U.S. patent application number 10/237151 was filed with the patent office on 2003-06-05 for multimodal miniature microscope.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Anslyn, Eric, Descour, Michael, Dupuis, Russell, Richards-Kortum, Rebecca.
Application Number | 20030103262 10/237151 |
Document ID | / |
Family ID | 23236449 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030103262 |
Kind Code |
A1 |
Descour, Michael ; et
al. |
June 5, 2003 |
Multimodal miniature microscope
Abstract
Apparatus for receiving and positioning optical components. The
apparatus includes a substrate, one or more mounting slots, and one
or more springs. The one or more mounting slots are formed in the
substrate, and each mounting slot includes a mounting slot wall. At
least one of the mounting slots is adapted to receive an optical
component. At least one of the mounting slots is coupled to one of
the springs.
Inventors: |
Descour, Michael; (Tucson,
AZ) ; Dupuis, Russell; (Austin, TX) ; Anslyn,
Eric; (Austin, TX) ; Richards-Kortum, Rebecca;
(Austin, TX) |
Correspondence
Address: |
FULLBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
23236449 |
Appl. No.: |
10/237151 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60318059 |
Sep 7, 2001 |
|
|
|
Current U.S.
Class: |
359/368 |
Current CPC
Class: |
B82Y 20/00 20130101;
G02B 21/0024 20130101; G02B 21/362 20130101; B82Y 10/00 20130101;
B82Y 5/00 20130101; G02B 21/34 20130101; G02B 7/003 20130101 |
Class at
Publication: |
359/368 |
International
Class: |
G02B 021/00 |
Goverment Interests
[0002] The government may own rights in the present invention
pursuant to the following grant: NSF BES-0086736 and NSF SGER
ECS-0074578.
Claims
What is claimed is:
1. An apparatus for receiving and positioning optical components,
the apparatus comprising: a substrate; one or more mounting slots
formed in the substrate, each mounting slot comprising a mounting
slot wall, and at least one of the mounting slots adapted to
receive an optical component; and one or more springs, wherein at
least one of the mounting slots is coupled to one of the
springs.
2. The apparatus of claim 1, wherein the substrate has a length of
less than about 10 mm, a width of less than about 5 mm, and a
thickness of less than about 3 mm.
3. The apparatus of claim 1, wherein the substrate is silicon.
4. The apparatus of claim 1, wherein the substrate is metal.
5. The apparatus of claim 1, wherein at least one of the mounting
slots further comprise one or more grooves formed in the mounting
slot wall.
6. The apparatus of claim 5, wherein each groove is adapted to
receive a protrusion formed on the optical component.
7. The apparatus of claim 6, wherein the springs are configured to
secure at least one of protrusions formed on the optical component
in at least one of the grooves formed in the mounting slot
wall.
8. The apparatus of claim 1, wherein the springs are silicon.
9. The apparatus of claim 1, wherein each spring comprises a first
elongated portion and a second elongated portion, the second
elongated portion coupled to the first elongated portion such that
an angle defined by the first elongated portion and the second
elongated portion is acute.
10. A microscope for generating images of tissue, comprising: a
substrate; a plurality of springs; a plurality of mounting slots
formed in the substrate, each of the plurality of mounting slots
adapted to receive an optical component, each of the plurality of
mounting slots comprising a mounting slot wall, and each mounting
slot having one of the plurality of springs coupled to the mounting
slot wall; a plurality of optical components; wherein one or more
of the plurality of optical components are adapted to be partially
contained in one or more of the plurality of mounting slots.
11. The microscope of claim 10, wherein each of the plurality of
mounting slots further comprise one or more grooves formed in the
mounting slot wall.
12. The microscope of claim 11, wherein the plurality of optical
components adapted to be at least partially contained in the
plurality of mounting slots further comprise one or more
protrusions that are adapted to be received in the one or more
grooves.
13. The microscope of claim 11, wherein the plurality of optical
components comprise: an illumination source configured to
illuminate the tissue with radiation; a detector configured to
collect radiation from the tissue; a beam splitter in operative
relation with the illumination source and the detector, the beam
splitter configured to select a first wavelength to be directed
from the illumination source to the tissue and configured to select
a second wavelength to be directed from the tissue to the detector;
and a lens in operative relation with the beam splitter.
14. The microscope of claim 13, wherein the illumination source,
detector, and beam splitter are adapted to be at least partially
contained in one of the plurality of mounting slots.
15. The microscope of claim 14, wherein the illumination source,
detector, and beam splitter each further comprise one or more
protrusions that are adapted to be received in the one or more
grooves.
16. The microscope of claim 13, wherein the illumination source is
located externally to the substrate.
17. The microscope of claim 13, wherein the plurality of optical
components further comprise: a collector mirror in operative
relation with the light source, the collector mirror; one or more
refractive lenses in operative relation with the beam splitter, the
one or more refractive lenses; a scanning grating in operative
relation with the beam splitter, the scanning configured to be used
for optical sectioning; and a CMOS active-pixel image sensor.
18. The microscope of claim 13, wherein the collector mirror, the
one or more refractive lenses, and the scanning grating are adapted
to be at least partially contained in one of the plurality of
mounting slots.
19. The microscope of claim 13, wherein the scanning grating is a
double-pass scanning grating.
20. The microscope of claim 13, wherein the scanning grating is
integral with the substrate.
21. The microscope of claim 13, wherein the illumination source
emits wavelengths from between 350 nm to 500 nm.
22. The microscope of claim 13, wherein the illumination source
emits wavelengths from between 600 nm to 1100 nm.
23. The microscope of claim 10, wherein the substrate has a length
of less than about 10 mm, a width of less than about 5 mm, and a
thickness of less than about 2.5 mm.
24. The microscope of claim 10, further comprising a
low-magnification imaging system.
25. A method for imaging tissue, comprising: obtaining a microscope
comprising: a substrate, a plurality of springs, a plurality of
mounting slots formed in the substrate, each of the plurality of
mounting slots adapted to receive an optical component, each of the
plurality of mounting slots comprising a mounting slot wall, and
each mounting slot having one of the plurality of springs coupled
to the mounting slot wall, a plurality of optical components,
wherein one or more of the plurality of optical components are
adapted to be partially contained in one or more of the plurality
of mounting slots; imaging the tissue with the microscope.
26. The method of claim 25, wherein the imaging comprises confocal
imaging.
27. The method of claim 25, where the confocal imaging comprises
confocal reflectance imaging and confocal fluorescence imaging.
28. The method of claim 25, wherein the imaging comprises
fluorescence imaging.
29. The method of claim 25, wherein the imaging comprises
reflectance imaging.
30. The method of claim 25, wherein the imaging comprises confocal
imaging, fluorescence imaging, and reflectance imaging.
31. The method of claim 25, wherein the imaging comprises imaging a
tumor in the tissue.
32. The method of claim 31, wherein the imaging comprises confocal
imaging.
33. The method of claim 31, where the confocal imaging comprises
confocal reflectance imaging and confocal fluorescence imaging.
34. The method of claim 31, wherein the imaging comprises
fluorescence imaging.
35. The method of claim 31, wherein the imaging comprises
reflectance imaging.
36. The method of claim 31, wherein the imaging comprises confocal
imaging, fluorescence imaging, and reflectance imaging.
37. The method of claim 31, further comprising marking the tumors
with a dye.
38. The method of claim 37, wherein the dye is conjugated to an
antibody.
39. The method of claim 38, wherein the dye is conjugated to an
antibody for cytokeratins.
40. The method of claim 39, wherein the dye is Nile Blue A.
41. The method of claim 39, wherein the dye is Texas Red.
42. The method of claim 31, further comprising marking the tumors
with a reflective nanoparticle.
43. The method of claim 42, the nanoparticle comprising gold.
44. The method of claim 31, further comprising marking the tumors
with a quantum dot.
Description
[0001] This application claims priority to provisional U.S. Patent
Application No. 60/318,059 entitled "Multimodal Miniature
Microscope," which was filed on Sep. 7, 2001. U.S. Patent
Application No. 60/318,059, in its entirety, is incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
diagnostic imaging. More particularly, it concerns a microscope
that utilizes the interaction of light with tissues in many
modalities to image morphology and biochemistry, thereby providing
better delineation of tumors. Even more particularly, it concerns a
miniaturized microscope capable of different imaging modalities
such as optical sectioning, 3-D spectral fluorescence imaging, and
reflectance imaging.
[0005] 2. Description of Related Art
[0006] The American Cancer Society estimates that 1,220,100 people
will have been diagnosed with cancer in 2000. In the same year,
552,200 persons were expected to succumb to cancer. Despite
significant advances in treatment, early detection of cancer and
its curable precursors remains the best way to ensure patient
survival and quality of life.
[0007] Pre-cancers are characterized by morphologic and biochemical
changes that include increased nuclear size, increased nuclear to
cytoplasmic ratio, hyperchromasia, pleomorphism, angiogenesis, and
increased metabolic rate. These changes currently can only be
assessed through invasive biopsy. Early detection of curable
pre-cancers has the potential to significantly lower cancer
mortality and morbidity. Many visual exam procedures, such as
colonoscopy and bronchoscopy, are routinely used to identify
pre-malignant changes and early cancers. However, these techniques
do not assess the microscopic and/or biochemical changes which are
the hallmark of pre-cancer. Thus, these techniques' sensitivity and
specificity are limited.
