U.S. patent application number 11/295162 was filed with the patent office on 2007-01-18 for systems and methods for in-vivo optical imaging and measurement.
Invention is credited to Clifford C. Hoyt, Richard Levenson, James Mansfield.
Application Number | 20070016078 11/295162 |
Document ID | / |
Family ID | 36578450 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070016078 |
Kind Code |
A1 |
Hoyt; Clifford C. ; et
al. |
January 18, 2007 |
Systems and methods for in-vivo optical imaging and measurement
Abstract
Disclosed are methods and systems for determining information
about a position of the entity within the sample using spectral
techniques. For example, the sample may be a living animal, such as
a mouse, and the embedded object may be a tumor.
Inventors: |
Hoyt; Clifford C.;
(Wellesley, MA) ; Mansfield; James; (Acton,
MA) ; Levenson; Richard; (Brighton, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
36578450 |
Appl. No.: |
11/295162 |
Filed: |
December 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60633511 |
Dec 6, 2004 |
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60702925 |
Jul 27, 2005 |
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60707497 |
Aug 11, 2005 |
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60634154 |
Dec 8, 2004 |
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60720080 |
Sep 23, 2005 |
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60697617 |
Jul 8, 2005 |
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Current U.S.
Class: |
600/476 |
Current CPC
Class: |
A61B 5/1075 20130101;
A61B 2562/0242 20130101; G01N 21/6428 20130101; A61B 2503/40
20130101; A61B 2562/0238 20130101; G01N 21/763 20130101; A61B
5/0073 20130101; G01N 2021/6419 20130101; A61B 5/0059 20130101;
G01N 21/4795 20130101; G01N 2021/6421 20130101; G01N 21/6456
20130101 |
Class at
Publication: |
600/476 |
International
Class: |
A61B 6/00 20060101
A61B006/00 |
Claims
1. A method comprising: providing a sample having an entity
embedded therein that is labeled with multiple fluorescence
compounds having different emission spectra; illuminating the
sample to cause emission from the labeled entity; measuring an
intensity of the emission at each of multiple emission wavelengths;
and determining information about a position of the entity within
the sample based on the measured emission at each of the different
emission wavelengths.
2. The method of claim 1, wherein the sample is a biological
sample.
3. The method of claim 2, wherein the sample is a living
animal.
4. The method of claim 1, wherein the entity is a tumor.
5. The method of claim 1, wherein the emission is measured at each
of three or more different emission wavelengths.
6. The method of claim 1, wherein the emission is measured at each
of four or more different emission wavelengths.
7. The method of claim 1, wherein the information about the
position of the entity within the sample is determined based
further on information about the relative absorption of the
different emission wavelengths in the sample and the relative
emission intensity of the entity at each of the different emission
wavelengths.
8. The method of claim 7, wherein the position information is
determined to account for scaling of the relative emission
intensity of the entity at each of the different emission
wavelengths caused by differential absorption of the emission
wavelengths in the sample when the emission propagates through the
sample.
9. The method of claim 1, wherein the multiple fluorescent
compounds labeling the entity produce emission peaks over a range
larger than 60 nm.
10. The method of claim 9, wherein the multiple fluorescent
compounds labeling the entity produce emission peaks over a range
larger than 100 nm.
11. The method of claim 1, wherein the multiple fluorescent
compounds labeling the entity produce emission peaks large enough
to produce a differential attenuation between at least two of the
emission peaks that is larger than two times for every centimeter
of depth in the sample material surrounding the entity.
12. The method of claim 1 1, wherein the multiple fluorescent
compounds labeling the entity produce emission peaks large enough
to produce a differential attenuation between at least two of the
emission peaks that is larger than five times for every centimeter
of depth in the sample material surrounding the entity.
13. The method of claim 1, wherein the position information is
determined based further on calibration data for the emission
spectra of the fluorescent compounds labeling the entity.
14. The method of claim 1, wherein the intensity of the emission at
each of multiple emission wavelengths is measured from each of
multiple sides of the sample, and wherein the position information
is based on the measured emission at each of the different emission
wavelengths from each of the multiple sides of the sample.
15. A system comprising: a light source system configured to
illuminate a sample, wherein the sample has an entity embedded
therein that is labeled with multiple fluorescence compounds having
different emission spectra and wherein the illumination of the
sample causes emission from the labeled entity; and a detector
system configured to measure an intensity of the emission at each
of multiple emission wavelengths; and an electronic processor
coupled to the detector system, wherein the electronic processor is
configured to determine information about a position of the entity
within the sample based on the measured emission at each of the
different emission wavelengths and calibration data about the
different emission spectra of the fluorescence compounds labeling
the entity embedded in the sample.
16. The apparatus of claim 15, wherein the electronic processor is
configured to determine information about a position of the entity
within the sample based further on calibration data about the
differential absorption of material in the sample surrounding the
embedded entity for the different emission wavelengths.
17. A method comprising: illuminating a sample at each of at least
two different excitation wavelengths; measuring radiation emitted
from an entity embedded in the sample in response to each of the
excitation wavelengths; and determining information about the
position of the entity in the sample based on the measured
radiation corresponding to the illumination at each of the
different excitation wavelengths.
18. The method of claim 17, wherein the sample is a biological
sample.
19. The method of claim 18, wherein the sample is a living
animal.
20. The method of claim 17, wherein the entity is a tumor labeled
with a fluorescent material.
21. The method of claim 17, wherein the radiation is directed to
the sample at each of three or more different excitation
wavelengths, and wherein the radiation emitted from the entity is
measured in response to each of the three or more different
excitation wavelengths.
22. The method of claim 17, further comprising, for each excitation
wavelength for which the radiation emitted from the entity is
measured, measuring the relative intensity of emitted radiation at
two or more emission wavelengths.
23. The method of claim 22, further comprising using the measured
relative intensities of the emitted radiation at the two or more
emission wavelengths to reduce the contribution of autofluorescence
from the measured radiation used to determine the entity depth.
24. The method of claim 23, wherein the reduction of the
contribution of autofluorescence is based on a linear decomposition
of the measured relative intensities in terms of spectral
signatures for the entity and one or more other components of the
sample.
25. The method of claim 21, further comprising, for each excitation
wavelength for which the radiation emitted from the entity is
measured, measuring the relative intensity of emitted radiation at
two or more emission wavelengths.
26. The method of claim 25, further comprising using the measured
relative intensities of the emitted radiation at the two or more
emission wavelengths to reduce the contribution of autofluorescence
from the measured radiation used to determine the entity depth.
27. The method of claim 26, wherein the reduction of the
contribution of autofluorescence is based on a linear decomposition
of the measured relative intensities in terms of spectral
signatures for the entity and one or more other components of the
sample.
28. The method of claim 17, wherein the position information is
determined based further on information about the relative
absorption of the different excitation wavelengths in the sample
and the relative emission intensity of the entity at each of the
different excitation wavelengths.
29. The method of claim 28, wherein the position information is
determined based further to account for scaling of the relative
emission intensity of the entity at each of the different
excitation wavelengths caused by differential absorption of the
excitation wavelengths caused by material in the sample through
which the excitation wavelengths pass to be incident on the
entity.
30. The method of claim 17, wherein the excitation wavelengths are
in the range of about 540 nm to 650 nm.
31. The method of claim 17, wherein the emitted radiation is in the
range of about 750 nm to 900 nm.
32. The method of claim 17, wherein the sample is sequentially
illuminated on each of multiple sides of the sample, wherein the
emitted radiation is measured in response to each of the excitation
wavelengths for the illumination of each of the sides, and wherein
the position information is determined based on the radiation
measured by the detector system at each of the different excitation
wavelengths for the illumination of each of the sides.
33. A system comprising: a light source system configured to
illuminate at a sample at each of at least two different excitation
wavelengths; a detector system configured to measure radiation
emitted from an entity embedded in the sample in response to each
of the excitation wavelengths; and an electronic processor
configured to determine information about the position of the
entity within the sample based on the radiation measured by the
detector system corresponding to the illumination at each of the
different excitation wavelengths.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to: U.S. Provisional
Application No. 60/633,511 entitled "METHOD AND SYSTEM FOR
CAPTURING MULTIPLE VIEWS OF AN EXTENDED, LUMINESCING SPECIMEN" by
Clifford C. Hoyt and Peter Domenicali, filed on Dec. 6, 2004; U.S.
Provisional Application No. 60/702,925 entitled "METHOD AND SYSTEM
FOR CAPTURING MULTIPLE VIEWS OF AN EXTENDED, LUMINESCING SPECIMEN
AND SEQUENTIAL ILLUMINATION THEREOF" by Clifford C. Hoyt and Peter
Domenicali, filed on Jul. 27, 2005; U.S: Provisional Application
No. 60/707,497 entitled "METHOD AND SYSTEM FOR CAPTURING MULTIPLE
VIEWS OF AN EXTENDED, LUMINESCING SPECIMEN AND SEQUENTIAL
ILLUMINATION THEREOF" by Clifford C. Hoyt and Peter Domenicali,
filed on Aug. 11, 2005; U.S. Provisional Application No. 60/634,154
entitled "METHOD FOR DETERMINING THE DEPTH OF FLUORESCENCE ENTITIES
INSIDE 0OBJECTS OR ORGANISMS" by Clifford C. Hoyt and James
Mansfield, filed on Dec. 8, 2004; U.S. Provisional Application No.
60/720,080 entitled "METHOD AND SYSTEM FOR DETERMINING THE DEPTH OF
FLUORESCENCE ENTITIES INSIDE OBJECTS OR ORGANISMS" by Clifford C.
Hoyt and Peter Domenicali, filed on Sep. 23, 2005; and U.S.
Provisional Application No. 60/697,617 entitled "METHOD AND SYSTEM
FOR ESTIMATING BIOMASS TUMORS USING OPTICAL IMAGING" by Clifford C.
Hoyt, filed on Jul. 8, 2005. The contents of each of the foregoing
provisional applications are incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to optical imaging and in particular,
to optical imaging in biological specimens.
BACKGROUND
[0003] Small animal optical imaging is an increasingly popular
technique for studying tumors and other physiological conditions
in-vivo in life sciences research and pharmaceutical drug
development. Optical imaging techniques can provide low cost,
rapid, and quantitative measurements compared to more conventional
medical imaging techniques such as MRI, CAT, PET, and SPECT.
[0004] Optical imaging techniques typically capture images of a
specimen using light in the ultraviolet, visible, and near-infrared
(near-IR) regions of the electromagnetic spectrum. It can be
difficult, however, to acquire accurate three-dimensional
information via optical imaging when tissue, which is a turbid
medium, is highly scattering and/or absorbs light at these
wavelengths. Some imaging techniques detect spatial distributions
of scattered, emitted and transmitted photons (or combinations
thereof) emanating from a specimen. Further information about the
internal structure of the specimen can be obtained from
time-of-flight emission measurements, fluorescence lifetimes,
and/or the spectral properties of emitted, scattered, and
transmitted photons. In general, many different approaches are
known and used for the detection of these photons.
[0005] Information about the distribution of light emanating from a
specimen can be used as an input to a light diffusion algorithm in
order to construct a 3D model of in-vivo entities based on the
spatial light distribution, see for example U.S. patent application
Ser. No. 10/606,976 entitled "METHOD AND APPARATUS FOR 3-D IMAGING
OF INTERNAL LIGHT SOURCES" by Daniel G. Steams et al., filed on
Jun. 25, 2003, the contents of which are incorporated herein by
reference. The accuracy of light diffusion algorithms, in general,
is enhanced by light distribution information acquired from
multiple views of the specimen. In consequence, measurement systems
that provide multiple-view capability may be more sensitive and
provide higher accuracy than single-view systems.
[0006] Systems that capture multiple views of a specimen can do so
in various ways. Four techniques for recording multiple views of a
specimen are shown in FIGS. 1A-D. FIG. 1A is a schematic diagram of
an imaging system where multiple CCD cameras 4 are oriented about a
specimen 2 in order to acquire multiple views of the specimen. FIG.
