U.S. patent number RE36,529 [Application Number 08/996,497] was granted by the patent office on 2000-01-25 for spectroscopic imaging device employing imaging quality spectral filters.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Ira W. Levin, Edgar N. Lewis, Patrick J. Treado.
United States Patent |
RE36,529 |
Lewis , et al. |
January 25, 2000 |
Spectroscopic imaging device employing imaging quality spectral
filters
Abstract
Techniques for providing spectroscopic imaging integrates an
acousto-optic tunable filter (AOTF), or an interferometer, and a
focal plane array detector. In operation, wavelength selectivity is
provided by the AOTF or the interferometer. A focal plane array
detector is used as the imaging detector in both cases. Operation
within the ultraviolet, visible, near-infrared (NIR) spectral
regions, and into the infrared spectral region, is achieved. The
techniques can be used in absorption spectroscopy and emission
spectroscopy. Spectroscopic images with a spectral resolution of a
few nanometers and a spatial resolution of about a micron, are
collected rapidly using the AOTF. Higher spectral resolution images
are recorded at lower speeds using the interferometer. The AOTF
technique uses entirely solid-state components and requires no
moving parts. Alternatively, the interferometer technique employs
either a step-scan interferometer or a continuously modulated
interferometer.
Inventors: |
Lewis; Edgar N. (Gaithersburg,
MD), Levin; Ira W. (Rockville, MD), Treado; Patrick
J. (Pittsburgh, PA) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
27398890 |
Appl.
No.: |
08/996,497 |
Filed: |
December 23, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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236655 |
Apr 29, 1994 |
5377003 |
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846824 |
Mar 6, 1992 |
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Reissue of: |
363363 |
Dec 23, 1994 |
05528368 |
Jun 18, 1996 |
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Current U.S.
Class: |
356/456;
250/339.02; 250/458.1; 356/301 |
Current CPC
Class: |
G01J
3/2823 (20130101); G02B 21/33 (20130101) |
Current International
Class: |
G02B
21/33 (20060101); G01J 3/28 (20060101); G01B
009/02 (); G01J 003/44 (); G01N 021/35 (); G01N
021/64 () |
Field of
Search: |
;336/300,301,346
;250/339.01,339.02,339.07,339,458.1,459.1,461.1,461.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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214129 |
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Dec 1983 |
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JP |
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80821 |
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May 1985 |
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JP |
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265131 |
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Oct 1989 |
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JP |
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Other References
SPIE, vol. 1347 "Optical Information Processing Systems and
Architectures II" (1990); article entitled Acousto-Optic Tunable
Filter (AOTF) Imaging Spectrometer for NASA Applications: Systems
Issues by Jeffrey Y, Tien Hsin Chao and Li-Jen Cheng. .
SPIE, vol. 1347 "Optical Information Processing Systems and
Architectures II" (1990); article entitled Acousto-Optic Tunable
Filter (AOTF) Imaging Spectrometer for NASA Applications:
Breadboard Demonstration by Tien-Hsin Chao, Jeffrey Yu, Li-Jen
Cheng & Jim Lambert. .
Jun. 7, 1985 Article by Goetz et al., Imaging Spectrometry for
Earth Remote Sensing, Science, vol. 228, No. 4704. .
1986 Book by Taylor et al., Applications of Fluorescence in the
Biomedical Sciences, Liss, New York. .
1990 Article by Levin et al., Fourier-Transform Raman Spectroscopy
of Biological Materials, Anal. Chem., 62(21). .
1989 Article by Treado et al., Multichannel Hadamard Transform
Raman Microscopy, Appl. Spectrosc., 44(2). .
Nov. 1987 Article by Kurtz et al., Rapid scanning fluorescence
spectroscopy using an acousto-optic tunable filter, Rev. Sci.
Instrum., vol. 58, No. 11. .
1987 Article by Bilhorn et al., Spectrochemical Measurements with
Multi-Channel Integrating Detectors, Applied Spectroscopy 41, 1125.
.
Gottlieb et al., SPIE, vol. 232, 1980 International Optional
Computing Conference (1980), pp. 33-41. .
NOI Bulletin Summary, National Optics Institute, vol. 4, No. 2, p.
1, Nov. 1993. .
Spectral Diagnostics Brochure entitled "Spectral Bio-Imaging
Systems" (date unavailable)..
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Primary Examiner: Evans; F. L.
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part of U.S. patent application Ser. No.
08/236,655, filed Apr. 29, 1994, now U.S. Pat. No. 5,377,003, which
is a continuation of U.S. patent application Ser. No. 07/846,824,
filed Mar. 6, 1992, now abandoned.
Claims
We claim:
1. A spectroscopic imaging device employing imaging quality
spectral filter suitable for use in Raman emission microscopy
comprising:
a high intensity monochromatic light source;
means for directing said monochromatic light toward a subject to be
analyzed;
collimation means for directing light emitted from each of a
plurality of spatial locations within said subject in response to
said monochromatic light source impinging upon said subject at an
interferometer, the interferometer being of the type which has at
least one movable mirror which can be positioned to produce a
multiplexed spectral output of the light passing through the
interferometer at a plurality of select positions of the movable
mirror;
means operatively connected to the interferometer for positioning
said movable mirror of said interferometer, wherein said
interferometer maintains the image fidelity of the subject as said
emitted light passes through the interferometer; and
means for collimating and directing said emitted light passing
through the interferometer at a focal plane array detector
comprising a two-dimensional array of charge coupled devices,
wherein said charge coupled devices of said focal plane array
detector measures the intensity of emitted light from each of said
plurality of spatial locations of the subject at each of said
plurality of select positions of the movable mirror.
2. The spectroscopic imaging device of claim 1 further comprising
means for converting said intensity of said light from optical
retardation to wavelength.
3. The spectroscopic imaging device of claim 2 further comprising
means for displaying an image of said subject derived from said
intensity of said light at one or more wavelengths.
4. The spectroscopic imaging device of claim 1 further comprising
means for determining the position of said movable mirror of said
interferometer.
5. The spectroscopic imaging device of claim 4 wherein the means
for determining the position triggers the focal plane array
detector to obtain the image at each of the select positions.
6. The spectroscopic imaging device of claim 1 wherein the movable
mirror of the interferometer is a continuously movable mirror.
7. A spectroscopic imaging device employing imaging quality
spectral filters suitable for use in Fluorescence emission
microscopy comprising:
a substantially monochromatic light source;
means for directing said monochromatic light toward a subject to be
analyzed;
collimation means for directing fluorescent light emitted from each
of a plurality of spatial locations within said subject in response
to said monochromatic light source impinging upon said subject at
an interferometer, the interferometer being of the type which has
at least one movable mirror which can be positioned to produce a
multiplexed spectral output of the light passing through the
interferometer at a plurality of select positions of the movable
mirror;
means operatively connected to the interferometer for positioning
said movable mirror of said interferometer, wherein said
interferometer maintains the image fidelity of the subject as said
fluorescent light passes through the interferometer; and
means for collimating and directing said fluorescent light passing
through the interferometer at a focal plane array detector
comprising a two-dimensional array of charge coupled devices,
wherein said charge coupled devices of said focal plane array
detector measures the intensity of emitted light from each of said
plurality of spatial locations of the subject at each of said
plurality of select positions of the movable mirror.
8. The spectroscopic imaging device of claim 7 further comprising
means for converting said intensity of said light from optical
retardation to wavelength.
9. The spectroscopic imaging device of claim 7 further comprising
means for displaying an image of said subject derived from said
intensity of said light at one or more wavelengths.