[0008] Early detection would be particularly beneficial in the
treatment of several types of cancers. For instance, cancer of the
oral cavity is usually not diagnosed until it is in an advanced
stage. In the advanced stage, treatment is more disfiguring,
expensive, and prone to failure. Thus, early detection of
pre-cancer is the best method to improve patient quality of life
and survival. Certain lesions in the oral cavity have been
identified clinically to have the potential for malignant
conversion. These include leukoplakia (white plaques) and
erythroplakia (velvety, reddish lesions). Invasive biopsies are
often required to confirm the presence of pre-cancer. Thus, despite
the easy accessibility of the oral cavity to examination, there is
no satisfactory mechanism to adequately screen and detect
pre-cancers. The development of a noninvasive and accurate method
for real-time screening and diagnosis of oral cavity lesions would
have great potential to improve early detection of neoplastic
changes, and thereby improve the quality of life and survival rates
for persons developing carcinomas of the oral cavity.
[0009] Cervical cancer is the third most common cancer in women
worldwide and the leading cause of cancer mortality in women in
developing countries. The curable precursor to cervical cancer is
cervical intra-epithelial neoplasia. In the U.S. over $6 billion
are spent annually in the evaluation and treatment of low-grade
precursor lesions. Approximately 50 million Pap smears are
performed annually in the U.S. to screen for cervical cancer and
its precursor. The National Cancer Institute estimates 6-7% of
these tests to be abnormal. However, cervical cancer goes
undetected in developing countries because of the cost of the tests
and the lack of trained personnel and resources. In the U.S.,
resources are wasted on the evaluation and treatment of lesions
that are not likely to progress to cancer.
[0010] Optical technologies offer the ability to image tissue with
unprecedented spatial and temporal resolution using low-cost,
portable devices. As such, optical technologies represent an ideal
approach to imaging early neoplasia. Multiple in vivo optical
imaging and spectroscopic modalities have been explored recently as
diagnostic tools in medicine. These modalities include
multi-spectral fluorescence imaging, multi-spectral reflectance
imaging with unpolarized and polarized light, confocal microscopy,
reflectance, and fluorescence spectroscopy. In the ultraviolet (UV)
and visible regions of the spectrum, tissue reflectance spectra
provide information about the wavelength dependent scattering of
tissue as well as electronic absorption bands, primarily those of
oxy- and deoxyhemoglobin. The most common naturally occurring
fluorophores include the aromatic amino acids, the co-factors
NAD(P)H and FAD, crosslinks associated with collagen and elastin,
and porphyrins.
[0011] Furthermore, optical technologies may be used to complement
existing pre-cancer treatments, such as chemoprevention.
Chemoprevention refers to the use of chemical agents to prevent or
to delay the development of cancer in healthy populations or
patients with precancerous tissue changes. Despite their promise,
chemoprevention studies have several inherent problems. One is that
many patients hesitate to enroll in such trials because they
require multiple biopsies throughout the period when the
chemopreventive agent is given. Biopsies are processed to measure
morphologic and biochemical changes associated with cancer
progression and assess drug response. A second problem is that the
biopsy process itself can interrupt the natural progression of the
lesion. Many times these lesions are small enough that the biopsy
is the cure; frequent biopsies make it difficult to accurately
assess drug response. Thus, tools that non-destructively assess
quantitative morphologic and biochemical changes that do not
require biopsy could considerably improve chemoprevention
studies.
[0012] Both screening and detection could be vastly improved by in
vivo optical imaging technologies that improve the ability to
recognize and delineate pre-cancerous lesions in the cervix with
high sensitivity and specificity. A major challenge in implementing
quantitative optical tools for widespread screening is to develop
small, inexpensive imaging systems that provide both high
sensitivity and high specificity for the biochemical and
morphologic features of pre-cancer. A need therefore exists for
small, inexpensive imaging systems that may enhance or replace
traditional visual exam procedures to allow for more accurate
identification of pre-cancerous lesions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0014] FIG. 1 shows a schematic of an optical-sectioning
multi-modal miniature microscope device according to one embodiment
of the present disclosure.
[0015] FIGS. 2A and 2B shows a detailed schematic view of an
optical element mounted in a mounting slot etched in a silicon
micro-optical table ("MOT") substrate according to one embodiment
of the present disclosure.
[0016] FIG. 3 shows a magnetic microactuator that may be adapted to
the task of a scanning grating in one embodiment of the device of
the present invention. Specifically, a partially assembled
variable-reluctance magnetic linear microactuator is shown.
[0017] FIGS. 4A and 4B show lithographically patterned optical
elements. FIG. 4A shows a micro-optical element patterned in hybrid
sol-gel material on a 150 micron thick glass substrate.
[0018] FIG. 4B shows a hybrid sol-gel material patterned to a depth
of 34 microns.
[0019] FIGS. 5A and 5B show lithographically patterned optical
elements mounted and cemented in a silicon MOT substrate. FIG. 5A
shows the optical elements as seen under a microscope. FIG. 5B
shows the optical elements observed in an SEM.
[0020] FIGS. 6A and 6B show silicon-spring displacement and the
normal stress in the horizontal direction. FIG. 6A shows the
optical element in its penultimate position. FIG. 6B shows an image
of a fabricated silicon spring that retains an optical element in
the mounting slot. The width of the slot shown is 600 .mu.m, and
the length is 4,000 .mu.m.
[0021] FIG. 7 shows the deep X-ray lithography DXRL process. The
process steps shown are (1) exposure of a PMMA
(poly-methylmethacrylate) photoresist with x-rays generated from a
synchrotron light source through an x-ray mask, (2) PMMA
development to realize a plastic mold, (3) electro-deposition to
fill this mold with a metal material, (4) planarization to
accurately control part thickness, and (5) release and device
integration.
[0022] FIGS. 8A, 8B, and 8C show various aspects of one embodiment
of the device of the present invention. FIG. 8A shows a device that
contains only refractive microlenses and a folding mirror. FIG. 8B
shows a fabricated MOT silicon substrate comprising silicon-spring
mounting slots. FIG. 8C shows three 150-micron thick, unpatterned
glass substrates mounted in the MOT. The plates are separated by
800 microns.
[0023] FIG. 9 shows one design of a device that operates in the red
wavelength region.
[0024] FIG. 10A shows a cross-section of an interior wall of a
mounting slot, and two guide channels located on the wall. FIG. 10B
shows a one side of an optical component, and two protrusions on
the optical component.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0025] In one embodiment, the present invention is directed to a
class of microscopes, and in particular miniature microscopes, that
utilize the interaction of light with tissues in many modalities to
image morphology and biochemistry in vivo, yielding tools that
provide better delineation of tumors. It is contemplated that the
devices of the present invention may image microscopic and
molecular features of pre-cancer. The proposed miniature
microscopes are multi-modal because of their potential for enabling
different imaging modalities, which may include optical sectioning,
3-D spectral fluorescence imaging, and reflectance imaging. The
microscopes may be miniaturized by using a zero-alignment
microscopic optical-system. Specifically, a micro-optical table
("MOT") substrate may be used. Various mounting slots may be formed
in the MOT, and the mounting slots may be configured to receive and
secure various optical components.
[0026] The size and cost of these microscopes can be eventually
small enough so that they can aid in, for instance, guiding
diagnostic biopsy and to aid in margin detection during tumor
resection. The devices may have broad applicability in many organ
sites due to their very compact size and capability for imaging. It
has been previously demonstrated that the morphologic and
biochemical changes that accompany pre-cancer can be probed using
reflectance and fluorescence. Accordingly, the imaging devices of
the present invention may be designed to image both reflected light
and autofluorescence.
[0027] The multi-modal miniature microscopes proposed here
represent a fundamentally new way of approaching pre-cancer
detection. In one embodiment, the present devices integrate
micro-optical systems, micro-mechanical components, and image
sensors to achieve a high level of sensitivity and specificity in a
miniaturized, cost-effective package.
[0028] Micro-Optical Tables
[0029] In order to provide for the miniaturization of microscopes
in an effective manner, it is desirable to have a simple and
accurate method of building optical systems. A novel method of
constructing compact, three-dimensional imaging systems that
consist of various optical elements that may include, for example,
lenses and mirrors, micro-mechanical components, photo-detectors,
and light sources is disclosed.
[0030] These optical elements, both active and passive, may be
mounted on specially prepared MOT substrates, as shown in FIG. 1.
The substrates are referred to as micro-optical tables, in analogy
with the macroscopic version routinely used in optics laboratories.
Preferably, the MOTs are made of silicon, although those skilled in
the art will realize that other materials may also be used, such as
metal. The MOT is a zero-alignment microscopic optical-system
concept. In practical terms, the zero-alignment concept translates
into assembly errors that are preferably smaller than the
tolerances on the performance of the optical system.