1B is a schematic diagram of a measurement system having a source 6
and a detector 4. Both source 6 and detector 4 are scanned over the
surface of specimen 2 in order to capture multiple views of the
specimen's surface. The arrows in the figure illustrate the scan
directions. FIG. 1C is a schematic diagram of a measurement system
that uses multiple mirrors 8 to direct two different views of
specimen 2 to two detectors 4. FIG. 1D is a schematic diagram of a
measurement system that employs a compound mirror 10 to direct two
different side views of specimen 2 to an imaging lens 12, which
images these views, along with a front view of specimen 2, to
detector array 4.
[0007] Three-dimensional information about structures and entities
inside living organisms is useful in both research and clinical
applications. For example, in pharmaceutical pre-clinical trials,
tumors can be grown in immuno-compromised animal models and tracked
using imaging techniques such as fluorescence imaging. In some
cases, image contrast can be enhanced by labeling entities with
molecular fluorophores. The use of labels may also provide
information about the internal structure of a specimen, such as the
dimensions and/or density of particular internal features that have
specific molecular characteristics. Detection of fluorescence
emitted by a specimen is a preferred technique because the
biochemistry of target-specific fluorescence labeling is well
developed.
[0008] The advent of genetically engineered cell lines that express
fluorescent proteins for in-vivo measurements provides a means to
characterize an entity using a unique optical emission signal.
These techniques are described, for example, by M. Chalfie et al.
in "Green Fluorescent Protein as a Marker for Gene Expression,"
Science 263: 802-805 (1994), the contents of which are incorporated
herein by reference. Exogenous fluorophores can therefore be
introduced into internal structures such as tumors to enable
fluorescence imaging by inducing the tumors to express fluorescent
proteins, providing for a natural localization of the fluorescence
emission within a specimen. In some cases, fluorophores that bind
to a tumor can also be injected into a specimen.
[0009] The accuracy and resolution of in-vivo imaging of
fluorescent entities in living organisms can be limited by
scattering and absorption in tissues. These processes attenuate the
intensity of light passing through the tissue. The effects of
tissue scattering and absorption have been studied extensively, see
for example T. L. Troy and S. N. Thennadil, "Optical properties of
human skin in the near infrared wavelength range of 1000 to 2200
nm," Journal ofBiomedical Optics 6: 167-176 (2004), the contents of
which are incorporated herein by reference. At wavelengths from the
ultraviolet to the near-infrared, it has generally been found that
as the wavelength of incident light increases, both scattering and
absorption of the incident light by tissue decrease. As a result,
the effective "penetration depth" of incident light in tissue
varies with wavelength. In the range of wavelengths from about 400
nm to about 1100 nm, the greatest penetration depth occurs at about
800 nm .
[0010] Absorption and scattering properties of biological tissues
have been found to be substantially similar among animals, and this
finding has been used as a basis for numerous optical techniques
for probing turbid media with light. Computational techniques for
reconstructing 3D tissue models that take into account measured
spatial, temporal, and/or spectral information from light emitted
or scattered by specimen tissues are used to visualize the
specimen's internal structure. Monte Carlo methods may be used to
validate these structural models, see for example Q. Liu, C. Zhu
and N. Ramanujam, "Experimenal validation of Monte Carlo modeling
of fluorescence in tissues in the UV-visible spectrum," Journal of
Biomedical Optics 8: 223-236 (2003).
[0011] Depth or position information regarding entities within a
specimen can be provided by measuring wavelength shifts of light
emitted from the specimen, because the magnitude of the wavelength
shift varies as a function of the thickness of tissue between the
emitting entity and a detector. In particular, if a specimen emits
light in a portion of the spectrum where the scattering and/or
absorption properties have a substantially monotonic increase or
decrease as a function of tissue thickness, then the emission
spectrum of a labeled entity will shift as a function of the
entity's position within the specimen. The shift of the emission
spectrum may be small, i.e., a few nanometers, for significant
changes in tissue thickness, so measurement equipment used to
detect spectral shifts should have sensitivity that is sufficient
to detect small spectral changes in order to make precise thickness
estimates. Further, shifts of the emission spectrum can be produced
via other mechanisms such as biochemical processes.
SUMMARY
[0012] We disclose systems and methods for extracting information
about objects in dense or turbid media, e.g., tumors in biological
specimens such as mice. In general, the systems include a light
source and an imaging apparatus for capturing one or more views of
the specimen under study. In many embodiments, the light source can
be configured to cause the specimen or a structural entity therein
to fluoresce, and the imaging apparatus captures one or more
fluorescence images on a detector system.
[0013] In general, the systems are capable of operating in multiple
measurement modes in order to record different types of
information, thereby effecting various types of measurements. For
example, the systems are capable of operating in an alignment or
positioning mode, in which the position and orientation of a
specimen are optimized prior to recording data based on a
previously recorded reference image of the specimen. In addition,
the systems can operate in a structured illumination mode. For
example, one or more selected sides of a specimen can be
illuminated sequentially, and one or more spatial intensity
patterns can be imparted to the illumination light incident on any
selected side of the specimen. Emitted light from the specimen can
be detected in a single view of the specimen, or multiple views can
be measured, simultaneously or in a selected sequential pattern. In
some embodiments, the systems can be configured to measure a
spectral response from the specimen by providing excitation light
at multiple wavelengths and/or by resolving emission into various
emission wavelengths. To improve the accuracy of data obtained from
spectrally resolved measurements, multiple different fluorescence
labels can be attached to entities of interest. Single- or
multiple-view images that include spectral information can be used
to provide position information about fluorescing entities internal
to the specimen. In another mode of operation, the systems can be
configured to integrate the total emitted radiation from a
specimen, captured in one or more views or directions, and to
estimate a mass of a structural entity emitting the measured
radiation.
[0014] In each of the modes of system operation, various mechanisms
may produce the radiation captured in views or measurements of the
specimen. Incident light can be reflected, scattered, or
transmitted by the specimen. In addition, an important mechanism is
fluorescence, wherein light incident on a specimen induces the
specimen (or a structural entity therein) to fluoresce. In some
embodiments, the wavelength of the fluorescence is different (i.e.,
red-shifted) from the wavelength of the incident light, providing a
convenient means for separating the signals. Fluorescence can be
produced by chemical moieties naturally present in the specimen, or
the fluorescent moieties can be introduced through biological
(e.g., molecular genetic) or chemical (e.g., injection of
structurally-specific labels) techniques. Chemical moieties
naturally present in the specimen can produce autofluorescence and
it may be necessary, in some cases, to distinguish an
autofluorescence signal from fluorescence emission by a labeled
entity in order to obtain accurate measurements. In addition, some
specimens may exhibit bioluminescence, and light sources may not be
required in order to measure luminescence images.
[0015] We now generally summarize different aspects and features of
the invention.
[0016] In general, in one aspect, a method is disclosed including
using multiple reflective surfaces of an optical component to
direct two or more (and in some preferred embodiments, three or
more) side views of an extended specimen (such as a small animal,
like a mouse) to a detector system. The relative orientation of the
specimen meets one or more of the following conditions: i) a long
axis of the extended specimen is oriented parallel to an optical
axis connecting the optical component to the detector system; ii)
each view of the specimen is imaged onto the detector system via
the optical component and defines chief rays, and the specimen
oriented with respect to the optical component so that the chief
rays for the different views emerge from the specimen substantially
perpendicular to a long axis of the specimen; iii) each view of the
specimen is imaged onto the detector system via the optical
component, and the specimen is oriented with respect to the optical
component so that optical paths between the specimen and the
detector system for different views are substantially equal; and
iv) a long axis of the extended specimen is oriented nominally
collinear with a symmetry axis of the optical component.
[0017] Embodiments of the method may include any of the following
features.
[0018] Each reflective surface of the optical component is a mirror
configured to direct one view of the sample to the detector system.
For example, the optical component may include a pyramidal
arrangement of the mirrors.
[0019] The detector system may includes a CCD camera.
[0020] The method may further include using one or more lenses to
image the multiple views onto the detector system.
[0021] The multiple reflective surfaces may be curved to image the
multiple views onto the detector system.
[0022] The method may further including using one or more lenses
between the optical component and the detector system to image the
views from the specimen to the detector system. Alternatively, or
in addition, the reflective surfaces of the optical component may
be curved to image each view from the specimen to the detector
system.
[0023] In another, related aspect, a system is disclosed including:
a specimen holder configured to support an extended specimen (for
example, a small animal, like a mouse); a detector system; and an
optical component including two or more reflective surfaces (or in
some preferred embodiments, three or more reflective surfaces) each
configured to direct a different side view of the extended specimen
to the detector system. The orientation of the specimen holder is
set according to one or more of the following conditions: i) the
specimen holder is configured to orient a long axis of the extended
specimen parallel to an optical axis connecting the optical
component to the detector system; ii) the specimen holder is
configured to orient the extended specimen with respect to the
optical component so that so the optical paths between the specimen
and the detector system for different views are substantially
equal; iii) the specimen holder is configured to orient the
extended specimen with respect to the optical component so that
chief rays for the different views emerge from the specimen
substantially perpendicular to a long axis of the specimen; and iv)
the specimen holder is configured to orient the extended specimen
with respect to the optical component so that a long axis of the
specimen is oriented nominally collinear with a symmetry axis of
the optical component.
[0024] Embodiments of the system may include features corresponding
to any of the features described above in connection with the
related method.
[0025] In general, in another aspect, a method is disclosed
including: (i) sequentially illuminating different sides of a
sample; and (ii) using multiple reflective surfaces of an optical
component to direct multiple side views (for example, in some
preferred embodiments, three or more side views) of the sample to a
detector system in response to each sequential illumination.
[0026] Embodiments of the method may include any of the following
features.
[0027] The sequential illumination of the different sides of the
sample may be done via the multiple reflective surface of the
optical component.
[0028] The reflective surfaces of the optical component may have a
dichroic coating, and the sequential illumination of the sample may
pass through the dichroic coating and the multiple side views are
reflected by the coating.
[0029] Each illumination in the sequential illumination may use a
different one of the reflective surfaces to illuminate the
sample.
[0030] The sequential illumination may includes using a spatial
light modulator to selectively direct light to the different sides
of the sample.
[0031] The sequential illumination may includes using multiple
fiber bundles as the light source. For example, each fiber bundle
may be used to illuminate a corresponding one of the sides of the
sample.
[0032] One or more dichroic beamsplitters may be used to guide
light from the sample along a path different from that of light
used to illuminate the sample.
[0033] The method may further include positioning the spatial light
modulator in a plane conjugate to that of the detector system
[0034] The method may further include positioning the spatial light
modulator in a plane conjugate to that of the sample.
[0035] The method may further include adjusting the configuration
of the spatial light modulator to improve the uniformity of the
illumination at the sample The method may further including
adjusting the configuration of the spatial light modulator to
improve the uniformity of one or more of the side views as measured
by the detector system.
[0036] The relative orientation of the specimen may be set to meet
one or more of the following conditions: i) a long axis of the
extended specimen is oriented parallel to an optical axis
connecting the optical component to the detector system; ii) each
view of the specimen is imaged onto the detector system via the
optical component and defines chief rays, and the specimen oriented
with respect to the optical component so that the chief rays for
the different views emerge from the specimen substantially
perpendicular to a long axis of the specimen; iii) each view of the
specimen is imaged onto the detector system via the optical
component, and the specimen is oriented with respect to the optical
component so that optical paths between the specimen and the
detector system for different views are substantially equal; and
iv) a long axis of the extended specimen is oriented nominally
collinear with a symmetry axis of the optical component.
[0037] Each reflective surface of the optical component may be a
mirror configured to direct one view of the specimen to the
detector system. The optical component may include a pyramidal
arrangement of the mirrors. The detector system may include a CCD
camera.