10. The spectroscopic imaging device of claim 7 further comprising
means for determining the position of said movable mirror of said
interferometer.
11. The spectroscopic imaging device of claim 10 wherein the means
for determining the position triggers the focal plane array
detector to obtain the image at each of the select positions.
12. The spectroscopic imaging device of claim 6 wherein the movable
mirror of the interferometer is a continuously movable mirror.
13. A spectroscopic imaging device employing imaging quality
spectral filters suitable for use in near-infrared and infrared
absorption microscopy comprising:
a source of broadband light;
means for directing said broadband light toward a subject to be
analyzed;
collimation means for directing light transmitted or reflected from
each of a plurality of spatial locations within said subject in
response to said broadband light source impinging upon said subject
at an interferometer, the interferometer being of the type which
has at least one movable mirror which can be positioned to produce
a multiplexed spectral output of the light passing through the
interferometer at a plurality of select positions of the movable
mirror;
means operatively connected to the interferometer for positioning
said movable mirror of said interferometer, wherein said
interferometer maintains the image fidelity of the subject as said
transmitted or reflected light passes through the interferometer;
and
means for collimating and directing said transmitted or reflected
light passing through the interferometer at a focal plane array
detector comprising a two-dimensional array of charge coupled
devices, wherein said charge coupled devices of said focal plane
array detector measures the intensity of said transmitted or
reflected light from each of said plurality of spatial locations of
the subject at each of said plurality of select positions of the
movable mirror.
14. The spectroscopic imaging device of claim 13 further comprising
means for converting said intensity of said light from optical
retardation to wavelength.
15. The spectroscopic imaging device of claim 13 further comprising
means for displaying an image of said subject derived from said
intensity of said light at one or more wavelengths.
16. The spectroscopic imaging device of claim 13 further comprising
means for determining the position of said movable mirror of said
interferometer.
17. The spectroscopic imaging device of claim 16 wherein the means
for determining the position triggers the focal plane array
detector to obtain the image at each of the select positions.
18. The spectroscopic imaging device of claim 13 wherein the
movable mirror of the interferometer is a continuously movable
minor. .Iadd.
19. A method for non-invasive spectroscopic imaging of a sample
comprising:
subjecting the sample to be imaged to the spectroscopic imaging
device of claim 1;
collecting the spectroscopic data; and
manipulating the data to reveal the molecular arrangement of the
sample analyzed..Iaddend..Iadd.20. The method of claim 19 wherein
said sample is a biological material..Iaddend..Iadd.21. The method
of claim 19 wherein said imaging device further comprising means
for converting said intensity of said light from optical
retardation to wavelength..Iaddend..Iadd.22. The method of claim 19
wherein said imaging device further comprises means for displaying
an image of said subject derived from said intensity of said light
at one or more wavelengths..Iaddend..Iadd.23. The method of claim
19 wherein said imaging device further comprises means for
determining the position of said movable mirror of said
interferometer..Iaddend..Iadd.24. The method of claim 23 wherein
the means for determining the position triggers the focal plane
array detector to obtain the image at each of the select
positions..Iaddend..Iadd.25. The method of claim 19 wherein the
movable mirror of the interferometer is a continuously movable
mirror..Iaddend..Iadd.26. A method for noninvasive spectroscopic
imaging of a sample comprising:
subjecting the sample to be imaged to the spectroscopic imaging
device of claim 7;
collecting the spectroscopic data; and
manipulating the data to reveal the molecular arrangement of the
sample analyzed..Iaddend..Iadd.27. The method of claim 26 wherein
said sample is
a biological material..Iaddend..Iadd.28. The method of claim 26
wherein said imaging device further comprises means for converting
said intensity of said light from optical retardation to
wavelength..Iaddend..Iadd.29. The method of claim 26 wherein said
imaging device further comprises means for displaying an image of
said subject derived from said intensity of
said light at one or more wavelengths..Iaddend..Iadd.30. The method
of claim 26 wherein said imaging device further comprises means for
determining the position of said movable mirror of said
interferometer..Iaddend..Iadd.31. The method of claim 30 wherein
the means for determining the position triggers the focal plane
array detector to obtain the image at each of the select
positions..Iaddend..Iadd.32. The method of claim 26 wherein the
movable mirror of the interferometer is a continuously movable
mirror..Iaddend..Iadd.33. A method for noninvasive spectroscopic
imaging of a sample comprising:
subjecting the sample to be imaged to the spectroscopic imaging
device of claim 13;
collecting the spectroscopic data; and
manipulating the data to reveal the molecular arrangement of the
sample analyzed..Iaddend..Iadd.34. The method of claim 33 wherein
said sample is a biological material..Iaddend..Iadd.35. The method
of claim 33 wherein said imaging device further comprises means for
converting said intensity of said light from optical retardation to
wavelength..Iaddend..Iadd.36. The method of claim 33 wherein said
imaging device further comprises means for displaying an image of
said subject derived from said intensity of said light at one or
more wavelengths..Iaddend..Iadd.37. The method of claim 33 wherein
said imaging device further comprises means for determining the
position of said movable mirror of said
interferometer..Iaddend..Iadd.38. The method of claim 37 wherein
the means for determining the position triggers the focal plane
array detector to obtain the image at each of the select
positions..Iaddend..Iadd.39. The method of claim 33 wherein the
movable mirror of the interferometer is a continuously movable
mirror..Iaddend.
Description
FIELD OF THE INVENTION
The present invention relates to devices used for spectroscopic
imaging, and more particularly to devices that non-invasively and
rapidly collect images of a sample at multiple, discrete
wavelengths in the ultraviolet, visible, near-infrared and infrared
regions of the optical spectrum. The character of the images
recorded is determined by a sample's chemical characteristics as
revealed by its intrinsic electronic and vibrational absorptions or
emissions.
BACKGROUND OF THE INVENTION
Spectroscopic imaging can be used to determine the spatially
distributed and chemically distinct species in heterogenous
materials. Spectroscopic imaging is an analytical tool that has
been applied at both the macroscopic and microscopic levels.
At the macroscopic level, chemical imaging of areas exceeding 20
square kilometers can be achieved using airborne and spaceborne
remote sensing, near-infrared imaging spectrometers. On a smaller
scale, diagnostic imaging of the human body is possible through
nuclear magnetic resonance imaging techniques (MRI).
At the microscopic level, fluorescence imaging is a technique
employed for chemical state microscopy. Fluorescence is the
emission of radiation (light) through which a molecule in an
electronically excited state is able to dissipate its excess
energy. Molecular fluorescence emission normally occurs at visible
wavelengths. Fluorescent intensities from fluorophores (analyte
molecules with fluorescent properties) which exhibit high quantum
efficiencies can be relatively strong. Fluorescence spectroscopy,
in general, is an extremely sensitive technique. In fact, single
molecule detection has been demonstrated utilizing fluorescent
spectroscopy. Fluorescence microscopy involves the labeling of a
specific component of interest with a fluorescent tag and the
subsequent viewing of the spatially resolved fluorescence emission.
Improved specificity can be provided by immuno-fluorescent tags in
which the analyte is an antigen that binds to a fluorescently
tagged antibody. Many types of fluorescent immunoassays have been
developed and are widely used in biomedical and biological imaging.
Additional background information on fluorescence microscopy can be
found in "Applications of Fluorescence in the Biomedical Sciences,"
D. L. Taylor, A. S. Waggoner, R. F. Murphy, F. Lanni, R. R. Birge
(eds.), Liss, New York (1986).