[0031] Various mounting slots are formed in the MOT. The mounting
slots may be formed in a variety of ways, such as through
conventional etching techniques. The accurate positioning of each
mounting slot relative to other mounting slots on the MOT may be
obtained by using a sub-micron-precision layout of the photomask
from which the MOT may be made. The mounting slots may be etched
into the MOTs to various depths. In one embodiment, the mounting
slots may extend all the way through the MOT.
[0032] Preferably, each MOT also contains a spring device that
serves to hold an optical element in place once it has been
inserted into the MOT. A preferred embodiment of the mounting slot
21 formed in a MOT 20 and a spring 22 is shown in FIGS. 2A and 2B.
In the embodiment shown in FIGS. 2A and 2B, the spring 22 has a
first elongated portion 23 and a second shorter portion 24 that
forms an angle with the first elongated portion. One of the
purposes of the spring is to press-fit the optical component 25
into a more accurate position than might be achieved by using a
simple slot alone. When an optical component 25 is inserted into
the mounting slot 21, the spring 22 presses against the optical
component and helps to position and secure the optical component in
the mounting slot.
[0033] FIGS. 2A and 2B, illustrate an optical component inserted in
a mounting slot. The insertion of the optical component causes the
spring to be moved to a deflected position. The spring presses
against the optical component, thereby helping to secure it in the
mounting slot. FIG. 8B shows mounting slots 83 in which no optical
components have been inserted. As no optical component has been
inserted into the mounting slots 83, the springs 84 in FIG. 8B are
not in a deflected position. FIG. 8C shows optical components 85
inserted into the mounting slots 83.
[0034] The shape of the spring shown in FIG. 2 was selected in part
by generating a two-dimensional finite-element model to determine
the displacement and stress fields in the silicon spring and the
optical element as the optical element is inserted into the
mounting slot. FIGS. 6A and 6B show the top view of a rectangular
segment of a silicon MOT substrate. Also shown in FIG. 6, the
silicon spring and an optical element are being positioned in the
mounting slot. The dashed outlines in FIG. 6 indicate the starting
positions of the silicon spring and the optical element. The filled
outlines in FIG. 6 indicate positions of the silicon spring and the
optical element that correspond to the maximum stresses in the
silicon spring.
[0035] The objective of the analysis was to develop a
silicon-spring design that would not fail during insertion of an
optical element into a mounting slot. The spring design shown in
FIG. 6A satisfies this criteria. Those skilled in the art will
realize that several methods may be used to select other acceptable
shapes for the spring.
[0036] The spring may be attached to the mounting slot in various
ways. As shown in FIGS. 2A and 2B, a preferred embodiment is to
attach the spring 22 to the mounting slot 21 at one point along the
interior wall 26 of the mounting slot 21. It will be apparent to
those skilled in the art that the spring may be attached to the
mounting slot in variety of ways, including attaching the spring to
multiple points within the mounting slot, or attaching the spring
to the surface of the MOT. As used herein, the term "spring" is
intended to refer to any component having a configuration suitable
to assist in securing an optical component in the mounting slot. As
previously stated, those skilled in the art will realize that there
are many suitable configurations for the spring. Preferably, the
spring is made of silicon, although those skilled in the art will
realize that the spring may be made of other suitable
materials.
[0037] Each mounting slot may also contain one or more guide
channels that are formed in one or more of the walls of the
mounting slot. FIG. 10A depicts one of the walls 1001 of a mounting
slot, and two guide channels 1002 that are formed in the wall 1001.
The guide channels may be a variety of shapes, including, for
instance, cylindrical, "v" shaped, or rectangular. The guide
channels are preferably positioned to match complementary
protrusions 1004 in the optical component 1005 that is inserted in
the mounting slot. Thus, when an optical component is inserted into
a mounting slot, the protrusions located on the optical component
will fit into the guide channels. The guide channels thereby assist
in guiding the optical component into the mounting slot.
[0038] The depth to which the optical element is inserted into the
mounting slot may be controlled by the length of the guide channel.
For instance, as shown in FIG. 10A, the guide channel may extend
some distance down the wall of a mounting slot to an end surface
1003. Once the leading surface 1006 of the complementary protrusion
1004 of the optical component reaches the end surface 1003 of the
guide channel, the optical component will not be able to slide any
further down into the guide channel. A mounting slot spring is
preferably configured to press the cylindrical protrusions of the
optical element into the guide channels, thereby achieving more
accurate positioning of the optical element.
[0039] Very low assembly errors may be achieved through positioning
features on each optical component and a silicon spring in each
component mounting slot. Optical-element alignment to an accuracy
of .+-.2 .mu.m in position and .+-.0.5 mrad in rotation are
achievable using the illustrated method. The accurate positioning
of each optical element relative to other optical elements on the
MOT may be guaranteed through the sub-micron-precision layout of
the photomask from which the MOT is made.
[0040] Because diverse optical components can be embedded in the
MOT, the optical components can be fabricated separately in
substrates other than the MOT substrate and by means of processes
other than the processes involved in MOT fabrication. For example,
a refractive lens can be patterned in a photosensitive hybrid
sol-gel material coated on a glass substrate, while the MOT may be
fabricated in silicon. The refractive lens may fabricated using a
grayscale photomask. The MOT, on the other hand, may be fabricated
using a binary photomask. Finally, the disclosed MOT concept allows
for replacement of individual optical elements without sacrificing
the entire MOT.
[0041] 4M Devices
[0042] One preferred embodiment of a multi-modal miniature
microscope ("4M device") is shown in FIG. 1. The device of FIG. 1
uses lithographically fabricated refractive optical elements
positioned vertically in silicon-spring mounting slots. The 4M
devices may include a light source 11 mounted on the MOT 10. The
embodiment shown in FIG. 1 also includes a collector mirror 12, a
scanning grating 13, a folding flat mirror 14, a dichroic beam
splitter 15, a plurality of lithographically patterned refractive
lenses 16, a folding flat mirror 17, a photodetector 18, and an
objective lens 19. The tissue 20 to be imaged is placed below the
objective lens. Those skilled in the art will realize that the
optical components of the microscope may be arranged in a variety
of ways, that components may be added or taken away.
[0043] Light Source
[0044] The device includes a light source that may be designed to
operate in any part of the spectrum from the ultraviolet to the
near-infrared (NIR). Depending on the light source used,
microscopes may be constructed to operate in the blue (for
autofluorescence imaging), in the near-infrared (for reflectance
imaging), or both in the blue and in the near infrared. The light
source of the microscope may be integrated on the MOT as shown in
FIG. 1. In another embodiment of the invention, such as is shown in
FIG. 8A, a light source may not be mounted on the MOT. Instead, the
light source may also be external to the MOT and connected via a
variety of well known means, such as a fiber optic cable.
Additionally, as new contrast agents continue to be developed, the
present devices may operate at wavelengths chosen to match the
excitation and emission spectra of the new contrast agents.
[0045] Autofluorescence spectroscopy, reflectance imaging, and
confocal imaging (both of reflected light and autofluorescence)
each provide information about tissue architecture and biochemical
composition in near real-time without the need for tissue removal.
Autofluorescence and confocal imaging provide tools to assess two
fundamentally different sources of contrast between normal and
neoplastic epithelium: differences in autofluorescence (which are
related to metabolic rate, angiogenesis and collagen cross-linking)
and differences in refractive index profiles (which are related to
morphologic differences, primarily in the nucleus).
[0046] Autofluorescence provides a sensitive and specific tool to
improve detection of neoplasia. Confocal imaging may be used to
resolve sub-cellular detail throughout the entire epithelial
thickness, providing sufficient contrast to enable quantitative
feature analysis such as nuclear to cytoplasmic ratio. For example,
confocal imaging may offer a clinically useful adjunct to standard
histopathologic techniques for amelanotic tissue.
[0047] Depending upon the type of cancer that it desired to be
imaged, different imaging wavelengths may be chosen. For instance,
wavelengths of 380 nm and 460 nm correspond to diagnostically
useful regions identified for detection of pre-cancers of both the
cervix and the oral cavity. At 380-nm excitation, the co-factor
NADH is the primary cellular fluorophore. At 460-nm excitation, FAD
is the primary cellular fluorophore. Collagen crosslinks fluoresce
at both excitation wavelengths.
[0048] It has previously been shown that using reflectance confocal
imaging, images at tissue depths of 400 microns, penetrating the
entire epithelium, can routinely be obtained. It is also believed
that images throughout the epithelium may also be obtained using
autofluorescence.
[0049] Additionally, the proposed illumination levels in the near
UV do not pose significant 2 safety concerns. For instance, at 380
nm illumination, exposure should not exceed 47 J/cm.sup.2 for light
sources and for lasers in this illumination region, exposure should
not exceed 1 J/cm.sup.2, with a limiting aperture of 3.5 mm
diameter. It is estimated that the 4M devices will deliver between
500 .mu.W and 1 mW of laser light to the tissue surface at 380 nm
illumination and that total imaging time per field will be less
than 1 minute.