[0038] The method may further include using one or more lenses to
image the multiple views onto the detector system.
[0039] The multiple reflective surfaces may be curved to image the
multiple views onto the detector system.
[0040] In a related aspect, a system is disclosed that includes:
(i) a specimen holder configured to support an extended specimen;
(ii) a detector system; (iii) an optical component including
multiple reflective surfaces each configured to direct a different
side view of the extended specimen to the detector; and (iv) an
illumination source configured to sequentially illuminate different
sides of the specimen.
[0041] Embodiments of the system may include any of the following
features.
[0042] The illumination source may be configured to sequentially
illuminate the different sides of the specimen via the multiple
reflective surfaces of the optical component.
[0043] The reflective surfaces of the optical component may have a
dichroic coating, and the sequential illumination from the
illumination source may pass through the dichroic coating and the
side views are reflected by the coating.
[0044] The multiple reflective surfaces may include three of more
reflective surfaces.
[0045] The orientation of the specimen holder may be set according
to one or more of the following conditions: i) the specimen holder
is configured to orient a long axis of the extended specimen
parallel to an optical axis connecting the optical component to the
detector system; ii) the specimen holder is configured to orient
the extended specimen with respect to the optical component so that
so the optical paths between the specimen and the detector system
for different views are substantially equal; iii) the specimen
holder is configured to orient the extended specimen with respect
to the optical component so that chief rays for the different views
emerge from the specimen substantially perpendicular to a long axis
of the specimen; and iv) the specimen holder is configured to
orient the extended specimen with respect to the optical component
so that a long axis of the specimen is oriented nominally collinear
with a symmetry axis of the optical component.
[0046] The system may include one or more lenses to image each view
onto the detector system.
[0047] Each reflective surface of the optical component may be
curved to image the corresponding view onto the detector
system.
[0048] The illumination source may be configured so that each
illumination in the sequential illumination uses a different one of
the reflective surfaces to illuminate the sample.
[0049] The illumination source may includes multiple fiber bundles.
For example, each fiber bundle may be used to illuminate a
corresponding one of the sides of the specimen.
[0050] The system may further include one or more dichroic beam
splitters to guide light from the specimen along a path different
from that of light used to illuminate the specimen.
[0051] The illumination source may includes light conditioning
optics including a spatial light modulator. The spatial light
modulator may be positioned in a plane conjugate to that of the
detector system or positioned in a plane conjugate to that of the
specimen holder.
[0052] The system may further include an electronic controller
coupled to the spatial light modulator and configured to adjust the
configuration of the spatial light modulator to improve the
uniformity of the illumination at the sample and/or to improve the
uniformity of one or more of the side views measured by the
detector system.
[0053] In general, in another aspect, a method is disclosed
including: (i) sequentially illuminating a specimen with different
spatial distributions of light, wherein each illumination causes an
object embedded in the specimen to emit radiation in response to
the light; (ii) for each different spatial distribution of
illumination light, imaging the radiation emitted from the specimen
from each of multiple sides of the specimen (for example, in
preferred embodiments, from three or more sides of the specimen);
and (iii) determining information about the object in the specimen
based on the imaged radiation from each of the multiple sides for
each of the different spatial distributions of illumination
light.
[0054] Embodiments of the method may include any of the following
features.
[0055] The emitted radiation may be fluorescence.
[0056] The different spatial distributions of illumination light
may correspond to light distributions that illuminate different
sides of the specimen. For example, each spatial distributions may
have a common shape with respect to its side of the specimen, or it
may have a different shape with respect to its side of the
specimen.
[0057] At least some of the spatial distributions may correspond to
different illumination patterns for a common side of the
specimen.
[0058] The different illumination patterns may be produced by one
or more of the following: (i) adjusting a position of an
illumination light beam on the common side of the specimen; ii)
using multiple fiber light sources; iii) using beam splitting
optics to separate portions of light produced by a light source;
and iv) using a spatial light modulator.
[0059] Imaging the radiation emitted from the specimen from each of
multiple sides of the specimen may include one or more of the
following: (i) using a multiple reflective surfaces of an optical
component to collect emission from corresponding sides of the
specimen; ii) using multiple detectors positioned to collect
emission from the respective sides of the specimen; and (iii) using
multiple optical fibers positioned to collect emission from the
respective sides of the specimen.
[0060] The multiple sides may be separated from one another by more
than 30 degrees. For example, they may be separated from one
another by approximately 90 degrees.
[0061] The information about the object in the specimen may
includes information about a relative position of the object within
the specimen. For example, the information about the object in the
specimen may further include information about an orientation and
shape of the object in the specimen. Also, the information about
the object in the specimen may include information about a size the
object in the specimen.
[0062] The specimen may be an animal (for example, a small animal,
such as a mouse). The animal may be living. The object in the
specimen may be tumor in the animal.
[0063] Determining information about the object in the specimen may
include constructing a self-consistent model of the object in the
specimen based on imaged radiation from the multiple sides of the
sample for each of the different spatial distributions of
illumination light.
[0064] For each different spatial distributions of illumination
light, the illumination of the sample may include illuminating with
a common set of different excitation spectra, and the information
about the object in the specimen is further based on the
differences in the imaged radiation for each of the different
excitation spectra for each of the different spatial distributions.
In such cases, the method may further including removing
autofluorescence from the imaged radiation.
[0065] In other embodiments, the method may further include
spectrally resolving the radiation emitted from each side of the
specimen, and wherein the information about the object in the
specimen is further based on the differences in the variations in
the spectral content of the imaged radiation.
[0066] In a related aspect, an apparatus is disclosed including:
(i) a source configured to sequentially illuminate a specimen with
different spatial distributions of light, wherein each illumination
causes an object embedded in the specimen to emit radiation in
response to the light; (ii) a detector system configured to image
the radiation emitted from the specimen from each of multiple sides
of the specimen for each of the different spatial distributions of
illumination light; and (iii) an electronic processor coupled to
the source and detector system, the processor configured to
determine information about the object in the specimen based on the
imaged radiation from each of the multiple sides for each of the
different spatial distributions of illumination light. The source
may include light conditioning optics for producing the different
spatial distributions of light. The detector system include may
include light collection optics for imaging the emitted radiation
to one or more detectors.
[0067] Embodiments of the apparatus may include features
corresponding to any of the features listed above in connection
with the related method.
[0068] In general, in another aspect, a method is disclosed
including: (i) illuminating a living specimen to cause an object
embedded in the specimen (e.g., a living animal) to emit radiation
in response to the illumination, wherein the specimen has eyes and
the illumination extends over a region of the specimen including
the eyes; (ii) shaping the illumination to prevent it from exposing
the eyes of the specimen; and (iii) measuring the emitted radiation
to determine information about the object embedded in the
specimen.
[0069] Embodiments of the method may include any of the following
features.
[0070] For example, the shaping may include positioning an optical
element between the source of the illumination and the eyes of the
specimen and/or using a spatial light modulator to prevent the
illumination from exposing the eyes of the specimen.
[0071] The shaping may prevent involuntary movement of the specimen
caused by illuminating the eyes.
[0072] The emitted radiation may be imaged to a detector from each
of multiple sides of the specimen.
[0073] In a related aspect, an apparatus is disclosed including:
(i) a source configured to illuminate a living specimen to cause an
object embedded in the specimen to emit radiation in response to
the illumination, wherein the specimen has eyes and wherein the
illumination extends over a region of the specimen including the
eyes; (ii) a means for shaping the illumination to prevent it from
exposing the eyes of the specimen; (iii) a detector system for
measuring the emitted radiation; and (iv) an electronic process
coupled to the detector system and configured to determine
information about the object embedded in the specimen.
[0074] Embodiments of the apparatus may include any of the
following features.
[0075] The means for shaping may be an optical element positioned
between the source of the illumination and the eyes of the
specimen. For example, the optical element may be a stop or mirror.
The optical element may be configured to be adjustably
positioned.
[0076] In another aspect, a method is disclosed including providing
a sample having an entity embedded therein that is labeled with
multiple fluorescence compounds having different emission spectra,
illuminating the sample to cause emission from the labeled entity,
measuring an intensity of the emission at each of multiple emission
wavelengths, and determining information about a position of the
entity within the sample based on the measured emission at each of
the different emission wavelengths.
[0077] Embodiments of the method may include any of the following
features.
[0078] The sample may be a biological sample, such as a living
animal.
[0079] The entity may be a tumor.
[0080] The emission may be measured at each of three or more
different emission wavelengths, or at each of four or more
different emission wavelengths.
[0081] The information about the position of the entity within the
sample may be determined based further on information about the
relative absorption of the different emission wavelengths in the
sample and the relative emission intensity of the entity at each of
the different emission wavelengths.
[0082] The position information may be determined to account for
scaling of the relative emission intensity of the entity at each of
the different emission wavelengths caused by differential
absorption of the emission wavelengths in the sample when the
emission propagates through the sample.
[0083] The multiple fluorescent compounds labeling the entity may
produce emission peaks over a range larger than 60 nm. The multiple
fluorescent compounds labeling the entity may produce emission
peaks over a range larger than 100 nm.
[0084] The multiple fluorescent compounds labeling the entity may
produce emission peaks large enough to produce a differential
attenuation between at least two of the emission peaks that is
larger than two times (e.g., larger than five, or even ten times)
for every centimeter of depth in the sample material surrounding
the entity.
[0085] The position information may be determined based further on
calibration data for the emission spectra of the fluorescent
compounds labeling the entity.
[0086] The intensity of the emission at each of multiple emission
wavelengths may be measured from each of multiple sides of the
sample, and the position information may be based on the measured
emission at each of the different emission wavelengths from each of
the multiple sides of the sample.
[0087] In a related aspect, a system is disclosed including: (i) a
light source system configured to illuminate a sample, where the
sample has an entity embedded therein that is labeled with multiple
fluorescence compounds having different emission spectra and
wherein the illumination of the sample causes emission from the
labeled entity; (ii) a detector system configured to measure an
intensity of the emission at each of multiple emission wavelengths;
and (iii) an electronic processor coupled to the detector system,
where the electronic processor is configured to determine
information about a position of the entity within the sample based
on the measured emission at each of the different emission
wavelengths and calibration data about the different emission
spectra of the fluorescence compounds labeling the entity embedded
in the sample.
[0088] Embodiments of the system may have any of the following
features.
[0089] The electronic processor may be configured to determine
information about a position of the entity within the sample based
further on calibration data about the differential absorption of
material in the sample surrounding the embedded entity for the
different emission wavelengths.
[0090] Embodiments of the system may further include features
corresponding to any of the features listed above in connection
with the related method.
[0091] In general, in another aspect, the invention a method is
disclosed that includes illuminating a sample at each of at least
two different excitation wavelengths, measuring radiation emitted
from an entity embedded in the sample in response to each of the
excitation wavelengths, and determining information about the
position of the entity in the sample based on the measured
radiation corresponding to the illumination at each of the
different excitation wavelengths.
[0092] Embodiments of the method may include any of the following
features.
[0093] The sample may be a biological sample, such as a living
animal.
[0094] The entity may be a tumor labeled with a fluorescent
material.
[0095] The radiation may be directed to the sample at each of three
or more different excitation wavelengths, and the radiation emitted
from the entity may be measured in response to each of the three or
more different excitation wavelengths.
[0096] For each excitation wavelength for which the radiation
emitted from the entity is measured, the relative intensity of
emitted radiation at two or more emission wavelengths may be
measured. The measured relative intensities of the emitted
radiation at the two or more emission wavelengths may be used to
reduce the contribution of autofluorescence from the measured
radiation used to determine the entity depth. The reduction of the
contribution of autofluorescence may be based on a linear
decomposition of the measured relative intensities in terms of
spectral signatures for the entity and one or more other components
of the sample. For example, for each excitation wavelength for
which the radiation emitted from the entity is measured, the
relative intensity of emitted radiation at two or more emission
wavelengths may be measured. The measured relative intensities of
the emitted radiation at the two or more emission wavelengths may
be used to reduce the contribution of autofluorescence from the
measured radiation used to determine the entity depth, and the
reduction of the contribution of autofluorescence may be based on a
linear decomposition of the measured relative intensities in terms
of spectral signatures for the entity and one or more other
components of the sample.