Another class of spectroscopic microscopy techniques employs
vibrational spectroscopy. Specifically, the methods of Raman and
infrared spectroscopy provide chemical selectivity without
requiring undue sample preparation or incurring sample alteration
or degradation. Raman spectroscopy, an inelastic light scattering
phenomenon, is commonly used to characterize a sample on the basis
of its molecular vibrational spectrum. The Raman scattering
phenomenon is observed by illuminating a sample with a high
intensity monochromatic source, such as a laser, and detecting the
fraction of light scattered at longer wavelengths. Typically, about
1 Raman photon is scattered per 10.sup.8 incident photons. The
frequency displacements of the Raman scattered light from the
incident laser radiation correspond to the vibrational frequencies
of the sample molecules. Since the vibrational spectral bandwidths
observed in Raman spectroscopy are usually 5-30 cm.sup.-1 (0.3-6 mm
at 750 nm), spectroscopic devices must be able to resolve, as well
as detect, these faint signals.
Traditionally, Raman spectroscopy has been performed using visible
wavelength lasers, optics and detectors coupled to monochromators
that employ diffraction gratings for spectral dispersion and
isolation. More recently, laser-referenced Michelson
interferometers have been employed in conjunction with solid-state
near-infrared (NIR) laser excitation, primarily Nd:YAG, for Fourier
transform (FT) Raman spectroscopy. Additional information can be
found in "Fourier-Transform Raman Spectroscopy of Biological
Materials," Anal Chem., 62(21) 1990, Ira W. Levin and E. Neil
Lewis. One advantage of FT-Raman spectroscopy is the improved
instrumental performance of FT interferometers over standard
monochromators. Specifically, the instrument provides intrinsic
throughput (Jacquinot's advantage) and multiplex (Fellgett's
advantage) characteristics, as well as high spectral precision
(Connes' advantage) and the capacity for high spectral resolution.
Commercial FT interferometer systems can typically provide 0.02-5
cm.sup.-1 (0.02-5 nm at 3300 nm) spectral resolution. Current
interferometer based instruments are optimized for maximum signal
throughput, but are designed without regard for maintaining image
fidelity through the device. An FT interferometer that retains
image fidelity would provide the inherent advantages of
interferometry and could also be suitable for spectroscopic
imaging.
Infrared (IR) spectroscopy involves the absorption of IR radiation,
generally between 770.degree.-10000.degree. nm (12,900-10.degree.
cm.sup.-1), by molecular species. Energies in the infrared region
of the spectrum are on the order of the energies of vibrational
transitions, and IR spectroscopy is complementary in its
information content to Raman spectroscopy. IR spectroscopic imaging
is applicable to a wide range of materials, but is especially well
suited to the study of polyatomic organic molecules, as vibrational
frequencies are well correlated with organic functional groups. In
particular, IR and Raman spectroscopy are suitable for the study of
biological materials. Almost all materials absorb infrared
radiation, except homonuclear diatomic molecules (O.sub.2, H.sub.2,
N.sub.2). Polyatomic molecules exhibit rich IR spectra. The spectra
include both the fundamental absorptions in the mid-IR
(2500.degree.-20000.degree. nm), but also the overtones and
combination bands, primarily of O--H, C--H and N--H absorptions, in
the near-IR (770-2500 mm). While the near-IR bands are
significantly weaker than the fundamental bands, the wavelengths at
which they are observed are compatible with quartz and germanium
refractive optics, making the near-IR region of the spectrum well
suited for high spatial resolution chemical state imaging studies
based on molecularly specific vibrational absorptions.
The sensitivity of Raman and infrared spectroscopy to even small
changes in molecular structure is well established, and these
techniques are capable of generating specific fingerprints for a
given molecular species. In general, systems capable of generating
infrared absorption or Raman emission images find wide use in a
variety of areas in science and technology. Materials amenable to
these types of analysis would include, but not be limited to
biological materials, polymers, superconductors, semiconductors and
minerals.
Currently, two primary methods are employed for image generation.
The first approach involves the systematic scanning of a sample.
Typically, this is achieved either by translating the sample
through a stationary field of view defined by the collection optics
and detector, or alternatively, by scanning the imaging source (or
detector) in a raster pattern across the surface of the stationary
sample. The scanning approach is typically utilized with a single
element detector. An example of a vibrational spectroscopic imaging
device employing the scanning method is a Fourier transform
infrared (FTIR) spectrometer coupled to a mid-infrared microscope
outfitted with an x-y mapping translation stage for imaging a
sample. The technique utilizes the infrared (vibrational)
absorption properties of molecular functional groups in the sample
to generate the image.
Scanning methods for vibrational spectroscopic imaging, while
workable, have certain drawbacks and deficiencies. Specifically,
the signal to noise ratios obtainable with FTIR microspectrometers
often requires substantial signal averaging at each spatial
position, thus making the FTIR systems inherently slow. As a
result, only crude spatial maps are generally obtained. In
addition, the near-infrared imaging spectrometers employed for
remote sensing typically use diffraction gratings for spectral
characterization which require that images be constructed a slice
at a time as the spectrometer scans the sample surface.
Furthermore, the numerous moving parts contained within these
systems limit the speed and reliability of these devices.
A second method of image generation involves wide field
illumination and viewing in conjunction with multichannel
detection. Direct viewing with a color video camera of a subject
illuminated by a broadband visible source is a simple example of
wide field illumination imaging. In such a case, colorometric
information based on the visible absorption of the sample is
obtained.
For greater specificity and selectivity, fluorescence microscopy
may be performed. In fluorescence microscopy, optical filtering of
an intense arc lamp illumination source to select strong plasma
lines can be employed to selectively excite a molecular fluorescent
label added to a sample. Alternatively, a laser is employed for
illumination having a wavelength output which falls within the
absorption range of the fluorescent label and selectively excites
the tag. The fluorescent light, which emits at longer wavelengths
than the excitation source, is commonly discriminated using
dielectric interference filters. Where several spectral regions are
to be viewed separately, filter wheels containing multiple filters
can be utilized. Fluorescent spectral linewidths are usually 10-100
nm wide. Where only a single type of fluorophore is present in a
sample, spectral filters providing relatively broad spectral
resolution, 5-25 nm, can be adequate. Where multiple similar
fluorophores are present simultaneously, multiple filters providing
spectral resolution of 1-2 nm may be necessary.
Wide field illumination methods employing glass or interference
filters, however, have certain drawbacks and deficiencies.
Specifically, the application of discrete notch filters for
spectral selectivity requires the use of a separate filter at each
desired wavelength, ultimately limiting operation to only several
wavelengths. In addition, the dielectric notch filters employed
provide resolution of approximately 10-100 nm, which is often an
inadequate spectral resolution for discriminating similar but
different species in multicomponent environments. The techniques
using filter wheels provide only limited spectral resolution and
spectral coverage, and also suffer from the constraints of moving
mechanical parts, limiting the speed and reliability of these
systems.
A hybrid spectroscopic imaging method combining wide field
illumination and multichannel detection with spatial multiplexing
has been developed. The technique is called Hadamard transform
spectroscopic microscopy and has been especially adapted for Raman
emission microscopy. Additional information can be found in
"Multichannel Hadamard Transform Raman Microscopy," Appl.
Spectrosc., 44(2) 1989, Patrick J. Treado and Michael D. Morris.
The technique employs a dispersion spectrograph as the spectral
filter and is capable of generating spectral images of a variety of
materials at sub-micron spatial resolution.