[0050] It is anticipated that actual image-acquisition time will be
much shorter, but this will enable the clinician or operator to
examine the image and ensure that the optimal area is in the field
of view. Averaging over the 3.5 mm limiting aperture, gives a total
illumination level of (1 mW)(60 seconds)/(0.096 cm.sup.2)=625
mJ/cm.sup.2. The proposed illumination levels are 1.6 to 75 times
less than the exposure levels allowed for lasers or light sources,
respectively.
[0051] Scanning Grating
[0052] The MOT concept allows for the inclusion of components that
provide functionality beyond that achievable with lenses alone. A
specific example is optical sectioning of a three-dimensional (3D)
specimen. Optical sectioning may be accomplished using structured
illumination with a scanning amplitude grating that is projected
into the 3D specimen. Images of the 3D specimen may be taken at
three lateral positions of the scanning grating. The three images
may next be processed in a simple manner to provide an
optical-section image of the 3D specimen. The method is based on
the simple principle that the amplitude grating appears in-focus
only over a limited axial range. Outside that range, the amplitude
grating may be out of focus and blurred. The out-of-focus blurring
results in loss of modulation. Lateral motion of the amplitude
grating therefore results in modulation of light that originates
only within a thin section of a 3D specimen.
[0053] In one embodiment, the present invention uses a continually
oscillating amplitude grating instead of a grating stepped to three
discrete positions. The continuous motion of the amplitude grating
will modulate in time the fluorescence signal from a thin section
of the specimen. This approach may also include a custom CMOS
active-pixel image sensor. Each active pixel may include a narrow
temporal-frequency band-pass filter. Consequently, the proposed
CMOS active-pixel image sensor only records signal that is
modulated at or near a center frequency that corresponds to the
scanning frequency of the amplitude grating.
[0054] A macroscopic amplitude grating will be translated
perpendicular to the optical axis and a minimum of three images
will be collected with a CCD camera to demonstrate functionality.
The images may be subsequently combined to determine the
autofluorescence distribution or reflectance variation at a fixed
working distance.
[0055] Preferably, the prime mover for the miniature scanning
grating is a variable reluctance magnetic microactuator constructed
via deep x-ray lithography (DXRL) and electroforming processing.
The basic DXRL and electroforming process flow is outlined in FIG.
7. With this process, arbitrarily shaped components with dimensions
from 1 micron to several centimeters may be fabricated with
sub-micron tolerances. The sidewall rms roughness of these
components is below 20 nm. Materials that may be used in the
process include an array of electroformed metals such as copper,
nickel, nickel-iron, and gold as well as molded plastics and
ceramic materials. Microactuator-drive designs may use a DXRL
assembly plate that is mounted to the MOT substrate and
accommodates all microactuator-drive components via press-fit
joining. Press-fit joints have been demonstrated in a number of
similar devices. DXRL processing enables extremely well controlled
press-fit joints.
[0056] Design information from previously fabricated magnetic
microactuators as outlined herein may be extrapolated and used to
formulate designs appropriate for the 4M scanning grating drive.
Extensive magnetic computer-aided design (CAD) tools exist and may
be used to design a permanent-magnet-assisted linear magnetic
microactuator with a resonant frequency of 100 Hz and motion range
of .+-.100 .mu.m. The microactuator resonant frequency may readily
be increased to 400 Hz at the expense of increased power required
to drive the microactuator. The microactuator resonant frequency
may also be increased if 1/f noise in the image sensor requires
such a change.
[0057] In one embodiment, a clamping scheme for
sub-micron-precision positioning of the grating component that will
be fabricated independent of the magnetic microactuator may be
integrated. A flexure that supports a platform on which the grating
is positioned may be fabricated with high yield strength
electroplated material in order to provide good spring-like
behavior. In the case of the 100-Hz microactuator, the anticipated
power requirement, assuming a worst-case efficiency of 10%, is
expected to be on the order of 100 microWatts (.mu.W). The low
drive impedance of a magnetic-microactuator drive will enable
driving voltages of near 0.5 Volts with currents of a fraction of a
milliAmpere (mA). The entire scanning-grating drive is expected to
fit well within the MOT component design footprint, which in one
embodiment may be approximately 2 mm.times.3 mm.
[0058] These microactuators have been used previously for
positioning of variable wire-grid infrared filters and optical
fiber switches, and are well suited for the task of scanning the
grating in a 4M device. FIG. 3 shows a partially assembled example
of this type of magnetic microactuator. In FIG. 3, a folded spring
flexure is shown that supports a center driven plunger. The
microactuator is capable of providing forces in the milli-Newton
range with motion ranges of several hundred microns. Variable
reluctance magnetic microactuators also can be operated in
resonance in a closed-loop mode with high efficiency. Typical
resonance frequencies are near 100 Hz and input powers of a few
milliWatts are sufficient to drive the microactuator to the full
extent of its motion.
[0059] One possible design of the scanning amplitude grating calls
for a grating period of 15 microns. An amplitude grating of this
period can be readily fabricated as chrome on glass using
commercial microlithography processing. In the context of 4M
devices, the trade-off involved in making the choice of grating
period is between increasing the axial resolution and decreasing
signal-to-noise ratio. Full-volume, optically-section imaging may
be achieved by translation of the tissue relative to the 4M device.
Additionally, the epithelium may be translated through the device
focal plane using suction-based devices.
[0060] As will be understood by those having ordinary skill in the
art with the benefit of the present disclosure, the scanning
grating mechanism for translation of the grating may, in one
embodiment, be fabricated as part of the substrate instead of
having the scanning grating components being integrated into a
separate substrate.
[0061] As previously stated, the 4M devices may achieve optical
sectioning by structured illumination. One challenge associated
with the structured-illumination method of optical sectioning is
the expected high level of background signal when imaging turbid
media. If the level of background signal precludes acquisition of
image data with a useful signal-to-noise ratio, e.g., greater than
10, then instead of only projecting the scanning grating into a 3D
medium, imaging may also be done through the grating. This approach
is analogous to a Nipkow disk. Instead of a disk, however, a
scanning grating is used. A dichroic filter (in case of
autofluorescence imaging) may be placed behind the grating, unlike
the configuration shown in FIG. 1. The background should be reduced
by a factor of two in the case of a scanning grating with equally
wide open and closed sections (i.e., "50% duty cycle"). An
additional set of optics may be used to relay the image at the
plane of the grating to the CMOS image sensor.
[0062] In one embodiment, a scanning grating system may be used in
which illumination light passes through the grating twice. First,
the illumination light passes when the grating is projected into
the tissue, and second, illumination light passes when light is
reflected or emitted from the tissue and propagating towards the
image sensor. This "double-pass" grating system arrangement is
similar in concept to a spinning Nipkow disc except that, in this
embodiment, a grating may be translated. One advantage of this
approach is that it achieves an increased suppression of background
signal from the object as compared to a "single-pass"
arrangement.
[0063] Patterned Refractive Lenses
[0064] As shown in FIG. 1, the 4M device may also comprise one or
more lithographically patterned, refractive optical elements and a
glass objective lens that may be mounted in the MOT silicon wafer.
In one embodiment, the glass objective lens is spherical in shape
and may be adapted from a commercially available ball lens. In one
specific embodiment, the glass objective lens has a clear aperture
of 800 microns and a thickness of 500 microns. The objective lens
may be positioned in the MOT silicon wafer in a simple round
aperture 81 etched through the silicon wafer 82, as shown in FIG.
8B. Alternatively, the lens may be positioned in the MOT silicon
wafer by means of a multiple spring, circular self-centering mount
that may be etched through the silicon wafer. All remaining
components can be mounted in the MOT substrate as shown in FIG.
2.
[0065] Fabrication of Optical Elements
[0066] A preferred approach to fabrication of micro-optical and
opto-mechanical structures is based on the sol-gel technique. The
sol-gel technique has the unique potential for simultaneous
fabrication of micro-optical and opto-mechanical structures by UV
patterning in a single lithography step. No etching of patterned
structures is required when using the sol-gel technique.
Diffractive optical elements using binary and grayscale photomasks
[see FIG. 4A and FIG. 5A] have previously been patterned. More
recent patterning experiments demonstrate a doubled film thickness
(34 .mu.m) and an rms surface roughness of 20 nm, as shown in FIG.
4B. Reduced rms surface roughness means a reduction in undesired
scattering from lithographically fabricated optical elements.
[0067] In one lithography step and using a grayscale photomask, a
thick layer of hybrid sol-gel material may be patterned with a
diffractive, a reflective, a refractive optical surface, or a
combination thereof. In grayscale lithography, a standard spherical
optical surface is as simple to fabricate as an arbitrary aspheric
optical surface. To make a convex refractive lens, for instance,
the hybrid sol-gel material may be spin-coated to form a 60-.mu.m
thick film on borosilicate glass substrates. After spinning, the
films may be prebaked at 95.degree. C. for 10 minutes to decrease
the amount of solvents in the film. The baking step reduces the
effect of photomask adhesion to the film and also improves the
adhesion of the film to the glass substrate. Ultraviolet (UV)
exposure may be accomplished by using a mercury UV lamp at a
wavelength of 365 nm and a grayscale photomask.