[0097] The position information may be determined based further on
information about the relative absorption of the different
excitation wavelengths in the sample and the relative emission
intensity of the entity at each of the different excitation
wavelengths. The position information may be determined based
further to account for scaling of the relative emission intensity
of the entity at each of the different excitation wavelengths
caused by differential absorption of the excitation wavelengths
caused by material in the sample through which the excitation
wavelengths pass to be incident on the entity.
[0098] The excitation wavelengths may be in a range of about 540 nm
to about 650 nm, and the emitted radiation may be in a range of
about 750 nm to about 900 nm.
[0099] The sample may be sequentially illuminated on each of
multiple sides of the sample, and the emitted radiation may be
measured in response to each of the excitation wavelengths for the
illumination of each of the sides, and the position information may
be determined based on the radiation measured by the detector
system at each of the different excitation wavelengths for the
illumination of each of the sides.
[0100] In a related aspect, a system is disclosed including a light
source system configured to illuminate a sample at each of at least
two different excitation wavelengths, a detector system configured
to measure radiation emitted from an entity embedded in the sample
in response to each of the excitation wavelengths, and an
electronic processor configured to determine information about the
position of the entity within the sample based on the radiation
measured by the detector system corresponding to the illumination
at each of the different excitation wavelengths.
[0101] Embodiments of the system may further include features
corresponding to any of the features listed above in connection
with the related method.
[0102] In general, in another aspect, a method is disclosed that
includes collecting radiation emitted from an object embedded in a
biological sample from multiple sides of the sample, and estimating
the size of the object based on the collected radiation.
[0103] Embodiments of the method may include any of the following
features.
[0104] Collecting the radiation may include collecting radiation
emitted from the object through substantially all surfaces of the
sample.
[0105] The collected radiation may only be a fraction of the total
flux of radiation emitted from the object through substantially all
surfaces of the sample. The collected radiation may be used to
determine an index for the total flux, and the size of the object
may be estimated based on the index and calibration information
that correlates the index to the object size. Further, the index
may be determined by integrating the radiation collected from the
multiple sides of the object.
[0106] The object may be spaced from all of the surfaces of the
sample by more than a millimeter.
[0107] The emitted radiation may be fluorescence or
bioluminescence.
[0108] The object may be a tumor and the sample may be an
animal.
[0109] The object may be labeled with a compound that causes the
emitted radiation to be in a selected range of wavelengths. For
example, the emitted radiation may be in the near-infrared region
of the spectrum, such as from about 700 nm to about 900 nm.
[0110] Estimating the size of the object may include integrating
the emitted radiation and estimating the size of the object based
on the integrated radiation. Estimating the size of the object may
further include estimating the mass of the object from the
integrated radiation and estimating the size of the object based on
the estimated mass of the object. Further, estimating the size of
the object may include determining spatially resolved information
about the sample from at least some of the collected information
and using the spatially resolved information to improve the
estimation of the size of the object.
[0111] Collecting the emitted radiation may include using spectral
unmixing techniques to remove autofluorescence from the sample. For
example, using spectral unmixing techniques may include measuring
the relative intensity of the collected radiation at two or more
emission wavelengths, using the measured relative intensities of
the collected radiation at the two or more emission wavelengths to
adjust them to account for autofluorescence, and using the adjusted
intensities to estimate the size of the embedded entity. The two or
more emission wavelengths may include, for example, three or more
emission wavelengths, or four or more emission wavelengths.
Accounting for the autofluorescence, for example, may be based on a
linear decomposition of the measured intensities in terms of
spectral signatures for the object and one or more other components
of the sample.
[0112] Illumination of the sample may be used to induce the
emission of the radiation from the object.
[0113] Collecting the radiation emitted from the object from the
multiple sides of the sample may include imaging the radiation
emitted through each side of the object to a detector system. An
optical element having multiple reflective surfaces may be used to
image the multiple sides of the sample to the detector system.
[0114] In a related aspect, a system is disclosed including optics
for collecting radiation emitting from an object embedded in a
biological sample from multiple sides of the sample, a detector
system for receiving the radiation collected by the optics, and an
electronic processor coupled to the detector for estimating the
size of the object based on the collected radiation.
[0115] Embodiments of the system may include any of the following
features.
[0116] The collecting optics may be configured to collect radiation
emitted from the object through substantially all surfaces of the
sample.
[0117] The collecting optics may be configured to collect only a
fraction of the total flux of radiation emitted from the object
through substantially all surfaces of the sample. Further, an
electronic processor may be configured to use the collected
radiation to determine an index for the total flux and estimate the
size of the object based on the index and calibration information
that correlates the index to the object size. For example, the
electronic processor may be configured to determine the index based
on the integrated radiation collected from the multiple sizes of
the object.
[0118] A mount may be used to secure the biological sample relative
to the optics.
[0119] The system may include an illumination source.
[0120] The collecting optics may include a pyramidal arrangement of
mirrors and an imaging lens.
[0121] The detector system may be a multi-element detector.
[0122] The detector system may include multiple detectors
corresponding to different sides of the sample.
[0123] The detector system may be configured to measure the
relative intensity of the collected radiation at two or more
emission wavelengths, and the processor may be configured to adjust
the measured relative intensities of the collected radiation at the
two or more emission wavelengths to adjust for autofluorescence
from the sample and use the adjusted intensities to estimate the
size of the embedded entity. The two or more emission wavelengths
may include, for example, three or more emission wavelengths, or
four or more emission wavelengths. For example, the processor may
be configured to adjust for the autofluorescence based on a linear
decomposition of the measured intensities in terms of spectral
signatures for the object and one or more other components of the
sample that produce the autofluorescence.
[0124] In general, in another aspect, a method is disclosed that
includes positioning a specimen inside an optical measurement
system according to a reference image of the specimen indicative of
its position and orientation during an earlier measurement using
the optical measurement system, and measuring radiation emitted
from the positioned specimen to provide information about an object
embedded in the specimen.
[0125] Embodiments of the method may include any of the following
features.
[0126] The reference image may be recorded more than one hour
(e.g., more than one day) prior to the present positioning of the
specimen.
[0127] The reference image may be recorded based on a light
reflected or scattered from the specimen, or based on a fluorescent
light emitted from the specimen.
[0128] The emitted radiation may be fluorescence or
bioluminescence.
[0129] The positioned specimen may be illuminated to cause the
emitted radiation.
[0130] The emitted radiation may include radiation emitted from the
object embedded in the specimen.
[0131] The specimen may be a living animal, and the object may be a
tumor embedded in the animal.
[0132] Information about the object may be compared to information
derived from the earlier measurement of the specimen.
[0133] At a later time, the specimen may be positioned inside the
optical measurement system according to the reference image for a
subsequent measurement using the optical measurement system to
provide information about the object embedded in the specimen at a
later time.
[0134] The information about the object may include information
about the size, position, or shape of the object.
[0135] The earlier measurement may be performed and the reference
image may be recorded.
[0136] The specimen may be positioned to match the position and
orientation of the reference image.
[0137] A live image of the specimen may be obtained while
positioning the specimen, and the specimen may be positioned based
on a comparison between the live image and the reference image. The
live image may be processed to highlight edges or other features of
the specimen to aid in the positioning. The live image and the
reference image may be simultaneously displayed to aid in the
positioning of the specimen. The live image and the reference image
may further be superimposed to aid in the positioning of the
specimen. The comparison may include providing a numerical index,
based on image processing techniques, to indicate the degree to
which the same orientation is achieved in live and reference
images. A display element may be provided, which changes in
accordance with the value of the numerical index. Determining
whether to measure the emitted radiation may be based on the value
of the numerical index, and the determination may be made
automatically by the optical measurement system. For example, the
optical measurement system may illuminate the sample to cause the
emitted radiation when it determines that the value of the
numerical index indicates a sufficient match between the live image
and the reference image.
[0138] The reference image may be processed to highlight edges or
other features to aid in the repositioning.
[0139] The reference image may be adjusted to account for changes
in the size or shape of the specimen since the earlier measurement
by the optical measurement system.
[0140] In a related aspect, a system is disclosed that includes an
optical measurement system configured to measure radiation emitted
from a specimen, where the optical measurement system includes an
adjustable stage for positioning the specimen and an electronic
processor configured to determine information about an object
embedded in the specimen based on the measured radiation emitted
from the specimen, and where the electronic processor further
stores a reference image of the specimen indicative of its position
and orientation during an earlier measurement using the optical
measurement system.
[0141] Embodiments of the system may include any of the following
features.
[0142] The reference image may be recorded based on a light
reflected or scattered from the specimen, or based on a fluorescent
light emitted from the specimen.
[0143] The optical measurement system may be configured to
illuminate the specimen to cause the emitted radiation. The emitted
radiation may include radiation emitted from the object embedded in
the sample.
[0144] The processor may be configured to compare the determined
information about the object to information derived from the
earlier measurement of the specimen.
[0145] The information about the object may include information
about the size, position, or shape of the object.
[0146] The optical measurement system may be configured to perform
the earlier measurement and record the reference image.
[0147] The optical measurement system may be configured to obtain a
live image of the specimen while positioning the specimen on the
stage. The optical measurement system may further include a display
coupled to the electronic processor and configured to
simultaneously display the live image and the reference image to
aid in the positioning of the specimen. The processor may be
configured to process the live image to highlight edges or other
features of the specimen to aid in the positioning, and the
processor may be configured to compare the live image to the
reference image and produce a numerical index, based on image
processing techniques, to indicate the degree to which the same
orientation is achieved in live and reference images. Further, the
optical measurement system may include a display coupled to the
electronic processor for providing a display indication which
changes in accordance with the value of the numerical index. The
electronic processor may be configured to determine whether to
measure the emitted radiation based on the value of the numerical
index. The optical measurement system may be configured to
illuminate the specimen to cause the emitted radiation when it
determines that the value of the numerical index indicates a
sufficient match between the live image and the reference
image.
[0148] The processor may be configured to process the reference
image to highlight edges or other features to aid in the
repositioning.
[0149] The electronic processor may be configured to adjust the
reference image to account for changes in the size or shape of the
specimen since the earlier measurement by the optical measurement
system.
[0150] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict with documents incorporated herein by reference, the
present specification will control.
[0151] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0152] FIG. 1 is a schematic diagram of several different
techniques for acquiring multiple views of a specimen.
[0153] FIG. 2 is a schematic diagram of a measurement system for
in-vivo biological imaging and measurement applications.
[0154] FIG. 3 is a flow chart that includes steps for reproducibly
positioning a specimen prior to making measurements with the system
of FIG. 2.
[0155] FIG. 4 is a schematic diagram of one embodiment of light
collecting optics for acquiring multiple views of a specimen.
[0156] FIG. 5 is a schematic diagram showing two different
orientations of multiple views of a specimen imaged onto the
surface of a detector.
[0157] FIG. 6A is a plot showing the variation of the absorption
coefficient for biological tissues with the wavelength of incident
radiation.
[0158] FIG. 6B is a plot showing the results of a simulation of a
multiple excitation wavelength fluorescence measurement.
[0159] FIG. 7 is a flow chart that includes steps for making depth
or tissue thickness measurements using multiple excitation
wavelengths.
[0160] FIG. 8 is a schematic diagram showing wavelength-dependent
attenuation of fluorescence radiation emitted by a labeled
specimen.
[0161] FIG. 9 is a flow chart that includes steps for making depth
or tissue thickness measurements using multiple fluorescent labels
in a specimen.