The multichannel/spatial multiplex method, however, also has
certain limitations. Specifically, the number of spectral features
that can be collected simultaneously is determined by the
inherently limited spectral coverage of the spectrograph. Where
survey spectral images are to be collected, the Hadamard imager is
not optimal. In addition, artifacts can arise in the spectral
images due to systematic spatial encoding errors. These artifacts
ultimately compromise the spatial resolution of the technique.
SUMMARY OF THE INVENTION
Accordingly, a general object of the present invention is to
provide devices for rapidly filtering light sources in the
ultraviolet, visible, near-infrared and infrared regions of the
optical spectrum for utilization in absorption microscopy while
retaining image fidelity. Another general object of the present
invention is to provide devices for spectrally filtering emitted
radiation from samples encountered in emission microscopy,
specifically fluorescence and Raman microscopy, while retaining
image fidelity.
An additional object of the present invention is to provide a
solid-state spectroscopic imaging device that contains no moving
parts and that is capable of rapidly generating 1-2 nm spectral
resolution, and diffraction-limited spatial resolution images of
the molecular species comprising the desired sample in the
ultraviolet, visible, near-infrared and infrared regions of the
optical spectrum.
Yet another object of the present invention is to provide the
ability to collect spectroscopic data sets and manipulate the data
sets to reveal the precise molecular arrangements of the samples
analyzed, including particularly subtle molecular arrangements not
easily determinable by other analytical imaging techniques.
The foregoing and other objects are accomplished by the present
invention, using an acousto-optic tunable filter (AOTF) in
conjunction with a two dimensional array detector to non-invasively
and rapidly collect spectral images of a sample under conditions of
high spatial resolution. The images are derived from the intrinsic
electronic and vibrational absorptions and emissions of the
material.
In accordance with one aspect of the present invention, absorption
spectroscopy is achieved by filtering a light source using an AOTF.
The AOTF filters broadband light at pre-selected wavelengths. The
filtered light is then directed toward the sample of interest. For
thin samples, the filtered light may be transmitted through the
sample; alternatively, for thick samples and opaque materials, the
filtered light is preferably diffusely reflected by the sample. The
transmitted or reflected light is directed toward the two
dimensional array detector which measures the intensity of the
light at multiple locations within the sample. After retrieving the
spectroscopic information from the two dimensional array detector,
the data is analyzed, manipulated, and displayed using
spectroscopic, chemometric and image processing techniques.
An advantage to the present invention includes the ability to
rapidly and simultaneously record and analyze thousands of
absorption spectra with high spatial resolution. Unlike other
techniques there is no compromise in the amount of time required to
record data with adequate spectral characteristics versus high
image fidelity. Typically image clarity is limited only by the type
of imaging detector (CCD) employed.
In accordance with another aspect of the present invention,
emission spectroscopy is achieved by directing a monochromatic
light source (e.g. a laser), or nearly monochromatic light source
(e.g. a filtered arc lamp) toward a sample to be analyzed. The
resulting sample emission due to fluorescence and/or Raman
scattering is filtered by the AOTF and directed to the two
dimensional array detector which measures the intensity of the
emitted light at the AOTF selected frequency.
Another object of the present invention is to provide a
spectroscopic imaging device capable of generating 0.01-1.0 nm
spectral resolution, and diffraction-limited spatial resolution
images of the molecular species making up the sample. To that end,
an imaging quality, laser referenced, Michelson interferometer is
used as a spectral filter in conjunction with a two dimensional
array detector to non-invasively and rapidly collect spectral
images of a sample under conditions of high spatial resolution. The
images are derived from the intrinsic electronic and vibrational
absorptions and emissions of the material. In the present
invention, the operation of the imaging interferometer is identical
for both absorption and emission spectroscopic imaging.
To achieve absorption microscopy in accordance with the present
invention using the interferometer, a broadband light source is
directed toward the sample of interest. For thin samples, the light
may be transmitted through the sample, or alternatively, for thick
samples and opaque materials, the filtered light may be diffusely
reflected by the sample. The transmitted or reflected light is
collected and magnified with the microscope objective and projected
towards the interferometer which acts as the spectral filter. The
multiplexed spectral image output of the interferometer is directed
to the two dimensional array detector which measures the intensity
of the transmitted or reflected light at multiple locations within
the sample. Multiple images are recorded while moving a mirror in
one of the arms of the interferometer. This motion is achieved in a
step-scan manner rather than a continuous manner with images
recorded at different, static mirror retardations. The step scan
ensures that each of the pixels of an image are recorded at the
same retardation. Alternatively, a continuously modulated
interferometer may be used provided that an instantaneous or
near-instantaneous image is obtained rapidly enough relative to the
speed of the travelling mirror such that the image data maintains
its fidelity.
The relative positions of the two mirrors and hence the amount of
optical retardation is measured interferometrically using a
helium-neon laser. After retrieving the spectroscopic information
from the two dimensional array detector, the data is transformed
and stored as a series of images at different wavelengths. This is
achieved by a numerical technique known as a Fourier transform
(FT), which transforms the data from intensity versus mirror
displacement to intensity versus wavenumbers or wavelength. In
addition, the digital spectral images are further manipulated and
displayed using known spectroscopic, chemometric and image
processing techniques.
For emission spectroscopy, monochromatic light (e.g. from a laser),
or nearly monochromatic light (e.g. from a filtered arc lamp) is
directed toward a sample to be analyzed. The resulting sample
emission due to fluorescence and/or Raman scattering is multiplexed
by the interferometer and directed to the two dimensional array
detector which measures the intensity of the spectrally multiplexed
emitted light as a function of mirror displacement. Again, using a
Fourier transform technique, the data is transformed and stored as
a series of images at different wavelengths. The stored data and
images are then manipulated and displayed using known
spectroscopic, chemometric and image processing techniques.
An advantage to the present invention includes the ability to
rapidly and simultaneously record and analyze thousands of
absorption spectra with diffraction limited spatial resolution and
high spectral resolution.
While the invention will be described in connection with certain
preferred embodiments, it is not intended that the invention be
limited to those specific embodiments but rather that it be
accorded the broad scope commensurate with the appended claims,
consistent with the prior art.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a preferred embodiment of the
present invention--an imaging spectroscopic microscope--showing the
arrangement of some of its major elements and the AOTF illumination
scheme for both transmission and reflectance measurements.
FIG. 2 is a block diagram of the electronic control system suitable
for use in the practice of the present invention.
FIG. 3 is a schematic diagram of a typical image data set which may
be acquired by the imaging spectroscopic microscope of the
preferred embodiment shown in FIG. 1, for certain wavelengths of
filtered light transmitted by or reflected from a sample.
FIG. 4a is a graph of intensity plotted against wavelength for a
series of transmission values extracted from different images from
FIG. 3 at a fixed XY coordinate within the sample.
FIG. 4b is a graph of intensity plotted against wavelength for a
series of transmission values extracted from different images from
FIG. 3 at a fixed XY coordinate within the sample, different than
the fixed coordinate of FIG. 4a.
FIG. 5a is a diagram representing a spectroscopic image (taken at
520 nm) of a cross-section of human epithelia cells stained with a
visible dye, hematoxylineosin using the spectroscopic imaging
device of the present invention.
FIG. 5b is a diagram representing a spectroscopic image (taken at
710 nm) of a cross-section of human epithelia cells stained with a
visible dye, hematoxylineosin using the spectroscopic imaging
device of the present invention.
FIG. 6 is a schematic diagram of a preferred embodiment of the
present invention--an imaging spectroscopic microscope--showing the
arrangement of some of its major elements and the AOTF illumination
scheme for emission spectroscopy.