[0068] The grayscale photomask may be designed to impart to the
film an arbitrary surface. Lithographically fabricated optical
elements may be characterized interferometrically to measure the
accuracy of their surface figure and to determine their surface
roughness. The accuracy of the surface figure determines the
optical elements' first-order properties, e.g., the focal length,
and the aberrations introduced by the optical elements. The surface
roughness determines the fraction of light that is scattered by the
optical elements, leading to reduced throughput and reduced
contrast due to stray light. The scattering properties of the
patterned hybrid sol-gel material may be further characterized in
terms of a bi-directional scattering distribution function (BSDF).
The BSDF determines the angular distribution of scattered light.
The BSDF data may be used to increase the accuracy of modeling of
4M systems and to improve 4M designs by controlling stray
light.
[0069] As previously indicated, in one embodiment the optical
elements may be patterned to a depth of 60 microns. For a given
lens-aperture size, the patterned depth establishes a lower limit
on the focal length of a lithographically patterned optical
element. Finally, it is preferable that the precursors used in
hybrid sol-gel material processing be filtered to avoid the
introduction of impurities that could give rise to autofluorescence
of the fabricated optical elements.
[0070] CMOS Active-Pixel Arrays
[0071] One challenge associated with the structured-illumination
method of optical sectioning is the expected high level of
background signal when imaging turbid media. In the nominal
structured-illumination approach, computational methods may be used
to remove out-of-focus light. Consequently, system performance may
ultimately be limited by the noise associated with fluorescence
generated at out-of-focus planes.
[0072] One way to reduce the effect of the out-of-focus plane
fluorescence background is to replace a standard CCD camera with a
custom CMOS active-pixel image sensor with a narrow (.DELTA.f=1 Hz)
tuned temporal-frequency band-pass filter at each pixel. The
band-pass filter will block the unmodulated background fluorescence
or reflected light that originates at planes below and above the
optical section. This is based on the assumption that the noise
spectral power density is constant as a function of temporal
frequency, i.e., the noise is white. The band-pass filter will
therefore also reduce the integrated noise power.
[0073] There are several additional methods by which the signal can
be better discriminated from background. For instance, the DC
background signal can be reduced by a factor of two by illuminating
and imaging through a scanning grating. Additionally, the
oscillation frequency of the micro-mechanical scanning grating,
i.e., the carrier frequency, may be increased. From a theoretical
viewpoint, the increase in the oscillation frequency (i.e. from 30
Hz to 100 Hz) should result in at least a three-fold decrease in
noise power. Furthermore, every pixel in the image sensor may also
contain a 1-Hz band-pass filter to further limit the integrated
noise power.
[0074] CMOS active-pixel image sensors have become serious
competitors to CCDs (and any other image sensing technique) in
virtually all imaging applications. CMOS is especially appropriate
in the present application due to the system advantage of signal
processing in each pixel such as band-pass filtering. In one
preferred embodiment, the filter's band-pass is centered at 100 Hz,
which is the modulation frequency of the reflectance or
autofluorescence signal resulting from the motion of the scanning
grating. The images recorded with this kind of custom active-pixel
image sensor will correspond directly to the reflectance variation
or autofluorescence distribution at the object depth and no
post-processing of multiple images will be necessary.
[0075] Readily available, sub-micron CMOS fabrication processes
will support implementation of the photo-diode and signal
processing circuits within a pixel area of 15 .mu.m.times.15 .mu.m
or less. A 100.times.100 pixel array with peripheral support
circuits should easily fit on a chip of 2 mm.times.2.5 mm or less.
These chip dimensions are compatible with the requirements of the
embodiment of the 4M device shown in FIG. 1.
[0076] In one embodiment of the present invention, the image
recorded on the CMOS active-pixel image sensor may be magnified
electronically rather than optically for viewing by eye, i.e., the
image will be displayed scaled up. Such electronic magnification is
equivalent to the optical function performed by an eyepiece: In
each case, the user perceives the final image at a comfortable
viewing distance, e.g., 250 mm.
[0077] Additionally, the field of view of the 4M devices may be
expanded in many ways. This is desirable because it allows for more
efficient imaging. The field of view may be expanded, for example,
by introducing additional, low-magnification imaging systems on one
MOT substrate alongside the miniature microscope. This may be
accomplished without significantly increasing the size of the
microscope. Alternatively, "contact" imaging may be possible
whereby the bottom surface of the microscope device is itself a
low-resolution image sensor.
[0078] Even with a limited field of view, an imaging device capable
of sub-cellular resolution has important clinical roles. First,
clinicians already use their visual recognition skills to decide
where to obtain diagnostic biopsies. Using the present microscope
devices to interrogate these areas may reduce the costs of
detecting pre-cancer by better guiding biopsy during such
procedures as colposcopy or visual examination of the oral cavity.
Second, the microscopes may be similarly used at the time of tumor
resection to aid in margin detection. Third, 4M devices may be used
to facilitate chemoprevention studies in the cervix and the oral
cavity.
[0079] Studies
[0080] Reflectance Imaging Preliminary Studies
[0081] The preliminary imaging studies presented here emphasize
imaging of tissue sections that are perpendicular to the planes
that will be imaged with the 4M devices. However, these preliminary
data may be useful in interpreting the image data to be acquired
eventually by 4M devices.
[0082] The use of high-resolution, in vivo confocal imaging may
offer a clinically useful adjunct to standard methods for the
diagnosis and screening of epithelial pre-cancers. A
reflectance-based confocal microscope was used to image cervical
cells and colposcopically normal and abnormal cervical biopsies.
Images were obtained before and after the application of 6% acetic
acid. The confocal microscope resolved sub-cellular details
throughout the entire epithelial thickness. Normal and abnormal
cervical tissue were clearly differentiable. Addition of acetic
acid enhanced nuclear signal in all acquired images. Confocal
images of a short-term tissue culture of cervical tissue show the
increase in nuclear-to-cytoplasmic ratio throughout the epithelium
(see FIG. 15). These preliminary studies show that high-contrast,
reflected-light images of cervical tissue are attainable in near
real-time using a conventional confocal microscope.
[0083] Autofluorescence Imaging Studies
[0084] While a number of clinical studies have demonstrated that
fluorescence spectroscopy can provide highly sensitive, specific,
and cost-effective diagnosis of cervical precancers, the underlying
biochemical mechanisms responsible for differences in fluorescence
spectra of normal and dysplastic tissue are not fully understood.
It has recently been demonstrated that short-term tissue cultures
of normal and neoplastic tissue could be used to assess differences
in autofluorescence of normal and dysplastic tissue and to
understand the biological basis for these differences. Short-term
tissue cultures represent a novel, biologically appropriate model
for understanding epithelial autofluorescence.
[0085] Transverse, short-term tissue cultures were prepared from
colposcopically normal biopsies in a 31-patient study and from
normal and abnormal biopsies in a 34-patient study.
Autofluorescence images were acquired at 380 and 460 nm excitation.
At both excitation wavelengths, measurable epithelial and stromal
autofluorescence was detected. The autofluorescence of both tissue
layers was found to be age and hormone-status dependent.
Fluorescence images were placed into groups: (Group 1) bright
epithelial and weak stromal fluorescence, (Group 2) similar
epithelial and stromal fluorescence, and (Group 3) weak epithelial
and bright stromal fluorescence. The average ages of women in the
groups were 30.9, 38.0, and 49.2 years. Epithelial fluorescence
intensity was similar in Groups 1 and 2, but weaker in Group 3.
Stromal intensity was similar in Groups 2 and 3, but weaker in
Group 1. The ratio of epithelial to stromal fluorescence intensity
was significantly different for all groups. These results suggest a
biological basis for the increased fluorescence seen in older,
postmenopausal women.
[0086] With the development of dysplasia, statistically significant
increases in epithelial fluorescence intensity were observed at 380
nm excitation in pre-cancerous tissue [106.+-.39 in arbitrary units
(AU)] relative to normal tissue (85.+-.30 AU). The fluorophore
responsible for this increase is likely NADH. Stromal fluorescence
intensities in the dysplastic samples decreased at both 380 nm
[102.+-.34 (pre-cancer) vs. 151.+-.44 (normal)] and 460 nm
excitation [93.+-.35 (pre-cancer) vs. 137.+-.49 (normal)], i.e.,
wavelengths at which collagen is excited. A tissue's metabolic
state is sometimes described by calculating the "redox ratio," a
quantity obtained by dividing the fluorescence of FAD by the summed
fluorescence of FAD and NADH. The redox ratio which typically
decreases in cancer, is sensitive to changes in metabolic rate and
vascular oxygen supply. In principle, the Blue 4M device may be
adapted to simultaneously record both fluorescence signals, to
measure the redox ratio directly.
[0087] Decreased redox ratio (17% to 40% reduction), indicative of
increased metabolic activity, was observed in the pre-cancerous
samples. These results provide valuable insight into the biological
basis of differences in fluorescence of normal and pre-cancerous
cervical tissue. Furthermore, the results show that short-term
tissue cultures provide a novel biological system to explore the
optical changes that accompany the development of pre-cancer in
human tissue. This model system can be used to further explore the
capabilities of 4M devices in both autofluorescence and reflectance
mode, assessing the devices' ability to discriminate the changes in
morphology and biochemistry that accompany the development of
pre-cancer in human tissue.