[0162] FIG. 10 is a schematic diagram of one embodiment of a
measurement system operating in whole specimen integration
mode.
[0163] FIG. 11 is a flow chart that includes steps for estimating
the mass of a structure, such as a tumor internal to a specimen,
from integrated fluorescence measurements.
[0164] FIG. 12 is a schematic diagram showing an embodiment of a
measurement system that includes a structured illumination
source.
[0165] FIG. 13 is a schematic diagram showing an embodiment of a
measurement system that includes a structured illumination
source.
[0166] FIG. 14 is a schematic diagram showing an embodiment of a
measurement system that includes a structured illumination
source.
[0167] FIG. 15 is a schematic diagram showing an embodiment of a
measurement system that includes a structured illumination
source.
[0168] FIG. 16 is a schematic diagram showing an embodiment of a
measurement system that includes a structured illumination source
and a spatial light modulator.
[0169] FIG. 17 is a flow chart that includes steps for refining a
3D specimen model using measurement data acquired from structured
illumination of the specimen.
[0170] FIG. 18 is a schematic diagram of an embodiment of an
illumination stage with a shield to prevent incident radiation from
impinging upon the eyes of a specimen.
[0171] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0172] We disclose systems and methods for extracting information
about objects in dense or turbid media, e.g., tumors in biological
specimens such as mice. In general, the systems include a light
source and an imaging apparatus for capturing one or more views of
the specimen under study. In many embodiments, the light source can
be configured to cause the specimen or a structural entity therein
to fluoresce, and the imaging apparatus captures one or more
fluorescence images on a detector system.
[0173] In general, the systems are capable of operating in multiple
measurement modes in order to record different types of
information, thereby effecting various types of measurements. For
example, the systems are capable of operating in an alignment or
positioning mode, in which the position and orientation of a
specimen are optimized prior to recording data based on a
previously recorded reference image of the specimen. In addition,
the systems can operate in a structured illumination mode. For
example, one or more selected sides of a specimen can be
illuminated sequentially, and one or more spatial intensity
patterns can be imparted to the illumination light incident on any
selected side of the specimen. Emitted light from the specimen can
be detected in a single view of the specimen, or multiple views can
be measured, simultaneously or in a selected sequential pattern. In
some embodiments, the systems can be configured to measure a
spectral response from the specimen by providing excitation light
at multiple wavelengths and/or by resolving emission into various
emission wavelengths. To improve the accuracy of data obtained from
spectrally resolved measurements, multiple different fluorescence
labels can be attached to entities of interest. Single- or
multiple-view images that include spectral information can be used
to provide position information about fluorescing entities internal
to the specimen. In another mode of operation, the systems can be
configured to integrate the total emitted radiation from a
specimen, captured in one or more views or directions, and to
estimate a mass of a structural entity emitting the measured
radiation.
[0174] In each of the modes of system operation, various mechanisms
may produce the radiation captured in views or measurements of the
specimen. Incident light can be reflected, scattered, or
transmitted by the specimen. In addition, an important mechanism is
fluorescence, wherein light incident on a specimen induces the
specimen (or a structural entity therein) to fluoresce. In some
embodiments, the wavelength of the fluorescence is different (i.e.,
red-shifted) from the wavelength of the incident light, providing a
convenient means for separating the signals. Fluorescence can be
produced by chemical moieties naturally present in the specimen, or
the fluorescent moieties can be introduced through biological
(e.g., molecular genetic) or chemical (e.g., injection of
structurally-specific labels) techniques. Chemical moieties
naturally present in the specimen can produce autofluorescence and
it may be necessary, in some cases, to distinguish an
autofluorescence signal from fluorescence emission by a labeled
entity in order to obtain accurate measurements. In addition, some
specimens may exhibit bioluminescence, and light sources may not be
required in order to measure luminescence images.
Measurement Systems
[0175] A system 100 for capturing one or more views of a specimen
is shown schematically in FIG. 2. The system includes a light
source 102, light conditioning optics 106, an illumination stage
110, light collecting optics 114, a detector system 118, and an
electronic control system 122. Light source 102 provides light 104
which is directed into light conditioning optics 106. Light
conditioning optics 106 can include, for example, one or more
optical elements configured to direct light towards illumination
stage 110. In addition, light conditioning optics 106 can include
optical elements configured to modulate one or more properties of
light 104, such as the spectral properties or the spatial intensity
distribution of light 104. The action of light conditioning optics
106 on light 104 produces illumination light 108, which is further
directed by light conditioning optics 106 to be incident on a
specimen (not shown) that is mounted on illumination stage 110.
[0176] Illumination light 108 can interact with the specimen in
various ways to produce emitted light 112. For example,
illumination light 108 can be scattered from the specimen,
reflected by the specimen, transmitted by the specimen, or absorbed
by the specimen. In many embodiments, light absorbed by the
specimen may cause the specimen to fluoresce, producing additional
light that can be included in emitted light 112. Further, some
specimens may include bioluminescent structural entities therein,
which emit light even in the absence of the action of illumination
light 108.
[0177] Emitted light 112, which can include light produced by any
of the foregoing mechanisms, is collected by light collecting
optics 114. Light collecting optics 114 can be configured, for
example, to capture one or more views of the specimen, the views
corresponding to images of the specimen taken from different
spatial observation points and providing different perspective
views of the specimen. The views 116 of the specimen are directed
by light collecting optics 114 to be incident on a detector system
118 configured to record each of the one or more views and to
convert each to an electronic signal 120. An electronic control
system 122 coupled to the detector system receives the electronic
signals 120 and provides for further processing and system
control.
Positioning Mode
[0178] Time-series measurements of structures or entities within a
specimen can provide valuable information to researchers and
clinicians. For example, time-series measurements of tumor size can
be used to determine the rate of tumor growth and, in some
applications, the effectiveness of pharmaceutical agents employed
to counteract tumor growth. Successive measurements of the same
specimen, however, can be separated by periods of hours, days,
weeks, or months. Therefore, these time-series measurements can be
susceptible to variance that appears due to measurement error
introduced by non-reproducible positioning and orientation of a
specimen with respect to the measurement apparatus. If the
variability of the measured data over time is too large, for
example, the value of the data for research purposes can be
reduced.
[0179] In a first measurement mode, the system shown schematically
in FIG. 2 can be operated in a positioning mode in order to
reproducibly position and orient a specimen prior to beginning a
measurement in order to reduce errors due to non-repeatable
positioning and/or orientation in time-series measurements. In
particular, variable placement of the specimen over a sequence of
measurements can result in spurious errors due to spatial variation
in illumination intensity, from simple shadowing caused by the
specimen's shape, and from changes in specimen posture which
rearranges internal structures and overlying tissues. In order to
produce high quality data, reproducing the posture and position of
the specimen from one measurement to the next (within a reasonable
tolerance) can be an important consideration. The measurement
systems disclosed above therefore provide for operation in a
positioning mode.
[0180] FIG. 3 is a flow chart 150 showing the steps involved in
operating the system in a positioning mode. The first step 152 is
to capture and store a reference image of the specimen. This step
can be performed in conjunction with a fluorescence measurement,
i.e., before or after a first fluorescence measurement in a
time-series of measurements. The reference image can be taken with
white light or another broadband source, or with a narrowband
source. The reference image can, in general, be captured in any
imaging modality, such that it records the orientation and position
of the specimen in sufficient detail so that the recorded
information can be used prior to future measurements in order to
re-position the specimen. The reference image functions as a guide
regarding the positioning and orientation of the specimen for
future measurement sessions.
[0181] In general, the captured reference image can be a gray-scale
or color image. The reference image can be further processed using
image processing techniques to highlight edges and/or significant
anatomical features to aid the operator in the future positioning
of the specimen.
[0182] The second step 154 of flow chart 150 is performed when a
subsequent measurement session with the specimen is initiated. The
reference image is recalled and used as a guide for specimen
positioning. For example, the reference image can be projected onto
a display device that is part of electronic control system 122
while the operator arranges the specimen. A live view of the
specimen can be shown beside or superimposed upon the reference
image. In some embodiments, the reference and live images may be
displayed in different colors, so that when the images are
superimposed, a third color is produced, highlighting the portions
of the two images which do not overlap. For example, the reference
image can be shown in red and the live image in green. When the two
images are superimposed, the portions where the reference and live
images overlap appear in a color that is the additive sum of the
colors of the two separate images.
[0183] Further, when measurement system 100 is configured to
capture multiple views of the specimen, both the reference and the
live images can display the multiple views, providing extra
information for the operator to aid in positioning the
specimen.
[0184] The third step 156 in flow chart 150 involves positioning
and orienting the specimen in order to match the reference image.
It may not be possible to exactly match the current specimen
position and/or orientation with the reference image for various
reasons. For example, the specimen may have grown in size during
the intervening time period. Therefore, the operator may decide
upon a matching tolerance within which specimen placement is deemed
acceptable.
[0185] This decision can be aided in the fourth step 158 of the
procedure by estimation or computation of a positioning quality
metric by electronic control system 122. The computational step is
not necessary and may be omitted in some embodiments, such as when
an operator judges goodness-of-fit visually based on a display.
When employed, electronic control system 122 may, for example, use
the reference and live images to compute a goodness-of-fit metric
to quantify the degree of alignment between the two images and the
accuracy of the positioning. The computed accuracy metric can be
displayed to the operator using a color indicator, a numerical
readout, a bar indicator, or the like. A suitable goodness-of-fit
metric can be as simple as a normalized correlation measure for the
live image and the reference image, for example. Alternatively, or
in addition, a biometric approach may be taken, wherein points of
reference such as the toes, eyes, ears, and skin folds are
identified and their locations compared, where the goodness-of-fit
metric is a mathematical function of the displacement of one or
more selected reference points between the two images.
[0186] Step 158 may further incorporate a threshold condition
indicating whether specimen positioning is sufficient for recording
new measurements. For example, electronic control system 122, based
on the results of a computed goodness-of-fit metric, can be
configured to prevent further measurements from occurring until the
specimen is sufficiently well-positioned. This feature can be used
to ensure the integrity of measured data.
[0187] In response to a computed quality metric, or simply to
visual interpretation of matching between the reference and live
images, repositioning of the specimen by the operator may be
required. In general, steps 156 and 158 illustrated in flow chart
150 can continue in cyclic fashion until the computed metric is
satisfied or the operator determines that the alignment of the
specimen is sufficiently precise.
[0188] In general, it is advantageous to record both reference and
live images of the specimen using the same imaging optical elements
(i.e., lenses, filters, detectors) in order to ensure that optical
system parameters do not change between measurements. In the event
that optical system parameters do change, however, or in the event
that the specimen size or shape changes over time, the reference
image may be warped using image processing techniques in order to
produce a reference image that more closely matches the present
condition of the imaging system and the specimen.
[0189] It may also be advantageous in some embodiments to use
structured light to record the reference and live images of the
specimen. As discussed further below, the use of structured light,
where the spatial intensity profile of the illumination source
varies, may provide additional information that can be used in
order to ensure a more optimum orientation and position of the
specimen. For example, structured light can be used to produce a
light "grid" overlaying the surface of a specimen in a reference
image. In subsequent re-positioning steps, the same grid can be
reproduced using structured light and the specimen arranged such
that the overlay of the grid on its surface matches the grid
overlay in the reference image.
[0190] In some embodiments, positioning and orientation of the
specimen is performed in a low-light environment. For example,
detector system 118 can be configured, in other operating modes, to
capture low intensity measurement signals such as fluorescence
signals. In order to prevent stray light from damaging detector
system optics, portions of measurement system 100 (or the entire
system) may be enclosed within a light-tight box. The light source
used for positioning can be chosen to be a light source that is
also used to induce fluorescence emission from the labeled
specimen. In the current operating mode, the light source provides
radiation at a wavelength that passes through an emission barrier
filter. Alternatively, in some embodiments, the emission barrier
filter can be replaced by a standard fluorescence emission filter.