FIG. 7 is a block diagram of the electronic control system suitable
for use in the practice of the embodiment of the present invention
shown in FIG. 6.
FIG. 8 is a schematic diagram of a preferred embodiment of the
present invention--an imaging spectroscopic microscope--showing the
arrangement of some of its major elements and the interferometer
illumination scheme for both transmission and reflectance
measurements, as well as emission spectroscopy.
FIG. 9 is a block diagram of the electronic control system suitable
for use in the practice of the embodiment of the present invention
shown in FIG. 8.
FIG. 10a is a graph of intensity plotted against mirror retardation
of the interferometer of the preferred embodiment shown in FIG. 8,
for a series of values extracted from a series of images at a fixed
XY coordinate within the sample collected before applying the
Fourier transform technique to the data.
FIG. 10b is a schematic diagram of typical data sets of images
which may be acquired by the preferred embodiment shown in FIG. 8,
for certain retardation values of the interferometer before
applying the Fourier transform technique to the data.
FIG. 11a is a graph of intensity plotted against wavelength for a
fixed XY coordinate in the sample after applying the Fourier
transform technique to the data in FIG. 10a.
FIG. 11b is a schematic diagram of typical data sets of
spectroscopic images at discrete wavelengths which may be observed
after applying the Fourier transform technique to the data in FIG.
10b.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1, there is shown a preferred embodiment of the
present invention in the form of an imaging spectroscopic
microscope. The spectroscopic microscope consists generally of a
light source 10, an AOTF 14, a microscope 4, a focal plane array
detector 46, and a single point detector 44. The microscope 18 used
in the illustrated embodiment of the present invention may be a
modified Olympus BH-2 metallurgical microscope which includes a 10X
(N.A. 0.30) plan achromat objective 32 and 2.5X projection
eyepieces 40 and 42. The eyepiece 40 is used for collection,
magnification and presentation to the focal plane array detector
46. The eyepiece 42 is used for collection, magnification and
presentation to the video camera 50. The light source 10 may be a
standard 50 Watt quartz tungsten halogen light source. The
illustrated embodiment of the present invention also includes a
single point detector 44 consisting of dual detectors of Silicon
(Si) and Germanium (Ge), and a beamsplitter 38, which is a 50/50
cube beamsplitter. The Silicon and Germanium detectors may be of
the type made by Oriel. The video camera 50 may be of the type made
by Javelin and may be attached to a video monitor of the type made
by Ikegami. While the above elements used in the illustrative
embodiment of the present invention have been specifically
identified above, many variations and substitutions, known to those
skilled in the art, can be employed without deviating from the
intended scope of the present invention.
Certain aspects of this embodiment are known in the art, including
the AOTF. Briefly, an AOTF is an electronically tunable spectral
bandpass filter which can be operated from the ultra-violet through
the visible and into the infrared regions of the optical spectrum.
The AOTF functions by the interaction of light with a travelling
acoustic wave through an anisotropic medium. An acoustic transducer
is bonded to an end of an acousto-optic crystal and an acoustic
absorber is bonded to the other. The transducer converts a high
frequency rf signal into a pressure wave which propagates laterally
through the crystal. The acoustic absorber at the opposite end of
the crystal eliminates acoustic reflections which would corrupt the
primary acoustic waveform. The conservation of momentum between the
incident and diffracted photon wave vectors and the acoustic wave
vector determines the wavelength of the diffracted light. Optical
tuning is achieved by selecting the rf frequency signal. The
specific operation of an AOTF is illustrated more fully in Kurtz et
al., "Rapid scanning fluorescence spectroscopy using an
acousto-optic tunable filter" Rev. Sci. Instrum. Vol. 58, No. 11,
November 1987, the entire description found therein being
incorporated herein by reference.
In the present invention, the AOTF may be a Brimrose TEAF-.6-.1.2L.
The Brimrose crystal is constructed from tellurium dioxide
(TeO.sub.2), and the rf frequencies vary between 40-170 Mhz,
generating wavelengths between 400-1900 nm. For any given crystal,
the resolution is essentially fixed but may be varied by changing
the acousto-optic interaction distance at the time of manufacture.
The AOTF is entirely solid-state, containing no moving parts.
Random-access filtering is possible in the time it takes for the
acoustic wave to propagate the full length of the crystal,
typically around 5 micro seconds. The AOTF in the illustrated
embodiment of the present invention has a resolution of
approximately 2.5 nm and an achievable tunability of approximately
0.1 nm with a wavelength repeatability of greater than 0.05 nm.
Focal plane array detectors, used in the present invention, are
also well known in the art. The focal plane array detector 46 of
the illustrated embodiment of the present invention contains
silicon (Si) Charge Coupled Devices (CCDs), and may be a Spectrum
One, made by Spex. Using a two dimensional array (576.times.384
pixels) of CCDs, the focal plane array detector 46 can measure the
intensity of light incident upon it at multiple discrete locations,
and transfer the information received to a computer, or similar
device, for storage and analysis. The specific operation of a focal
plane array detector is illustrated more fully in R. B. Bilhorn, P.
M. Epperson, J. V. Sweedler and M. B. Denton, "Spectrochemical
Measurements with Multi-Channel Integrating Detectors", Applied
Spectroscopy 41, 1125, (1987), the entire description found therein
being incorporated herein by reference
FIG. 1 shows the path of light traveling through a preferred
embodiment of the present invention illustrated therein. Light
containing a wide range of wavelengths originates from the light
source 10, and comes into contact with a collimation optic lens 12.
The collimation lens 12 deflects the rays of light, making the rays
parallel to each other, and directs the parallel rays toward the
AOTF 14. The AOTF 14 is used to filter the collimated light at a
selected wavelength. The wavelength, or range of wavelengths, is
selected by the user under computer control. (A control system is
discussed more fully below.) After light of a selected wavelength
is emitted by the AOTF 14, it passes through a spatial filter 16.
The spatial filter 16 blocks the 0 Order Beam of broad-band light
and allows the light of the selected wavelength to pass.
At this point, the path of the spatially filtered light is
determined by the desired mode of operation. The operator
predetermines either transmission or reflectance, depending upon
the thickness or opacity of the sample, or other
considerations.
In the case of reflectance, epi-illumination is employed where the
nearly monochromatic spectrally filtered light is reflected by a
swing away mirror 18 toward a fixed mirror 20. The light is then
reflected by the fixed mirror 20 and passed through a collection
lens 22. The collection lens 22 assures that the intensity of the
light that reaches a sample 35 is equivalent at each point within
the sample 35. After passing through the collection lens 22, the
light is reflected by a 50/50 beamsplitter 24 toward an XY stage 26
upon which a glass microscope slide 33 holding the sample 35 rests.
The light then reflects away from the sample 35 and through the
objective 32, which magnifies the light. The intensity of the light
at this point depends upon the opacity and the absorption
characteristics of the sample 35.
In the case of transmission, instead of the spectrally reflected
light being reflected by the swing away mirror 18 toward the fixed
mirror 20, the swing away mirror 18 is retracted away from the path
of the light so that the light is reflected off a fixed mirror 28
toward a collection lens 30. The collection lens 30 assures that
the intensity of the light hitting the sample 35 is equivalent at
each point within the sample 35. The light then passes through a
condenser 34 toward the XY stage 26 upon which the glass microscope
slide 33 holding the sample 35 rests. The light that is not
reflected or absorbed then passes through the sample 35 and then
through the objective 32, which magnifies the light. Again, the
intensity of the light at this point depends upon the opacity and
the absorption characteristics of the sample 35.