[0088] Testing
[0089] Potential configurations of 4M device can be tested using
ANSYS to predict the mechanical and thermal properties of the
planned 4M devices. Thermal analysis using ANSYS will predict the
effects of power-dissipation due to the light source, the scanning
grating, and the CMOS image sensor. This analysis may be used to
control the cumulative effect of power dissipation on the imaging
function of a fully integrated 4M device. In addition, detailed
simulations of the 4M-device optics can be performed using ASAP, a
non-sequential ray tracing (NSRT) program. NSRT analysis may be
used to quantitatively determine the contrast-reducing effects of
light scattering from the lithographically fabricated optical
elements and any other sources of stray light within the 4M device.
Most significantly, NSRT analysis can be used to suppress any these
effects, by means of micro-baffles, for instance. 4M devices may be
tested, for example, in three biologically appropriate models of
normal and neoplastic oral-cavity epithelium. Tissue-engineering
methods may be used to develop three dimensional organotypic
cultures of normal and neoplastic oral cavity and cervix. Secondly,
short-term tissue cultures of normal and neoplastic oral cavity and
cervical tissue from tissue biopsies can be prepared. Third, an
animal model of oral-cavity neoplasia, the hamster cheek pouch
model of carcinogenesis, may be used. These model systems will
provide data from biologically relevant specimens of normal and
neoplastic epithelium that will allow for the testing of 4M
devices.
[0090] Organotypic Cultures
[0091] Growing cells as an adherent monolayer in plastic dishes or
in suspension culture is technically simple. Therefore, it is the
major method that cell biologists use to study animal and human
normal and original phenotypic characteristics. Cells are separated
from different types (e.g., mesenchymal cells are separated from
epithelial cells) to prevent one type from dominating another when
their growth rates or growth requirements vary. The maintenance of
the various tissue components in their normal anatomical
relationship is important for regulation of growth and
differentiation. Tumor cells, stromal fibroblasts, or endothelial
cells, may express a set of genes in situ that only partially
overlaps the set of genes expressed by each cell type in isolation
from the others in primary cultures.
[0092] In addition, the mesenchymal cells may secrete factors that
the tumor cells can use as mitogens. Organotypic cultures have been
developed initially for skin and then adapted for a variety of
epithelial cancers as an approach to provide three dimensional
growth with epithelial cell-epithelial cell interactions that are
major features of solid carcinomas and are lost partially in
monolayer cultures. The method is based on the growth of epithelial
cells at the air-liquid interface on top of a reconstituted
collagen gel containing fibroblasts.
[0093] This organ culture provides conditions that preserve tissue
architecture, growth, and function. It can be prepared with
different cell layers and can be analyzed as a tissue without
restrictions involved in obtaining actual surgical specimens from
patients or volunteers. Organ cultures are also more reproducible
than tissues obtained from different individuals. It is believed
that pre-clinical research would benefit from analysis of novel
diagnostic approaches directly in organotypic cultures. The results
are likely to be more informative and can be extrapolated to the in
vivo situation with greater confidence than work with cell lines in
monolayer cultures. Therefore, short-term organotypic cultures of
oral cancer cells may be used to determine the efficacy of new
diagnostic approaches such as those proposed here.
[0094] Organotypic cultures of normal cervix, cervical neoplasia,
and oral-cavity neoplasia may be examined using 4M devices designed
to measure autofluorescence. Using 380 and 460 nm excitation,
analysis can be done as to how well the 4M device separates
fluorescence of the epithelial cells from the supporting stroma and
how effectively signals from normal and neoplastic samples can be
separated. Similar tests can also be performed to record
reflected-light optical-section images. The performance of 4M
devices can be characterized based on autofluorescence and
reflectance in terms of signal-to-noise ratio, penetration depth,
and the ability to separate normal and neoplastic samples.
[0095] Short-Term Tissue Cultures
[0096] While organotypic cultures allow for the examination of
autofluorescence and reflectance in a three-dimensional geometry,
there may be differences in the fluorescence of the cell lines used
in this model system and the pre-cancerous epithelial cells found
in lesions in vivo. The second testing model overcomes this
limitation. Short-term cultures of normal and neoplastic biopsies
obtained from patients can be prepared. For instance, biopsies (2
mm.times.4 mm.times.1 mm) of the oral cavity and the cervix may be
obtained from patients. Preferably, cervical biopsies will be
obtained from women being seen for colposcopy because of an
abnormal Pap smear. Preferably, biopsies of the oral cavity will be
obtained from patients suspected to have an oral-cavity cancer.
Biopsies should be obtained from a normal-appearing area and an
area suspected for dysplasia. The biopsies may placed in chilled
culture medium (DMEM without phenol red), and embedded in 4%
agarose for slicing. A Krumdieck Tissue Slicer (Alabama Research
and Development MD1000-A1) may then be used to obtain 200 .mu.m
thick fresh tissue slices, cut perpendicular to the epithelial
surface. Fluorescence and reflectance images can then be obtained
from tissue slices within 1.5 to 5 hours of biopsy. Control
experiments show that fluorescence intensities are stable to within
.+-.10% for up to 5.5 hours after preparation of the slices.
[0097] Animal Models
[0098] Organotypic cultures and short-term tissue cultures do not
allow for the monitoring of lesion progression over time or to
examine the effects of angiogenesis. Thus, an animal model may be
used for further testing of 4M devices. The hamster cheek pouch
carcinogenesis model, using chronic treatments of
dimethylbenz[.alpha.]anthracene (DMBA) may be used as a model
system to investigate changes in epithelial tissue fluorescence
throughout the dysplasia-carcinoma sequence. Images may be taken
weekly using both autofluorescence at 380 and 460 nm excitation and
reflected light at 800 nm from both DMBA treated animals and
control animals. Histopathology may be obtained at regular
intervals throughout the study.
[0099] Previous studies that have investigated the autofluorescence
of this model at weekly intervals and found that diagnostic
algorithms based on autofluorescence can separate neoplastic and
non-neoplastic tissue with 95% sensitivity and 93% specificity. The
greatest contributions to diagnostic algorithms were obtained with
excitation in the 370-380 nm wavelength range. This result was
similar to that found in an in vivo study of both cervical and
human oral-cavity neoplasia. Consequently, the hamster-cheek-pouch
model is very well suited to characterize the performance of the 4M
devices. Changes in fluorescence intensity are apparent as early as
three weeks following initial treatment with DMBA, while
morphologic changes associated with dysplasia occur on average at
7.5-12.5 weeks following initial treatment. Performance of 4M
devices in imaging autofluorescence and reflected light in these
models may be compared in terms of SNR, penetration depth, and the
ability to separate normal and neoplastic samples (quantified in
terms of sensitivity and specificity as compared to
histopathology).
[0100] Contrast Agents
[0101] The techniques of this disclosure may be used in conjunction
with any type of contrast agent. For instance, any type of dye may
be used, including a dye conjugated to any type of antibody. For
instance, a dye may be conjugated to an antibody for cytokeratins.
Such a dye may be, for instance, Nile Blue A and/or Texas Red.
Further, in different embodiments, one may use reflective
nanoparticles to aid in imaging. For example, in one embodiment,
gold nanoparticles may be used to increase imaging contrast. In
another embodiment, quantum dots may be used.
EXAMPLES
[0102] NA=0.4 Red 4M Device
[0103] The proposed 4M device shown in FIG. 1 is water-immersion,
has a numerical aperture of NA=0.4, a working distance of WD=250
.mu.m, a field of view 300 .mu.m in diameter, a transverse
magnification of m=-4, and is designed for monochromatic operation
at 800 nm. Due to the wavelength choice, this is called the "Red"
4M device. The value of the NA is bounded by a 60-.mu.m maximum
thickness of the UV-patternable hybrid sol-gel material that we
expect to reach in Year One. The Red 4M device shown in FIG. 1 is
designed for use with an array of photodetectors that are spaced by
10 .mu.m.
[0104] The lateral resolution of this Red 4M device at the tissue
level is expected to be approximately 5 microns. Those skilled in
the art will realize that other 4M devices may be based on a
configuration similar to that shown in FIG. 1. The primary
difference will be an increased NA and a lateral resolution at the
tissue level of approximately 3 microns. Additionally, other light
sources may be used that operate over a variety of wavelengths.
Additionally, a plurality of light sources could be used, and each
light source could operate in various wavelength ranges.
[0105] The 4M device shown in FIG. 1 has been analyzed in terms of
fabrication and assembly tolerances. Table 1 lists the top four
tightest design tolerances. These tolerances have to be met for the
optics of the 4M device in FIG. 1 to remain diffraction-limited in
imaging performance. The expected lens-positioning accuracy of
.+-.2 .mu.m and rotation accuracy of .+-.0.5 mrad compare very
favorably with the position and rotation tolerances required by the
4M device. The radius-of-curvature tolerance is a "precision"
tolerance and will be achieved by fabricating multiple replicas of
each lens and then selecting those lenses that are within the
specified tolerance. The index-of-refraction tolerance is a loose,
"commercial" tolerance.