In general, light source 102 provides radiation at a particular
wavelength for operation in positioning mode so that detection
system 118 measures a signal that arises from direct reflection or
transmission of illumination light, rather than fluorescence
emission by the specimen. Operation in positioning mode can further
entail a reconfiguration of light conditioning optics 106 and/or
light collecting optics 114 in order to provide for detection of
appropriate images by detector system 118. For example, filters and
other elements for manipulating the wavelength spectrum of light
can be repositioned in order to create a suitable spectral
configuration for illumination light 108.
[0191] In positioning mode, light source 102 can be a broadband
light source, i.e., a white light source, or light source 102 can
have a narrower bandwidth. Light source 102 can be a
multi-wavelength source, for example, where one of the wavelengths
is used when the system operates in positioning mode, and one or
more other wavelengths are used in other modes of operation.
Similarly, detection system 118 can include a single detector for
multiple modes of operation, including positioning mode, or
detection system 118 can include one or more detectors for
positioning mode operation, and one or more additional detectors
for other operating modes.
[0192] Light conditioning optics 106 can include elements such as
bandpass filters that are employed together with light source 102
in order to produce light used in positioning mode that is outside
the detector system's fluorescence detection region and/or does not
induce fluorescence in the specimen. For example, detector systems
configured to measure fluorescence signals can be very sensitive
due to the relatively weak intensity of many fluorescence
emissions. Positioning mode light at a wavelength in the
fluorescence measurement region of the detector system may be too
intense and may damage optical elements in the detector system.
Positioning mode light that induces fluorescence of a specimen may
produce background signal in reference images and may also produce
fluorescence emission that saturates the detector system.
Accordingly, filters such as neutral density filters can be used to
attenuate the intensity of the light incident on detector system
118. Alternatively, a less-intense light source element can be used
in light source 102 to provide the light used in positioning
mode.
[0193] Light conditioning optics 106 can farther include optical
elements designed to modify the spatial intensity profile of the
light used in positioning mode, in order to provide for structured
illumination. These optical elements are discussed in more detail
below in connection with the structured illumination mode of
operation.
Multiple Viewing Mode and Detection System
[0194] Light collecting optics 114 can be configured to capture one
or more views of a specimen, and can include an optical element
having multiple reflective surfaces, the element positioned and
oriented with respect to the specimen in order to capture the
multiple views. One embodiment of this optical element is a
four-sided pyramid 200 shown in FIG. 4. In the embodiment shown,
the pyramid is nominally a 90-degree, four faceted pyramid with
internal surfaces that function as mirrors to project four side
views of a specimen 2 to a lens and, eventually, to detector system
118. As shown in FIG. 4, the pyramid is oriented so that its axis
of symmetry and a long axis of an extended specimen coincide. A
single imaging lens 204 transfers the multiple side views 116a-d of
the specimen to detector system 118. Imaging lens 204 and detector
system 118 are nominally oriented such that they view the extended
specimen from an end-on direction, along the symmetry axis of
pyramid 200.
[0195] In some embodiments, the apex of pyramid 200 may be removed
to provide for back-illumination of the specimen. For example,
white light illumination of the specimen from a position opposite
detector system 118 can be used for profilometry or topographic
imaging of the specimen. Removal of the pyramid's apex can also
provide an access port for insertion of the specimen, for
example.
[0196] In general, the angle of pyramid 200 does not need to be 90
degrees, and could be greater or less than 90 degrees to
accommodate different measurement modes and optical elements in
light conditioning optics 106 and light collecting optics 114.
Pyramid 200 may be fabricated from any material suitable for
fabrication of optical components such as, for example, BK7 glass
or fused silica. In some embodiments, one or more surfaces of
pyramid 200 may be coated with a material in order to enhance the
reflectivity of the coated surfaces and/or to impart dichroic
properties to the coated surfaces.
[0197] Pyramid 200 may, in general, have more than 4 angled,
reflecting sides (e.g., 5 or more sides, 6 or more sides, 10 or
more sides). If pyramid 200 includes more than 4 sides, measurement
system 100 may be configured to capture more than 4 views of a
specimen, since each reflective side of pyramid 200 can capture a
view of the specimen. In general, the sides of pyramid 200 can also
be curved, and the curvature may enhance the ability of pyramid 200
to direct light toward detector system 118. In some embodiments, a
pyramid 200 having curved sides may obviate the need for one or
more imaging lenses such as lens 204 in light collecting optics
114. Curved pyramid surfaces can also provide light collecting
interfaces that correspond better to the surface of a specimen than
flat light collecting surfaces would, and can therefore provide
more sharply focused specimen views on a detector.
[0198] A general feature of embodiments of pyramid 200 is that the
multiple views 116 of a specimen captured by pyramid 200 can be
focused to a common focal plane at the position of detector system
118 by a lens or lens system such as lens 204, because the optical
path lengths of the light from each of the views 116 are about the
same due to the symmetry of the specimen position with respect to
pyramid 200. This feature provides for higher resolution imagery
and greater accuracy than would otherwise be possible in a
measurement system where the optical path lengths of multiple views
differed substantially.
[0199] In some embodiments, light conditioning optics 106 and light
collecting optics 114 can share one or more common elements. For
example, pyramid 200 can be used in both of these measurement
system components. In one aspect, the reflective sides of pyramid
200 can be used to direct illumination light from a source to be
incident on a surface of the specimen, for example. In another
aspect, the reflective sides of pyramid 200 can be used to capture
and direct multiple views of the specimen to a detector system.
[0200] Light collecting optics 114 transfer one or more views 116
to detector system 118, which captures and records each of the
views simultaneously or sequentially. Detector system 118 may also
convert each of the views to electronic signals 120. Detector
system 118, in general, can include one or more detectors. If more
than one detector is present, then in some embodiments, each one of
the detectors can be configured to capture one of the multiple
views 116 of a specimen, and each detector can be positioned in the
image plane for the particular view it captures.
[0201] In some embodiments, detector system 118 may include imaging
detectors such as CCD arrays and non-imaging photodetectors such as
photodiodes and/or photomultiplier tubes. The non-imaging
photodetectors can be used for light integrating measurement modes,
such as whole specimen integration mode (to be described
subsequently), and the imaging detectors can be used to capture
views of the specimen.
[0202] In some embodiments, detector system 118 may include a
single imaging detector such as a CCD array, and all of the
measured views of the specimen can be projected thereon by light
collecting optics 114. For example, FIG. 5 shows an embodiment
wherein a single CCD detector is used to capture four side views
116a-d and an end-on profile view 206 of a specimen.
[0203] The views may be captured simultaneously or in any
sequential arrangement. The profile view 206 can be used to provide
additional information, such as boundary conditions, to 3D
reconstruction models for the determination of specimen
morphology.
[0204] Detector system 118 can generally be configured to record
images produced from collected light that originates from different
sources and/or emission mechanisms. For example, measurements may
include projecting multiple views of a specimen onto one or more
detectors, the images derived from illumination of the specimen
with a white light or other broadband light source. Measurements
can also include fluorescence emitted by the specimen in response
to illumination light from a different light source such as a
narrowband light source, or bioluminescence emitted by the
specimen. Detector system 118 can be configured to simultaneously
record measurement signals derived from multiple-source
illumination of the specimen. Further, detector system 118 can be
configured to monitor a specimen and provide a live view of the
specimen during insertion, positioning and orientation, and removal
from the measurement system. The live view of the specimen can be
displayed for a system operator, for example, when operating in
positioning mode as discussed above.
[0205] In some embodiments, detector system 118 may further be
time-gated in order to measure temporal information from a
specimen, such as time-of-flight scattering or fluorescence
emission lifetime. This information can complement other
measurement data and further guide 3D reconstruction algorithms.
For example, static imaging measurements (i.e., with no
time-gating) can be performed using a coherent or incoherent
continuous wave (CW) light source element such as a CW laser, a
photodiode, or a lamp. Dynamic, or time-domain, measurements can be
performed, in some embodiments, using a coherent or incoherent
pulsed light source element that is temporally synchronized with an
electronic gating signal provided to detector system 118.
[0206] Electronic signals 120 corresponding to measurement signals
and views captured by detector system 118 may be further processed
using, for example, one or more mathematical algorithms implemented
in electronic control system 122 to derive information about a
specimen. In general, electronic control system 122 is
electronically coupled to detector system 118 and implements
algorithms such as 3D reconstruction algorithms to which the
measured information serves as input. Reconstruction algorithms
may, for example, use the information contained in multiple views
of a specimen to construct a model of the internal structure of the
specimen. Algorithms may use threshold or edge detection
projections, along with other known image processing techniques, in
order to improve input information or extract particular
information from measured data.
Multiple-Wavelength Illumination Mode
[0207] In another mode of operation, measurement system 100 can be
configured so that illumination light 108 provides multiple
illumination wavelengths. In optical imaging of biological
specimens, it is often useful to acquire information about
structural entities located in the interior of a specimen. For
example, information about the size and position of a tumor within
an animal such as a mouse may be useful in both the diagnosis and
treatment of disease and the testing of pharmaceutical agents. In
particular, it may be especially advantageous to establish the
position of a sub-surface entity within the specimen.
[0208] Fluorescence imaging is a useful technique for acquiring
such depth or position measurements. Sub-surface entities can be
labeled with a fluorescent moiety using either molecular biological
or chemical techniques. Excitation light from a light source is
absorbed by the fluorescent moieties, which then emit fluorescence.
In general, fluorescence emission occurs at a wavelength different
from the wavelength of the excitation light, providing a spectral
emission optical signature that is different and separable from the
excitation light source. Two proteins that can be expressed in
biological structures of interest are green fluorescent protein
(GFP) and red fluorescent protein (RFP).
[0209] Depth or position information can also be used to correct
other measured data for scattering and absorption of emitted
radiation by specimen tissues. Therefore, acquisition of this
information can also be used to improve the accuracy of other
measurements made with measurement system 100.
[0210] In general, biological tissues are turbid media that
attenuate incident light by means of scattering and absorption. The
attenuation factor scales with the thickness of the tissue, and
therefore a measurement of emitted radiation from a specimen that
includes a tissue layer, when compared with a measurement of
emitted radiation from a similar specimen without the tissue layer,
can be used to determine the tissue layer thickness. For example,
if the specimen includes a sub-surface structural entity that is
labeled with a fluorophore, measurement of the emitted fluorescence
intensity from the structural entity and comparison to a calibrated
emission standard for the same fluorophore in the absence of
specimen tissue can be used (i.e., via a calibration table) to
determine the thickness of tissue through which the emitted
fluorescence radiation has passed, and therefore the depth of the
structural entity below the surface of the specimen. If the same
measurement is performed in two or more directions, the position of
the structural entity within the specimen can be determined.
[0211] Unfortunately, radiation--either incident or emitted--also
undergoes wavelength-dependent processes on passing through
biological tissues, which change the overall spectral distribution
of the radiation. It is often difficult to predict the magnitude of
this shift at specific wavelengths for a particular sample, as
different biological tissues produce different perturbative
effects. The use of two or more incident wavelengths provides a
means to correct measurement data for tissue thickness-dependent
wavelength shifts in order to obtain better estimates of tissue
thickness using the techniques described above.
[0212] An advantage of multiple-wavelength illumination, which
provides spectral resolution in the measurement data on the
excitation side, derives from the variation of the absorption
coefficient of specimen tissues with the wavelength of radiation
incident on the tissues, as shown in FIG. 6A. In general, better
resolution in measured data is achieved when measurements are
performed in spectral regions where the absorption coefficient
varies more strongly with wavelength, such as in the region from
about 600 nm to about 700 nm. One approach to acquiring spectral
information is to provide a single source and to spectrally resolve
the emitted fluorescence. However, light emitted via fluorescence
is red-shifted relative to excitation light, and therefore, in
order for the fluorescence emission to appear in a region from
600-700 nm, for example, the excitation light has a shorter
wavelength. Unfortunately, the tissue absorption coefficient at
shorter wavelengths, such as 550 nm for example, is considerably
larger, so that the excitation light is strongly attenuated before
reaching the fluorophores. As a result, the fluorescence signal can
be relatively weak.
