial confocal
fluorescence scanning microscope, based on a microlens carried on a
rapidly oscillating arm, the 418 Array Scanner.TM., available from
Genetic MicroSystems, Inc. By following a similar analysis for
other instruments one can arrive at suitable fluorescent levels for
those instruments, by considering the sets of data for all
instruments a standard calibration tool is obtainable.
APPENDIX
Analysis of Fluorescence Required for a Practical Wide Field of
View
Flying Objective Microscope With on-Axis Scanning (418 Array
Scanner available from Genetic MicroSystems, Inc.)
[0066] TABLE-US-00001 TABLE 1 (1) Illumination Power 3 mW on
specimen at 6 37 nm (2) Delivery efficiency to the PMT Detector
Collection Efficiency Geometric 13% 13 Dichroric Transmission .9 nm
Emission Filter 6 Approximate Delivery Efficiency to the PMT = 070
(3) 5 V/nW min Gain of PMT = sensitivity = S (4) Approximately 0 5
v full scale Typical PMT signal = C (5) Assume the weakest PMT is
saturated (eg. Hammamatsu PMT for detection of .SIGMA. = 637 nm
Response = R = C - S R = 5 V _ 5 V/nw = 1 nw @ PMT nw S = 1 nW _
070 = 1 4 nW @ microscope slide (6) Fluorescence Production Rate =
1 4 (10.sup.-9) _ 3 (10.sup.-3) of the order of 1 5 _ 3 (10.sup.-6)
= 5 (10.sup.-6)
[0067] In this table, illumination power represents the amount of
power that is typically delivered to the microscope slide for
exciting fluorescent emission. The delivery efficiency (2) is
defined by three values. The first is the geometric collection
efficiency of the lens, based upon the size of the confocal pinhole
and the distance to the microscope slide. For the instrument of the
example, 13% of the fluorescing light emitted is collected, i.e.,
the fluorescence light is emitted at the target with spherical
distribution, and the instrument collects 13% of that light. That
light passes through a dichroic mirror, necessarily involving a
lose factor, so that 90% of the light is passed and 10% is
reflected elsewhere in the system and is wasted. Finally, in front
of the photomultiplyer tube an emission filter passes about 60% of
the fluorescent light. The emission filter is a multi-layer optical
filter which resects the excitation light that accompanies the
fluorescent energy which is generally centered about 25-30
nanometers away from the wavelength of the excitation laser
energy.
[0068] The model 418 Array Scanner instrument operates at 532 and
637 nanometer. Another useful wavelength is 473 nanometer. At these
wavelengths, for this instrument, the surface layer of fluorescing
material in the calibration tool must produce fluorescence power
leaving its surface of the order of at least 1 millionth the
illumination power reaching a specimen.
[0069] The product of the three numbers discussed,
0.13.times.9.times.6, shows that the delivery efficiency is
approximately 070. Referring further to the table, line 3 relates
to the gain of the photomultiplyer tube modules employed in the 418
Array Scanner. The modules with the least gain have a gain of 5
volts per nanowatt, meaning typically around 637 nm .SIGMA., the
PMT produces a 5 volt signal for every nanowatt of light reaching
it.
[0070] At line 4, the 418 Array Scanner system is such that when a
full strength signal is obtained, the instrument produces 1/2 a
volt at the photomultiplyer tube.
[0071] Thus by assuming that a desired test material will saturate
the weakest photomultiplyer tube, an equation is produced that
shows the desired performance of the material. The response of the
photomultiplyer tube is equal to the gain times the signal (amount
of light falling on the photomultiplyer tube).
[0072] This gives a signal equal to 1/2 volt divided by 5 volts per
nanowatt, or 01 nanowatts of light are obtained at the
photomultiplyer tube By taking the 01 nanowatt and dividing it by
the efficiency of 07, one determines that at the microscope slide
14 nanowatts of fluorescent light are produced, or a little higher.
Thus, the fluorescence production rate is approximately 14 or
15.times.10.sup.-9 which is the nanowatts divided by
3.times.10.sup.-3, which is equal to 3 milliwatts This results in a
value of about 1/2.times.10.sup.-4 or a factor of 1 million,
meaning that for each fluorescing photon reaching the PMT,
approximately 1 million photons are required to impinge on the
surface layer of fluorescent material.
[0073] This shows that the fluorescent efficiency of the
fluorophore needs to be approximately 1.times.10.sup.-6 or higher.
It needs to receive 10.sup.6 photons for every photon it emits.
Accordingly, the conversion efficiency of a suitable fluorescent
reference material needs to be of the order of 1.times.10.sup.-6 or
higher.
* * * * *