1TABLE 1 Selected four tightest fabrication and assembly tolerances
associated with NA = 0.4 Red 4 M device. Tolerance Type Tolerance
Values Radius of curvature .+-.0.3% Hybrid sol-gel material index
of refraction .+-.0.001 Position of lithographically patterned
lenses .+-.10 .mu.m (lat.); .+-.10 .mu.m (vert.); -0/+3 .mu.m
(axial) Rotation of lithographically patterned lenses .+-.8
mrad
[0106] NA=0.6 Red 4M Device
[0107] The proposed 4M device shown in FIG. 9 is a water-immersion
Red 4M device designed to operate with a numerical aperture of
NA=0.6, a working distance of WD=250 .mu.m, a field of view 250
.mu.m in diameter, a transverse magnification of m=4. This 4M
device is designed for monochromatic operation at 800 nm. FIG. 9
shows a schematic diagram of this preliminary design. A significant
feature of the NA=0.6 design is that the relative positions of the
optical elements remain the same as in previously introduced 4M
devices. Consequently, the MOT silicon substrate shown in FIG. 8B
needs no modification and can be re-used for this
higher-performance miniature microscope. The relatively low-cost
optical elements, on the other hand, can be readily modified
according to the design's specifications.
[0108] NA=0.4 Blue 4M Device
[0109] The Blue 4M device needs to be designed for imaging over a
wavelength range extending from 380 nm to 500 nm. A design similar
in form and specifications to that shown in FIG. 9 has been
developed. There are two major differences between the Red and Blue
4M-device designs: (1) the ball lens from which the objective lens
may be fashioned in the Blue 4M device may be made from a
lower-dispersion glass than its counterpart in the Red 4M device
design, and (2) the lens labeled "1" in FIG. 9 is a combination
diffractive-refractive lens. The use of a properly selected
diffractive surface all but compensates for any longitudinal
(a.k.a. axial) and lateral chromatic aberrations that may be
encountered in the design.
REFERENCES
[0110] Each of the references listed below are hereby incorporated
by reference.
[0111] S. Silverman, M. Gorsky, and F. Lozada, "Oral leukoplakia
and malignant transformation. A follow up study of 257 patients,"
Cancer, 53, pp. 563-68 (1984).
[0112] The 1988 Bethesda System for reporting cervical/vaginal
cytologic diagnoses. National Cancer Institute Workshop. JAMA 1989;
262:931-934.
[0113] L. G. Koss, "The Papanicolaou test for cervical cancer
detection. A triumph and a tragedy" [see comment citation in
Medline]. JAMA 1989; 261:737-743.
[0114] S. Lam, T. Kennedy, M. Unger, Y. E. Miller, D. Gelmont, V.
Rush, B. Gipe, D. Howard, J. C. LeRiche, A. Coldman, and A. F.
Gazdar, "Localization of Bronchial Intraepithelial Neoplastic
Lesions by Fluorescence Bronchoscopy,"Chest 113 (2), 696-702
(1998).
[0115] S. Lam, C. MacAulay, J. Hung, J. LeRiche, A. E. Profio, and
B. Palcic, "Detection of Dysplasia and Carcinoma In Situ with a
Lung Imaging Fluorescence Endoscope Device," J of Thoracic &
Cardiovascular Surgery 105 (6), 1035-40 (1993).
[0116] B. W. Pogue, G. C. Burke, J. Weaver, D. M. Harper,
"Development of a Spectrally Resolved Colposcope for early
detection of Cervical Cancer," in Biomedical Optical Spectroscopy
and Diagnostics Technical Digest (Optical Society of America,
Washington D.C., 1998), 87-89.
[0117] S. L. Jacques, J. R. Roman, and K. Lee, "Imaging Superficial
Tissues with Polarized Light," Lasers Surg. Med. 26, 119-129
(2000).
[0118] C. Smithpeter, A. Dunn, R. Drezek, T. Collier, R.
Richards-Kortum, "Real Time Confocal Microscopy of In Situ
Amelanotic Cells: Sources of Signal, Contrast Agents and Limits of
Contrast," Journal of Biomedical Optics, 3:429-36, 1998.
[0119] L. T. Perelman, V. Backman, M. Wallace, G. Zonios, R.
Manoharan, A. Nusrat, S. Shields, M. Seiler, C. Lima, T. Hamano, I.
Itzkan, J. Van Dam, J. M. Crawford, and M. S. Feld, "Observation of
Periodic Fine Structure in Reflectance from Biological Tissue: A
New Technique for Measuring Nuclear Size Distribution," Physical
Review Letters, vol. 80, pp. 627-630, 1998.
[0120] J. R. Mourant, I. J. Bigio, J. Boyer, R. L. Conn, T.
Johnson, and T. Shimada, "Spectroscopic Diagnosis of Bladder Cancer
with Elastic Light Scattering," Lasers in Surgery and Medicine,
vol. 17, pp. 350-357, 1995.
[0121] I. J. Bigio, J. R. Mourant, J. D. Boyer, T. M. Johnson, T.
Shimada, and R. L. Conn, "Noninvasive Identification of Bladder
Cancer with Subsurface Backscattered Light," presented at Advances
in Laser and Light Spectroscopy to Diagnose Cancer and Other
Diseases, Los Angeles, Calif., 1994.
[0122] I. J. Bigio, J. D. Boyer, T. M. Johnson, J. Lacey, and J. R.
Mourant, "Detection of Gastrointestinal Cancer by Elastic
Scattering and Absorption Spectroscopies with the Los Alamos
Optical Biopsy System," presented at Advances in Laser and Light
Spectroscopy to Diagnose Cancer and Other Diseases II, San Jose,
Calif., 1995.
[0123] Sokolov K., Drezek R., Gossage K., and Richards-Kortum R.
Reflectance Spectroscopy with Polarized Light: Is it Sensitive to
Cellular and Nuclear Morphology.--Optics Express, 1999, v. 5, No.
13, pp. 302-317.
[0124] R. R. Alfano, G. C. Tang, A. Pradham, W. Lam, D. S. J. Choy,
and E. Opher, "Fluorescence Spectra from Cancerous and Normal Human
Breast and Lung Tissues," IEEE Journ. Quant. Electron., vol. QE-23,
pp. 1806-1811, 1987.
[0125] R. M. Cothren, R. Richards-Kortum, M. V. Sivak, M.
Fitzmaurice, R. P. Rava, G. A. Boyce, M. Doxtader, R. Blackman, T.
B. Ivanc, G. B. Hayes, M. S. Feld, and R. E. Petras,
"Gastrointestinal tissue diagnosis by laser-induced fluorescence
spectroscopy at endoscopy," Gastrointest. Endosc., vol. 36, pp.
105-111, 1990.
[0126] R. M. Cothren, R. Richards-Kortum, M. V. Sivak, M.
Fitzmaurice, R. P. Rava, G. A. Boyce, M. Doxtader, R. Blackman, T.
B. Ivanc, G. B. Hayes, M. S. Feld, and R. E. Petras,
"Gastrointestinal tissue diagnosis by laser-induced fluorescence
spectroscopy at endoscopy," Gastrointest. Endosc., vol. 36, pp.
105-111, 1990.
[0127] S. Lam, C. MacAulay, J. Hung, J. LeRiche, A. E. Profio, and
B. Palcic, "Detection of dysplasia and carcinoma in situ with a
lung imaging fluorescence endoscope device," J. Thorac. Cardiovasc.
Surg., vol. 105, pp. 1035-1040, 1993.
[0128] K. Svanberg, S. Andersson-Engels, R. Berg, J. Johansson, S.
Svanberg, and I. Wang, "Tissue Characterization in Different
Malignant Tumors Utilizing Laser-Induced Fluorescence," presented
at Advances in Laser and Light Spectroscopy to Diagnose Cancer and
Other Diseases II, San Jose, Calif., 1995.
[0129] T. Vo-Dinh, M. Panjehpour, B. F. Overholt, C. Farris, F. P.
Buckley, and R. Sneed, "In Vivo Cancer Diagnosis of the Esophagus
Using Differential Normalized Fluorescence (DNF) Indices," Las.
Surg. Med., vol. 16, pp. 41-47, 1995.
[0130] M. Follen Mitchell, S. B. Cantor, N. Ramanujam, G.
Tortolero-Luna, R. Richards-Kortum, "Fluorescence Spectroscopy for
Diagnosis Squamous Intra-Epithelial Lesions of the Cervix,"
Obstetrics and Gynecology, 93:462-70, 1999.
[0131] N. Ramanujam, M. Follen Mitchell, A. Mahadevan-Jansen, S. L.
Thomsen, G. Staerkel, A. Malpica, T. Wright, N. Atkinson, R.
Richards-Kortum, "Cervical Pre-Cancer Detection Using a
Multivariate Statistical Algorithm Based on Laser Induced
Fluorescence Spectra at Multiple Excitation Wavelengths,"
Photochemistry and Photobiology, 6:720-35, 1996.