[0213] In contrast, by providing spectral resolution on the
excitation side via a multiple-wavelength illumination source, the
multiple source wavelengths can be selected to be in a suitable
spectral region, such as the 600-700 nm region. The emitted
fluorescence radiation will appear in a wavelength region that is
red-shifted such as, for example, the near-infrared region. As a
result, the illuminating radiation will be subject to an absorption
coefficient in specimen tissues that is smaller than, for example,
the absorption coefficient at 550 nm and as a result, radiation of
higher intensity is delivered to the specimen, producing a stronger
fluorescence signal.
[0214] Typically, in fluorescence optical imaging, tissue
autofluorescence limits measurement sensitivity. Autofluorescence
exists even when tissue is excited at near-infrared wavelengths. In
the visible region, autofluorescence often overwhelms fluorescence
emission of interest from labeled internal structures. In some
cases, however, measured emission signals from a specimen can be
acquired and used to separate signals specific to the internal
entity under study from background signals due to tissue
autofluorescence using spectral analysis tools such as linear
spectral unmixing and principal component analysis. The
autofluorescence spectrum of a typical tissue sample is shown, for
example, in the inset of FIG. 6A.
[0215] A particular advantage of multiple wavelength illumination
of a specimen is the ability to remove the autofluorescence signal
from measured data. In general, a 2D fluorescence emission image of
a specimen includes a 2D array of pixels. The fluorescence spectrum
at any spatial position (i.e., any pixel location) in the image is
given, to a first approximation, by a weighted linear sum of a
fluorescence emission spectrum from a labeled entity of interest
within the specimen and an autofluorescence spectrum of the
specimen. If the two basis functions, i.e., the pure fluorescence
emission spectrum from the labeled entity and the pure
autofluorescence spectrum of the specimen are known, then a simple
matrix inversion can be used to determine the weighting
coefficients, and the autofluorescence spectrum can be
mathematically subtracted from the measured spectrum at each
pixel.
[0216] In some cases, only one of the basis functions--usually the
pure autofluorescence spectrum--may be known. Spectral unmixing
techniques can still be applied, even on a pixel-by-pixel basis, to
the measured data. One procedure for unmixing label fluorescence
and autofluorescence involves subtracting multiples of the
autofluorescence spectrum from the measured fluorescence spectrum,
until the lowest spectral intensity at any wavelength in the
measured spectrum reaches zero. The difference spectrum that
remains represents the pure fluorescence spectrum of the labeled
entity.
[0217] Under some conditions, neither of the fluorescence component
basis functions are known. Spectral unmixing techniques used in
these situations are disclosed, for example, in U.S. patent
application Ser. No. 10/669,101 entitled "SPECTRAL IMAGING OF DEEP
TISSUE" by Richard M. Levenson et al., filed on Sep. 23, 200, and
in PCT Patent Application PCT/US2004/0316 entitled "SPECTRAL
IMAGING OF BIOLOGICAL SAMPLES" by Richard M. Levenson et al., filed
on Sep. 23, 2004 and published as WO 2005/040769. Both of the
preceding applications are incorporated herein by reference.
[0218] FIG. 6B is a plot showing the results of a simulation of the
spectral shapes of various excitation and emission bands involved
in a typical multiple wavelength excitation mode fluorescence
measurement. The simulation uses the fluorescent label Alexa Fluorm
610 (Molecular Probes, 29851 Willow Creek Road, Eugene, Oreg.
97402). Curve 207a shows the wavelength dependence of the tissue
absorption coefficient. Curves 207b and 207c show the absorption
and emission bands of the label, respectively, with the emission
band red-shifted relative to the absorption band. Curves 207d and
207e show the wavelengths and bandwidths of the two excitation
sources used in the simulated measurement. The wavelengths of the
two excitation light sources are separated by about 50 nm. Curve
207f shows the combined fluorescence emission signal from the label
due to excitation at both wavelengths (i.e., with sources having
spectral bandshapes 207d and 207e) simultaneously. The emission
signal 207f is centered in a region of the spectrum where tissue
absorption is relatively weak, and therefore the intensity of
fluorescence band 207f can be relatively high in some
embodiments.
[0219] FIG. 7 is a flow chart 160 that summarizes steps in
measuring the depth or position of a structural entity, such as a
structure labeled with fluorophores in a specimen, using a multiple
wavelength illumination source. The first step 162 includes
measurement of a fluorescence spectrum from the specimen in
response to excitation light at a first wavelength. In an optional
second step 164, the autofluorescence spectrum may be removed from
the fluorescence spectrum measured in first step 162 in order to
correct the measured data. The third step 166 includes measuring a
fluorescence spectrum from the specimen in response to excitation
light at a second wavelength different from the first wavelength,
and may be performed in sequence with first step 162. The fourth
step 168, which is also optional, includes correction of the data
measured in step 166 by removing the component of the measured
spectrum that arises from tissue autofluorescence. In a fifth step
170, the two fluorescence signals are compared to produce a
spectrum of relative fluorescence strength.
[0220] In general, a number of different techniques can be used in
step 170 to compare and/or combine the fluorescence spectra
obtained at two or more different excitation wavelengths. Using the
example shown in flow chart 160 for two distinct excitation
wavelengths, two corrected spectra are obtained as a result of the
steps shown: I.sub.c(.lamda..sub.I), the corrected fluorescence
emission spectrum (i.e., with specimen autofluorescence removed)
for excitation at wavelength .lamda..sub.I; and
I.sub.c(.lamda..sub.2), the corrected fluorescence emission
spectrum for excitation at wavelength .lamda.. The ratio of the
spectral intensities at a specific wavelength .lamda. is denoted
I.sub.c(.lamda..sub.I; .lamda./I.sub.c(.lamda..sub.2; .lamda.). The
ratio of the spectral intensities in a pure sample of the
fluorescent label excited at wavelengths .lamda..sub.I and
.lamda..sub.2 (i.e., without any intervening scattering medium such
as tissue) is generally known or can be measured, and provides a
reference ratio denoted as I.sub.r(.lamda..sub.I;
.lamda./I.sub.r(.lamda..sub.2; .lamda.). Step 172 then involves
comparing the measured spectral intensity ratio
I.sub.c(.lamda..sub.i.lamda.)/I.sub.c(.lamda..sub.2;.lamda.) to the
known reference spectral intensity ratio I.sub.r(.lamda..sub.I;
.lamda.)/I.sub.r(.lamda..sub.2; .lamda.) in order to obtain a
measurement of the depth of the emitting structure below the
surface of the specimen or, in other words, a tissue thickness. In
some embodiments, for example, step 172 includes taking a ratio of
the quantities I.sub.c(.lamda..sub.1;
.lamda./I.sub.c(.lamda..sub.2; .lamda.) and I.sub.r(.lamda..sub.I;
.lamda./I.sub.r(.lamda..sub.2; .lamda.) for purposes of comparison.
A look-up table or other means such as a mathematical algorithm can
be used to transform the quantity I.sub.c(.lamda..sub.I;
.lamda.)/I.sub.c(.lamda..sub.2; .lamda.) into a thickness. The
measured and reference spectral intensity ratios may be compared at
a single wavelength .lamda. in order to determine a thickness, or
the intensity ratios at a number of selected wavelengths, or even
at all measured wavelengths in the spectra, may be compared and
averaged to obtain an estimate of tissue thickness. In some
embodiments, the properties of the specimen may dictate that
particularly accurate measurements are obtained by considering the
intensity ratios at a known subset of specific wavelengths, and
therefore the measured and reference intensity ratios at these
wavelengths may be preferentially selected for comparison in order
to estimate tissue thickness.
[0221] Frequently, depth or thickness measurements derived from
combining spectral data obtained at multiple excitation
wavelengths, as shown in step 170, are more accurate than
measurements derived from single excitation wavelength fluorescence
emission. The sequence of steps in flow chart 160 can be further
repeated for illumination along two additional directions, each
orthogonal to the other and orthogonal to the first illumination
direction, in order to determine the 3D position of the structural
entity within the specimen.
[0222] In general, the measurement procedure shown in FIG. 7 can
include more than two illumination wavelengths. For example, three
or more wavelengths (e.g., four or more wavelengths, five or more
wavelengths, ten or more wavelengths) can be used, and the measured
fluorescence signals due to excitation at each of the excitation
wavelengths may be combined in any desired manner in step 170 in
order to provide more accurate depth or tissue thickness
measurements. The excitation wavelengths may further be chosen to
be as far apart as desired, provided excitation light at each
wavelength induces a measurable fluorescence emission signal in the
specimen.
[0223] Measurement system 100 can be configured to provide for
multiple wavelength illumination of a specimen under study. Light
source 102 can include, for example, two different light source
elements, configured to produce light at different selected
wavelengths, where the wavelengths are chosen to provide accurate
measurement signals. In some embodiments, light source 102 can
include light source elements that provide light at three or more
excitation wavelengths (e.g., four or more wavelengths, five or
more wavelengths, ten or more wavelengths). The light source
elements that provide the excitation wavelengths can be separate
elements, each configured to provide light at a chosen wavelength.
Alternatively, in some embodiments, the light source can include a
single broadband light source element, and light conditioning
optics 106 can include a series of optical elements configured to
produce excitation light having different wavelength components.
For example, light conditioning optics 106 can include active
filter elements such as liquid crystal tunable filters, passive
filter elements such as bandpass filters, and beam directing optics
such as beamsplitters, mirrors, and the like. In some embodiments,
for example, the light conditioning optics 106 and light collecting
optics 114 may share some common optical elements; that is, the
optical paths traversed by the excitation light and by the emitted
light (i.e., fluorescence) may be partially collinear, the two
paths separated eventually by an element such as a dichroic
beamsplitter. Such a configuration is referred to as
epi-fluorescence measurement. In other embodiments, the optical
paths of the excitation light and the emitted fluorescence are not
collinear. Light collecting optics 114 can be configured to capture
one or more views of the specimen under study, and can include, for
example, imaging elements such as lenses and light gathering optics
such as pyramid 200.
Multiple Wavelength Emission Mode
[0224] Measurement system 100 can also be configured to operate in
a multiple wavelength emission mode. As discussed above in
connection with the multiple wavelength illumination mode,
radiation such as fluorescence emitted from a specimen can be
captured and spectrally resolved in order to provide an estimate of
the depth of an emitting structural entity below the surface of a
specimen or, in three dimensions, the internal position of the
entity within the body of the specimen.
[0225] Typically, fluorescence imaging techniques estimate
thicknesses of scattering tissues by measuring an attenuation
factor for emitted fluorescence radiation and comparing the
intensity of the emitted radiation to the known intensity for the
fluorescent label of interest in the absence of scattering tissues.
However, emitted radiation such as fluorescence also undergoes a
wavelength shift on passing through scattering media such as
specimen tissues, and because the absorption coefficient of the
tissue is wavelength dependent, tissue thickness-induced wavelength
shifts can produce errors in tissue thickness estimates that arise
from uncompensated variations in the absorptive properties of the
tissues.
[0226] Multiple wavelength emission mode provides a means for
correcting measured data to account for wavelength dependent
absorption properties of biological tissues. As discussed
previously, fluorescent labels or moieties can be introduced into a
specimen using molecular biological or chemical means and localized
in an internal structural entity of interest. In the present mode
of operation of system 100, multiple different fluorescent labels
of interest are introduced into the structural entity, each label
type having a different emission band. When the specimen is
illuminated with light from a source, fluorescence emission is
induced from each of the fluorescent labels and is captured using
light collecting optics 114 and detector system 118.