In the case of reflectance, after the light is incident upon the
sample 35, the light passes through the beamsplitter 24. In the
case of transmission, beamsplitter 24 is retracted and light passes
unaltered toward swing away mirror 25. In either case, at this
point, the path of the light is again determined by the desired
mode of operation. The operator predetermines either spectral
imaging through the use of the focal plane array detector 46 or
single point detection through the use of detector 44.
In the case of spectral imaging, the light is then reflected by the
swing away mirror 25 away from the single point detector 44 toward
a lens 36. The lens 36 forms an intermediate image of the collected
light which is then split by the beamsplitter 38. The beamsplitter
38 directs the light toward two identical 2.5X projection eyepieces
40 and 42. The eyepieces 40 and 42 provide additional magnification
and present flat-field spectral images to the focal plane array
detector 46 and to the video camera 50, respectively. The video
camera 50 is parfocal with the focal plane array detector 46 and
operates with the video monitor, not shown, to position and focus
the sample 35. The focal plane array detector 46 is cooled using
Liquid Nitrogen (LN.sub.2) to improve the operation of the
CCDs.
In the case of single point detection, instead of reflecting the
light using the swing away mirror 25 toward the lens 36, the swing
away mirror 25 is retracted away from the path of the light so that
the light is directed toward a focusing lens 43. The focusing lens
43 focuses the light on the single point detector 44.
Microspectroscopy, commonly used on unknown materials, is achieved
by the operator preselecting single point detection.
Microspectroscopy provides survey spectral characterization of a
selected region of interest of the unknown material, typically
prior to the imaging of a sample. The survey scan typically
provides the user with an indication of the bulk chemical
composition of the sample 35 and indicates a wavelength range or
series of wavelengths over which to collect images. By
incorporating fast, single point detection, the illustrated
embodiment of the present invention acquires survey spectra very
rapidly. The silicon photodiode of the single point detector 44 is
used to measure the intensity of the transmitted or reflected light
for visible wavelength operation (400-1100 nm), while the germanium
photodiode of the single point detector 44 is used for
near-infrared operation (800-1800 nm). In the illustrated
embodiment of the present invention, the single point detector 44
is operated uncooled, and accordingly, signal averaging is used to
improve the signal to noise performance. Typically, several hundred
averages are performed which increases the acquisition time of the
survey spectra to several seconds. Alternatively, an improvement in
the acquisition time can be achieved by using a more efficient
photodiode material or by operating the single point detector 44
cooled.
In accordance with the present invention, there is provided a
control system 102 which controls the operation of the device.
Referring to FIG. 2, there is shown a schematic diagram of the
control system 102 which is usable with the illustrated embodiment
of the present invention. The control system 102 consists generally
of a controller 100, a display 100 and a keyboard 120. The
controller 100 may be of the type referred to as a "Dell 310", an
80386-based computer. However, other microprocessor based
controllers may be substituted for the Dell 310 computer without
deviating from the invention. The display 110 may be a VGA monitor,
and the keyboard 120 may be a standard Dell 310 compatible
keyboard. Again, variations and substitutions, known to those
skilled in the art, can be employed, if desired.
Software for the controller 100 may, for example, be written in the
C programming language. The software allows the controller to,
among other things, control the AOTF 14; position the swing away
mirrors 18 and 25; position the beamsplitter 24; store the data
acquired from the focal plane array detector 46; store data
acquired from the single point detector 44; digitize and store the
image from the video camera 50; and perform data manipulation and
analysis. Contrast enhancement, pseudo-coloring and image
presentation may be performed using a commercial image processing
package, such as BioScan Optimas 3.0.
The controller 100 selects the wavelength of light emitted from the
AOTF 14 by generating a 32 bit binary word for a particular
wavelength. The 32 bit binary word is converted into an rf
frequency by a synthesizer board 130. The value of the 32 bit
binary word is based on the independent spectral calibration of the
AOTF 14. A Fourier transform interferometer (not shown), well known
in the art as a very accurate spectrometer, may be used to
calibrate the AOTF 14. By recording the resulting wavelengths of
light that the AOTF 14 emits for a test group of rf frequencies
(e.g. approximately 30), polynomial approximation techniques may be
used to calculate wavelength as a function of rf frequency.
The swing away mirrors 18 and 25 and the beamsplitter 24 are
connected to the controller 100 via an interrupt driven RS232
communications link RS232 communication links are well known in the
art. The mirrors 18 and 25 and the beamsplitter 24 are moved
repeatedly with high precision in and out of position depending
upon whether reflectance or transmission is employed and whether
spectral imaging or single point detection is desired. The movement
of the swing away mirrors 18 and 25 and beamsplitter 24 are
controlled by daisy-chained microstepper translation stages.
The Si CCDs of the focal plane array detector 46 store data as 16
bit binary integers. The controller 100 acquires the data from the
CCDs of the focal plane array detector 46 and stores the data as 32
bit floating point numbers. Using 32 bit floating point number
enhances the precision of the measured images. The Si and Ge
photodiodes of the single point detector 44 contain output analog
data signals. The controller 100 digitizes the analog signals to 16
bit precision using an analog to digital board 140 attached to the
controller 100. The analog to digital board 140 may be of the type
ATMIO-16, made by National Instruments. All data retrieved by the
controller is stored in a Spectral Image File Format (SPIFF) file
for later retrieval.
The controller 100 provides a means to capture a white light image
of the sample from the digital video camera 50. The white light
image is captured using a grabber board 142 attached to the
controller 100. This white light image is also saved in the SPIFF
file and is retrievable at a later time to provide the operator
with a white light picture of the sample with which to compare the
spectroscopic images and data. The grabber board 142 may be a
Scorpion board, made by Univision.
The controller 100 also controls the timing of the AOTF 14 with the
acquisition of data from either the focal plane array detector 46
or the single point detector 44. At a predetermined time, or as the
AOTF 14 incrementally changes the wavelengths of the emitted light,
the controller 100 obtains the data from either the focal plane
array detector 46 or the single point detector 44 corresponding to
the given wavelength.
Typically, the invention operates and is used as follows. First,
the operator places a sample to be analyzed on the XY stage 26 of
the imaging spectroscopic microscope. Next, the operator uses the
digital video camera 50 to survey the sample and focus and frame
the field of view for the subsequent collection of spectroscopic
data. Then, the operator uses the keyboard 120 to select the
various parameters which control the AOTF 14, the swing away
mirrors 18 and 25, the beamsplitter 24, and the timing of the focal
plane array detector 46 or single point detector 44 with the data
collection of the controller 100. The parameters which the operator
can choose in the illustrated embodiment of the present invention
include 1) reflectance or transmission, 2) spectral imaging or
single point detection. 3) wavelength range and wavelength
increment, if any, of the light filtered from the AOTF 14, 4)
integration time for imaging detection of each wavelength, 5) the
number of scans or sweeps of the AOTF 14. Of course, it is
appreciated that both the selection and the range of possible
parameters depend upon the application and the type of analysis
desired.
In the illustrated embodiment, integration times are varied to
correct for intensity variations in detector response as a function
of wavelength. Such response variations are inherent in any
spectroscopic system. In an alternate embodiment, a partial
correction for this response function is made automatically by
either pre-recording the response variation or determining the
response variation during run-time.