[0132] M. Follen Mitchell, S. B. Cantor, C. Brookner, U. Utzinger,
G. Staerkel, R. Richards-Kortum, "Receiver Operator Characteristic
Curve of Fluorescence for the Screening of SILs," 94:889-896,
Obstetrics and Gynecology, 1999.
[0133] D. Heintzelman, U. Utzinger, H. Fuchs, A. Gillenwater, R.
Jacob, B. Kemp, R. Richards-Kortum, "Optimal Excitation Wavelengths
for In Vivo Detection of Oral Neoplasia Using Fluorescence
Spectroscopy," Photochemistry and Photobiology, 72(1):103-113,
2000.
[0134] T. Collier, P. Shen, B. de Pradier, K. Sung, A. Malpica, M.
Follen, R. Richards-Kortum, "Near real time confocal microscopy of
amelanotic tissue: Dynamics of aceto-whitening enable nuclear
segmentation," Optics Express, 6, pp. 40-48 (2000).
[0135] G. J. Kelloff, C. W. Boone, J. A. Crowell, V. E. Steele, R.
Lubet, L. A. Doody, "Surrogate endpoint biomarkers for phase II
cancer chemoprevention trials," J Cell Biochem Suppl; 19:1-9
(1994).
[0136] M. B. Daly, "The chemoprevention of cancer: Directions for
the future," Cancer Epidemiol Biomarkers Prev;2:509-12 (1993).
[0137] D. S. Goodman, "Basic Optical Instruments," Ch. 4 in
Geometrical and Instrumental Optics, D. Malacara, ed. (Academic
Press, 1988).
[0138] Survey of microscope-objective patents from 1976 to 1990, M.
R. Descour, unpublished.
[0139] R. H. Webb and C. K. Dorey, "The pixilated image" in
Handbook of Biological Confocal Microscopy, J. B. Pawley, ed., Ch.
4 (1995).
[0140] R. Levy, M. R. Descour, R. J. Shul, C. L. Willison, M. E.
Warren, T. Kololuoma and J. T. Rantala, "A concept for
zero-alignment micro optical systems," Proc. of Micromachine
Technology for Diffractive and Holographic Optics, S. H. Lee and J.
A. Cox, eds., SPIE 3879-18 (September 1999).
[0141] M. A. A. Neil, R. Ju{haeck over (s)}kaitis, and T. Wilson,
"Method of obtaining optical sectioning by using structured light
in a conventional microscope," Opt. Lett., 22, No. 24, 1905 (Dec.
15, 1997).
[0142] M. Kufner and S. Kufner, Micro-Optics and Lithography, Ch. 9
(VUB Press, 1997)
[0143] H. Guckel, T. R. Christenson, T. Earles, K. J. Skrobis, J.
Klein, "Laterally Driven Electromagnetic Actuators," 1994
Solid-State Sensor and Actuator Workshop, Hilton Head Island, N.C.,
pp 49-52
[0144] T. R. Ohnstein, J. D. Zook, H. B. French, H. Guckel, T.
Earles, J. Klein, P. Mangat, "Tunable IR Filters with Integral
Electromagnetic Actuators," Tech. Digest of the 1996 Solid-State
Sensor and Actuator Workshop, Hilton Head Isl., SC, pp. 196-199
(1996).
[0145] .sup.1H. Guckel, HARMST Conference, Kisarazu, Japan, (June
1999).
[0146] A. K. Dunn, NMR Center, Massachusetts General Hospital,
Harvard Medical School, Charlestown, Mass. 02129 (private
communication, 2000).
[0147] S. Gaalema, Black Forest Engineering, Colorado Springs,
Colo. (private communication, 2000).
[0148] J. T. Rantala, R. Levy, L. Kivimki, and M. R. Descour,
"Direct UV patterning of thick hybrid glass films for
micro-opto-mechanical structures," Electronics Letters, 16, No. 6,
pp. 530-531 (Mar. 16, 2000).
[0149] ANSYS is a product of ANSYS, Inc., Canonsburg, Pa. 15317
(www.ansys.com).
[0150] C. J. Wilson and P. A. Beck, "Fracture testing of bulk
silicon microcantilever beams subjected to a side load", Journal of
Microelectromechanical Systems, 5, No.3, pp. 142-150 (1996).
[0151] R. R. Shannon, The Art and Science of Optical Design, Ch. 6,
p. 361 (Cambridge University Press, 1997).
[0152] K. Sokolov, J. Galvan, A. Myakov, A. Lacy, R. Lotan, R.
Richards-Kortum, "Realistic Three Dimensional Epithelial Tissue
Phantoms for Biomedical Optics," submitted, The Journal of
Biomedical Optics (2001).
[0153] P. yrs, J. T. Rantala, R. Levy, M. R. Descour, S. Honkanen,
and N. Peyghambarian, "Multilevel structures in sol-gel thin films
with a single UV-exposure using a gray-scale mask," Thin Solid
Films 352, 9 (1999).
[0154] J. T. Rantala, P. yrs, R. Levy, S. Honkanen, M. R. Descour,
N. Peyghambarian, "Binary phase zone-plate arrays based on hybrid
sol-gel glass," Optics Letters, 23, 1939 (Dec. 15, 1998).
[0155] J. T. Rantala, GuideOptics, Inc., San Jose, Calif., and
Espoo, Finland (private communication, 2000).
[0156] T. Christenson, Sandia National Laboratories, Albuquerque,
N. Mex. (private communication, 2000).
[0157] F. Laermer and A. Schilp, "Method of anisotropically etching
silicon," U.S. Pat. No. 5,501,893 (Mar. 26, 1996).
[0158] T. Collier, P. Shen, B. de Pradier, K. Sung, A. Malpica, M.
Follen, R. Richards-Kortum, "Near real time confocal microscopy of
amelanotic tissue: Dynamics of aceto-whitening enable nuclear
segmentation," Optics Express, 6:40-48, 2000.
[0159] J. Mourant, J. Freyer, A. Hielscher, A. Eick, D. Shen, and
T. Johnson, "Mechanisms of Light Scattering from Biological Cells
Relevant to Noninvasive Optical-Tissue Diagnostics," Applied
Optics, Vol. 37, 3585-3593, 1998.
[0160] H. W. Wang, J. Willis, M. I. Canto, M. V. Sivak, Jr., J. A.
Izatt, "Quantitative Laser Scanning Confocal Autofluorescence
Microscopy of Normal, Premalignant and Malignant Colonic Tissues,"
IEEE Trans BME, 46(19):1246-52 (1999).
[0161] C. L. Smithpeter, A. K. Dunn, A. J. Welch, R. R.
Richards-Kortum, "Penetration Depth Limits of in vivo Confocal
Reflectance Imaging," Applied Optics, 37:2749-54 (1998).
[0162] 1999 TLVs and BEIs, published by ACGIH, Cincinnati, Ohio, p.
154.
[0163] R. K. Kimmel and R. E. Parks, eds., "Surface Texture," Ch. 8
in ISO 10110 Optics and Optical Instruments, Optical Society of
America (1995).
[0164] L. Coghlan, U. Utzinger, R. Richards-Kortum, C. Brookner, A.
Zuluaga, I. Gimeniz-Conti, M. Follen, "Fluorescence Spectroscopy of
Epithelial Tissue Throughout the Dysplasia-Carcinoma Sequence in an
Animal Model: Spectroscopic Changes Precede Morphologic Changes,"
in press, Lasers in Surgery and Medicine (2001).
[0165] G. M. Morris and K. J. McIntyre, "Optical system design with
diffractive optics," in Diffractive Optics for Industrial and
Commercial Applications, J. Turunen and F. Wyrowski, eds., Ch. 3,
p. 95 (Akademie Verlag, 1997).
[0166] C. Brookner, M. Follen, I. Boiko, J. Galvan, S. Thomsen, A.
Malpica, S. Suzuki, R. Lotan, R. Richards-Kortum, "Tissue Slices
Autofluorescence Patterns in Fresh Cervical Tissue," Photochemistry
and Photobiology, 71:730-36, 2000.
[0167] R. Drezek, C. Brookner, I. Pavlova, I. Boiko, A. Malpica, R.
Lotan, M. Follen, R. Richards-Kortum, "Autofluorescence Microscopy
of Fresh Cervical Tissue Sections Reveals Alterations in Tissue
Biochemistry with Dysplasia," in press, Photochemistry and
Photobiology, 2000.
[0168] G. S. Kino, "Intermediate Optics in Nipkow Disk
Microscopes," in Handbook of Biological Confocal Microscopy, J. B.
Pawley, ed., Ch. 10 (Plenum Press, 1995).
[0169] F. R. Miller, D. McEachern, and B. E. Miller, "Growth
Regulation of Mouse Mammary Tumor Cells in Collagen Gel Cultures by
Diffusible Factors Produced by Normal Mammary Gland Epithelium and
Stromal Fibroblasts," Cancer Research, 49, pp. 6091-6097
(1989).
* * * * *