In the case of single point detection, the intensity at each
wavelength is digitized by the analog to digital board 140 and
stored in the SPIFF file and presented for display on the VGA
screen 110. In the case of spectral imaging, the data from the
focal plane array detector 46 is stored as a 32 bit floating point
number in the SPIFF file for later manipulation and analysis. In
addition to the data from the focal plane array detector 46, the
single point detector 44, and the video camera 50, the SPIFF file
also contains date, time, sample information, wavelength ranges,
integration times, and other variable information in its
header.
Once the data has been stored in the SPIFF file, a standard imaging
analysis software package is used to display the results. Referring
to FIG. 3, there is shown a schematic representation of typical
data sets of images acquired by the imaging spectroscopic
microscope for various wavelengths of filtered light transmitted or
reflected from a sample. FIG. 3 shows 4 different planar images of
the same sample for different wavelengths .lambda.0, .lambda.1,
.lambda.2 and .lambda.n-1. It should be appreciated that each
planar image represents a third dimension of imaging distinct from
a mere two dimensional spatial image. The spectroscopic images are
generated from data derived from the intrinsic optical and
vibrational absorptions, and chemical properties, of the
material.
Referring to FIG. 4a, there is shown a graph of a series of
transmission values extracted from different data sets of images
from FIG. 3 at a fixed coordinate within the sample. The graph
represents the intensity of the light transmitted through or
reflected away from a sample at a given fixed location
(X.sub.i1,j1) for each wavelength .lambda.0 through .lambda.n-1.
FIG. 4b is another graph of a series of transmission values
extracted from different data sets of images from FIG. 3 at a fixed
coordinate (X.sub.i2,j2) within the sample different than the fixed
coordinate of FIG. 4a. By way of example, at the point
(X.sub.i2,j2) in FIG. 4b for the wavelength .lambda.0, it can be
seen that the relative intensity of light transmitted through or
reflected away from a sample is lower than the intensity for
wavelength .lambda.1. In other words, more light of wavelength
.lambda.1 is absorbed than light of wavelength .lambda.1. This is
readily apparent from FIG. 3 where the point (X.sub.i2,j2) is much
darker for wavelength .lambda.0 than for wavelength .lambda.1.
Referring to FIG. 5a, there is shown a diagram representing a
spectroscopic image (taken at 520 nm) of a cross-section of human
epithelia cells stained with a visible dye. FIG. 5b is another
diagram renting a spectroscopic image (taken at 710 nm) of a
cross-section of human epithelia cells stained with a visible dye.
For FIG. 5a and FIG. 5b, total magnification provides a spatial
resolving element of approximately 2.0 microns per pixel. Images
were recorded over the wavelength range 400-800 nm at 2 nm
increments using a 15 millisecond exposure at each wavelength. The
total measurement time was 3 seconds. The 520 nm wavelength used to
obtain the measurements of FIG. 5 corresponds to the absorption
maximum of the histological stain. It is apparent that FIG. 5a
provides significantly higher visual clarity and structural
information than FIG. 5b. The apparent contrast in the image of
FIG. 5a demonstrates the chemical differences in the arterial wall
relative to the bulk material in this particular tissue type.
Referring to FIG. 6, there is shown an alternate embodiment of the
present invention in the form of an imaging spectroscopic
microscope 6. The referenced numerals in FIG. 6 correspond to the
components of FIG. 1 and function similarly. The AOTF 14 is
positioned between the sample 35 and the focal plane array detector
46 to filter the light after the light has impinged upon the sample
35. A collimation lens 23 is also positioned between the AOTF 14
and the sample 35. With the configuration shown in FIG. 6, in
addition to absorption spectroscopic imaging studies, emission
spectroscopic imaging may be achieved, including fluorescence and
Raman imaging. Holographic Raman filters 41 are positioned between
the AOTF 14 and the focal plane array detector 46. The filters 41
are removeable and used exclusively for measuring Raman emission.
The filters 41 are used to selectively block the laser radiation,
while allowing the weaker Raman scattered photons to pass through
to the focal plane array detector 46. The holographic filters
exhibit improved performance relative to dielectric interference
filters, namely, uniform transmission and sharper cut-on. The
operation of the embodiment shown in FIG. 6 is very similar to the
operation of the embodiment shown in FIG. 1.
To achieve absorption spectroscopy in the embodiment shown in FIG.
6, a dichroic beamsplitter 13 is retracted, and the broad-band
light source 10 directs light toward the sample to be analyzed.
After impinging upon the sample 35, the transmitted or reflected
broad-band light is then filtered by the AOTF 14 and directed
toward either the focal plane array detector 46 or the single point
detector 44, depending upon the desired operation as described
above for the embodiment shown in FIG. 1.
To achieve emission spectroscopy in the embodiment shown in FIG. 6,
a non-filtered light source 11 directs light toward the
beamsplitter 13. The non-filtered light source 11 may be a laser.
The beamsplitter 13 then directs the non-filtered light toward the
sample 35 to be analyzed. After the non-filtered light has impinged
upon the sample 35, the resulting emission from the sample 35 is
directed toward the AOTF 14. Then the AOTF 14 filters the emitted
light at a selected wavelength and directs the filtered light
toward either the focal plane array detector 46 or the single point
detector 44, again depending upon the desired mode of
operation.
A control system 104, shown in FIG. 7, is usable with the
embodiment shown in FIG. 6. The referenced numerals in FIG. 7
correspond to the components of FIG. 2 and function similarly. The
controller 100, among other things, controls the AOTF 14; positions
the swing away mirrors 18 and 25; positions the beamsplitters 13
and 24; stores the data acquired from the focal plane array
detector 46; stores the data acquired from the single point
detector 44; digitizes and store the image from the video camera
50; and performs data manipulation and analysis. Ultimately,
contrast enhancement, pseudo-coloring and image presentation may be
performed using a commercial image processing package, such as
BioScan Optimas 3.0.
Referring to FIG. 8, there is shown an alternate embodiment of the
present invention in the form of an imaging spectroscopic
microscope 8. The referenced numerals in FIG. 8 correspond to the
components of FIG. 6 and function similarly. An interferometer 60
positioned between the sample 35 and the focal plane array detector
46. The operation of the embodiment shown in FIG. 8 is very similar
to the operation of the embodiment shown in FIG. 6, except the
interferometer 60 shown in FIG. 8 essentially performs the function
of the AOTF 14 shown in FIG. 6. The embodiment shown in FIG. 8
achieves both absorption spectroscopic imaging and emission
spectroscopic imaging, including fluorescence and Raman imaging.
Holographic Raman filters 41 are positioned between the
interferometer 60 and the focal plane array detector 46. The
filters 41 are removeable and used exclusively for measuring Raman
emission. The filters 41 are used to selectively block the laser
radiation, while allowing the weaker Raman scattered photons to
pass through to the focal plane array detector 46. The holographic
filters exhibit improved performance relative to dielectric
interference filters, namely, uniform transmission and sharper
cut-on.
The function of the interferometer 60 is to spectrally filter the
light. To achieve absorption spectroscopy in the embodiment shown
in FIG. 8, a dichroic beamsplitter 13 is retracted, and the
broad-band light source 10 directs light toward the sample to be
analyzed. After impinging upon the sample 35, the transmitted or
reflected broad-band light is then directed toward the
interferometer 60. To achieve emission spectroscopy in the
embodiment shown in FIG. 8, a non-filtered light source 11 directs
light toward the beamsplitter 13. The non-filtered light source 11
may be a laser. The beamsplitter 13 then directs the non-filtered
light toward the sample 35 to be analyzed. After the non-filtered
light has impinged upon the sample 35, the resulting emission from
the sample 35 is directed toward the interferometer 60. For both
absorption and emission spectroscopy, after the light is directed
toward the interferometer 60, the operation of the interferometer
60 is essentially the same.
The interferometer 60 consists generally of a fixed mirror 62, a
moveable mirror 64, a beamsplitter 66, and a measuring laser 68.
The light directed toward the interferometer 60 is directed by the
beamsplitter 66 to the fixed mirror 62 and moveable mirror 64.
Initially, the fixed mirror 62 and moveable mirror 64 are located
an equal distance from the beamsplitter 66. In this initial
position, the light directed to the mirrors 62 and 64 is reflected
back toward the beamsplitter 66 and recombined, in phase, before
being directed toward the lens 36. Then, the moveable mirror 64 is
incrementally retarded away from the beamsplitter 66. The
incremental motion of the moveable mirror 64 is controlled by the
controller 100, shown in FIG. 9. The relative position of the two
mirrors, and hence the amount of optical retardation, is measured
interferometrically using the measuring laser 68. The measuring
laser 68 may be a helium-neon (HeNe) laser. A typical increment of
retardation is one-half the wavelength of the light of the
measuring laser 68.
With the moveable mirror incrementally retarded, the light that is
directed toward the fixed mirror 62 and the moveable mirror 64 is
reflected back toward the beamsplitter 66 and recombined before
being directed toward the lens 36. However, because the moveable
mirror 64 was incrementally retarded, the light that is recombined
by the beamsplitter 66 is slightly out of phase. By continuing to
incrementally retard the moveable mirror 64, and measuring the
intensity of light at each incremental step using the focal plane
array detector 46, a multiplexed spectral output at multiple
locations within the sample is obtained.
A control system 106, shown in FIG. 9, is usable with the
embodiment shown in FIG. 8. The referenced numerals in FIG. 9
correspond to the components of FIG. 7 and function similarly. The
controller 100, among other things, controls the moveable mirror 64
of the interferometer 60, positions the swing away mirrors 18 and
25; positions the beamsplitters 13 and 24; stores the data acquired
from the focal plane array detector 46; manipulates the data
acquired from the focal plane array detector 46 to create data sets
of spectroscopic images; digitizes and store the image from the
video camera 50, and performs data manipulation and analysis.
Ultimately, contrast enhancement, pseudo-coloring and image
presentation may be performed using a commercial image processing
package, such as BioScan Optimas 3.0. The controller 100 may also
control a single point detector 44 which may be added to the
embodiment shown in FIG. 8 if single point detection is desired. If
so, the single point detector 44 may be positioned where the video
camera 50 is located.
Alternatively, the interferometer 60 employed to spectrally filter
the light may be a continuously modulated interferometer. With a
continuously modulated interferometer, however, the image obtained
may be distorted as a consequence of the change in distance that
occurs during the time interval taken to read out the array of CCDs
of the focal plane array detector 46. Accordingly, with this
embodiment it is preferred to employ a rapid camera or focal plane
array detector that is capable of instantaneously or near
instantaneously scanning the entire image in a manner that
preserves image fidelity.
For example, a new class of focal plane array detectors
commercially available from Santa Barbara Focalplane, Model No.
IMAGIR, enables each CCD of the focal plane array detector 46 to
take a simultaneous "snap-shot" image of the scene at a given
instant. For each instantaneous snapshot, a position sensor such as
a helium-neon laser 68 is employed in a well-known manner to
precisely determine the position or positions of the
continuously-moving interferometer mirror 64 during the period when
the CCDs were being charged and report it to controller 100. The
fast analog-to-digital voltage converters of the focal plane array
detector enables the charges of the CCDs to be rapidly digitized
such that the fidelity of the data stream is maintained.
The controller 100, or other variable speed driving means (not
shown), controls the movement of the continuously modulated
interferometer 60. The acquisition of each image may be coordinated
with the position of the moving mirror 64, such as by having the
laser 68 report the retardation (i.e., relative distance) of the
mirror to the controller 100 to trigger the snapshot as the mirror
46 achieves select retardations. The image data is thereby obtained
at a number of selected positions. As a result, the data is
comparable to the data obtained with the previously described
step-scanning technique.
Indeed, even if there is some uncertainty in the way in which the
detector 46 is read out in the continuously modulated system, the
data maintains its fidelity as long as the well known Nyquist
sampling theorem for the desired wavelength range and scan speed is
not violated. In other words, provided that the entire detector
array 46 is read before the interferometer mirror 64 has moved to
the next desired sampling position, the continuous interferometer
60 yields a useable data stream. However, if it is determined that
a phase shift has occurred in the data obtained from different
pixels, this can be readily corrected after the data is collected
by knowing the readout rate of the camera.
Referring to FIG. 10a, there is shown a graph of the intensity of
the multiplexed spectral output plotted against the distance of
retardation of the interferometer 60 for a series of values
collected by the focal plane array detector 46 at a fixed
coordinate within the sample 35. Referring to FIG. 10b, there is
shown a schematic diagram of typical data sets of images acquired
by the focal plane array detector 46 for a certain retardation
values (i.e. 1, 2, 3, 2.sup.n) of the interferometer 60. The
symbols "H", "F", "E", and "T" shown in FIG. 10b are representative
of multiple sections of the sample 35, each section having its own
spectral characteristics. However, the spectral characteristics of
the sections can not be readily determined simply by displaying the
multiplexed spectral output of the interferometer 60 in FIGS. 10a
and 10b. Further data manipulation is required to display the
spectral characteristics of the sections of the sample 35.
After retrieving and storing the multiplexed spectral data from the
focal plane array detector 46, the controller 100 transforms the
data into spectroscopic image data by a well-known numerical
technique known as a Fourier transform (FT). The FT technique
transforms the data from intensity versus mirror retardation to
intensity versus wavenumbers or wavelength. Then, the spectroscopic
image data is further manipulated and displayed using known
spectroscopic, chemometric and image processing techniques.
Referring to FIG. 11a, there is shown a graph of intensity plotted
against wavelength acquired after applying the Fourier transform
technique to the data in FIG. 10a at the fixed coordinate within
the sample. Referring to FIG. 11b, there is shown a schematic
diagram of certain data sets of spectroscopic images at discrete
wavelengths observed after applying the Fourier transform technique
to the data in FIG. 10b. The spectral characteristics of the
sections of the sample 35 can now be readily seen.
Alternate embodiments of the present invention use indium
antinomide (InSb) or platinum silicide (PtSi), for use in the focal
plane array detector or single point detector, which deliver
enhanced infrared sensitivity to the photon energy of the light
emitted, transmitted through, or reflected away from the sample. By
using InSb detectors, the alternate embodiment of the present
invention can be used at higher wavelengths (up to 5500 nm), well
into the infrared region. In other embodiments, detectors could be
made of iridium silicide (IrSi), or other materials to allow
spectral imaging techniques to wavelengths of 12000 nm.
While the illustrated embodiment of the present invention includes
an imaging spectroscopic microscope, the present invention can be
applied to other traditional absorption or emission spectroscopic
approaches. These techniques may be applied in, for example, areas
involving biological materials, polymers, semi-conduct and
situations involving remote sensing.
For example, an alternate embodiment of the present invention
achieves macroscopic spectroscopic imaging by substituting a
macroscopic lens, such as a typical 50 mm camera lens, for the
modified microscope device shown in the illustrated embodiments. In
such a case, the macroscopic lens directs light, such as ambient
room light, that has been reflected or emitted from a sample toward
the focal plane array detector 46. The controller 10 then collects,
manipulates and displays the resulting macroscopic spectroscopic
images.
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