[0227] The fluorescence emission signals at multiple emission
wavelengths provide complementary data concerning the thickness of
tissues through which the fluorescent light has passed. When the
data are combined in order to correct for wavelength dependent
absorption properties of specimen tissues, a more accurate estimate
of tissue thickness than would otherwise result from using only a
single distinct fluorescent label may be obtained. When only one
type of fluorescent label is introduced into the structural entity
of interest, the differential absorption between two different
wavelengths in the emission band of the label is usually too small
to realize accurate depth measurements. Corrections to depth
measurements that are based on determining ratios of fluorescence
emission signals at different wavelengths are not as effective
because the wavelengths must necessarily both fall within the
emission band of the fluorescent label. In contrast, when multiple
fluorescent labels are used, the labels can be chosen such that
their emission bands are spectrally separated (e.g., 75 nm apart,
100 nm apart, 150 nm apart, 200 nm apart, 500 nm apart). For such a
large separation in emission wavelengths, the differential tissue
absorbance at the emission wavelengths is typically much larger
than in singly-labeled specimens, so that corrections to depth
measurements that are based on ratios of fluorescence emission
signals are more effective, and depth measurements are more
accurate.
[0228] The present mode of operation permits any number of distinct
fluorescent labels to be introduced into the specimen. For example,
structural entities within the specimen can be labeled using two or
more distinct fluorescent moieties (e.g., three or more distinct
fluorescent moieties, five or more distinct fluorescent moieties,
ten or more distinct fluorescent moieties). As an example, where
two distinct fluorescent moieties are introduced into a specimen,
the two fluorescent labels may be bound to or expressed by the same
target location (i.e., a structural entity of interest) in known
proportion. A fluorescence label mixture can be engineered to have
spectral fluorescence characteristics tailored to take advantage of
spectral variations of scattering efficiency and tissue absorption
in order to accurately determine the depth or position of the
structural entity.
[0229] FIG. 8 is a schematic diagram showing wavelength dependent
attenuation of emitted fluorescence signals from a specimen. A
light source (not shown) is first used, on the left hand side of
the figure, to illuminate a structural entity such as a tumor
located at a relatively shallow depth below the surface of the
specimen. The tumor is labeled with three different fluorophores
having fluorescence emission bands centered at 500 nm, 625 nm, and
750 nm, respectively. As discussed previously, absorption by
specimen tissues varies according to wavelength and is typically
stronger at shorter wavelengths. For a shallow tumor emitting
fluorescence 186 at each of these three wavelengths, the
attenuation of the fluorescence intensity due to tissue absorption
at each wavelength is relatively small. Band 180a at 500 nm is not
attenuated to a significantly larger extent than either band 182a
at 625 nm or band 184a and 700 nm. Illumination of a deep tumor,
located well below the surface of the specimen as shown on the
right hand side of the figure, induces emission of fluorescence
188. After propagating through a relatively larger thickness of
tissue, the intensity of band 180b at 500 nm is more strongly
attenuated than the intensity of band 182b at 625 nm or band 184b
at 700 nm, due to the larger tissue absorption coefficient at
shorter wavelengths. It is clear from the relative intensities of
the fluorescence emission bands that depth estimates based on only
a single emission wavelength will vary depending on the chosen
wavelength due to the wavelength dependent absorption properties of
specimen tissues.
[0230] FIG. 9 is a flow chart 190 that shows a series of
measurement steps for making multiple wavelength emission
measurements to determine the depth or position (i.e., tissue
thickness) of a light emitting structural entity within a specimen.
In a first step 192, a specimen labeled with multiple different
types of fluorescent labels is illuminated with light from a
source, inducing fluorescence emission from the labels. The labels
are typically chosen such that the maxima in their respective
emission bands are separated from one another spectrally. The
second step 194 includes measurement of the simultaneous
fluorescence emission signals from each of the distinct labels.
Step 194 provides a total fluorescence spectrum, I.sub.I(.lamda.),
that includes the fluorescence emission spectra of each of the
fluorescent labels scaled by the tissue absorption coefficient,
.sigma.(.lamda.). To a first approximation, the total fluorescence
emission spectrum for three distinct labels 1, 2, and 3 having
fluorescence emission maxima at .lamda..sub.I, .lamda..sub.2, and
.lamda..sub.3, respectively, may be written as
I.sub.t(.lamda.)=.sigma.(.lamda..sub.2)+.sigma.(.lamda..sub.2)
I.sub.2(.lamda.)+.sigma.(.lamda.) I.sub.3(.lamda.). In addition, a
reference fluorescence spectrum corresponding to emission from the
three labels in the absence of scattering tissue can be written as
I.sub.r(.lamda.)=I.sub.1(.lamda.)+I.sub.2(.lamda.)+I.sub.3(.lamda.).
The third step 196 includes comparison of the measured multiple
wavelength emission signal I.sub.t(.lamda.) with the reference
signal I.sub.r(.lamda.). For example, the two signals may be
ratioed at selected wavelengths or at all wavelengths in the
spectra in order to produce a reduced multiple wavelength emission
signal. If the two signals are ratioed or otherwise compared at
multiple wavelengths, the results may be averaged, for example.
[0231] From the expressions for I.sub.t(.lamda.) and
I.sub.r(.lamda.), it is evident that using a larger number of
fluorescence labels in a specimen may provide more accurate
measurement results, because the spectral intensity of the total
measured fluorescence signal will have a broader distribution of
high intensity regions.
[0232] The fourth step 198 includes providing an estimate of the
depth of the entity below the surface of the specimen or,
alternatively, the thickness of tissue through which the
fluorescence emission signals have propagated, based on the
comparison between the total measured and reference fluorescence
emission signals (e.g., an averaged ratio of the two signals). The
depth estimate can be obtained, for example, from a calibrated
look-up table or from a mathematical algorithm that relates the
ratio of the fluorescence signals to a calibrated depth
measurement. The measurement cycle shown in flow chart 190 can be
repeated from two additional, mutually orthogonal directions in
order to determine a 3D position of the structural entity within
the body of the specimen.
[0233] In order to provide an accurate calibrated depth
measurement, some information regarding the concentration ratio of
the various fluorescent labels in the specimen should be known.
Once this knowledge is gained, small changes over time in the
concentration ratios of the fluorescent labels have relatively
little effect on measurements of depth, which are typically much
more sensitive to tissue thickness.
[0234] In some embodiments, fluorophores are chosen to provide
fluorescence emission signals that, when ratioed, either increase
or decrease monotonically as a function of tissue thickness. This
is helpful in avoiding depth determination ambiguities which might
otherwise result from the dependence of the tissue absorption
coefficient as a function of wavelength.
[0235] In some embodiments, at least one fluorescent label
introduced into the specimen can have a fluorescence emission band
centered at about 980 nm. A label having fluorescent emission near
980 nm can be used to determine the thickness of specimen tissues
containing significant quantities of water.
[0236] In general, the techniques described in connection with
multiple wavelength illumination mode can also be used in
conjunction with multiple wavelength emission mode. For example,
multiple distinct fluorescent probes can be introduced into the
specimen, and multiple excitation wavelengths can be used to induce
fluorescence at each of the emission wavelengths of the
fluorophores. This may provide complementary information that can
be used to further refine depth/thickness estimates, for example.
In addition, the combination of both excitation- and emission-side
spectral resolution may permit autofluorescence removal from the
measured data, as discussed previously.
[0237] Measurement system 100 can be configured to provide for
operation in multiple wavelength emission mode. In particular,
light collecting optics 114 can include, for example, active
spectral filtering elements such as liquid crystal tunable filters
and/or passive filtering elements such as spectral bandpass
filters, along with other optical elements for collecting and
directing emitted light, such as dichroic beamsplitters, spectrally
neutral beamsplitters, mirrors, and the like.
Whole Specimen Integration Mode
[0238] Most 3D non-optical imaging techniques provide 3D internal
structure information in a specimen under study by resolving
physical or structural variation. The volumes and physical
dimensions of internal structures are often derived from detected
structural boundaries between regions internal to the specimen that
have specific differences in their physical characteristics.
[0239] Fluorescent labels specific to a particular structural
feature of interest in a specimen, such as a tumor, can be readily
introduced using previously discussed techniques. In the present
mode of operation, under appropriate conditions, the total
fluorescence radiation intensity emitted by a labeled structure can
be correlated with the mass of the structure. For example,
measurements of total fluorescence intensity from a tumor in a
specimen such as a mouse can be used to estimate the mass of the
tumor. In a measurement configuration designed to measure total
fluorescence intensity, therefore, the specimen can act as an
integrating sphere for emitted radiation.
[0240] Experimental work has shown that the mass of subcutaneous
tumors located near the surface of a specimen can be tracked
quantitatively by integrating the fluorescence emission signal on a
single 2D image of the tumor, see for example F. E. Dieln et al.,
"Noninvasive fluorescence imaging reliably estimates biomass
In-Vivo", Biotechniques 33: 1250-1255 (2002), the contents of which
are incorporated herein by reference. In these situations, for
example, the tumor is located a fraction of a millimeter from the
specimen surface, and almost all of the emitted fluorescence
escapes from the specimen in the immediate vicinity of the tumor,
thereby facilitating detection using a single 2D image.
[0241] In general, however, if the structural entity of interest
(i.e., a tumor) is positioned deeper inside the body of a specimen,
single-view 2D imaging is often much less effective due to
scattering of fluorescence emission in multiple directions. For
example, in orthotopic tumors, fluorescence radiation escapes in
all directions in difficult-to-predict ways due to inhomogeneous
scattering, and the apparent integrated fluorescence signal derived
from a single 2D image depends upon the angle of specimen
observation.
[0242] In order to use emitted fluorescence to accurately measure
the mass of a tumor or other labeled structural entity, the total
emitted radiation flux from the tumor or entity should be nearly
directly proportional to the mass of the tumor or entity. In
addition, fluorescence at the measurement wavelength should
generally be specific to the structure of interest, i.e., no other
internal structures should act as significant sources of emitted
light at the measurement wavelength. Further, the excitation and
emission wavelengths should be selected so that absorption of the
incident and emitted radiation by specimen tissue is sufficiently
non-perturbative that these effects can be overlooked. Fluorescent
moieties which satisfy these conditions include, for example,
Cyanine dyes (Amersham Biosciences Corp., 800 Centennial Ave.,
Piscataway N.J. 08855-1327) and Alexa dyes (Molecular Probes, 29851
Willow Creek Road, Eugene, Oreg. 97402).
[0243] Removal of fluorescence intensity due to specimen
autofluorescence can be especially important when operating in
whole specimen integration mode, because the entire surface of a
specimen will contribute a background autofluorescence signal,
whereas fluorescence emission derived from a labeled structural
entity is confined to a much smaller spatial volume region.
Autofluorescence removal can be performed using the spectral
decomposition techniques discussed previously, or using other
non-spectral methods such as time-gating of the detector
system.
[0244] Whole specimen integration mode, or whole mouse integration
mode (when the specimen of interest is a mouse) can provide a speed
advantage over other measurement techniques such as 3D structure
determination via computational models. In whole specimen
integration mode, the total emitted radiation flux from a
particular view of the specimen can be measured without spatially
resolving the intensity distribution. Thus, a single intensity
index is obtained for each view of the specimen. Time-consuming 3D
reconstruction algorithms are not employed to process measured
fluorescence data.
[0245] In addition, scattering of fluorescence emission by specimen
tissues poses fewer problems than in spatially-resolved measurement
modes. Since emitted light from all regions of the specimen is
integrated by the detector system, scattering events in tissue,
which typically redistribute light in space, have a relatively
small impact on the accuracy of the measured fluorescence
intensity. Only if scattering events in tissues increase the
average optical path length to the degree that tissue absorption
becomes important will scattering pose a significant problem.
[0246] Measurement system 100 can be configured to operate in whole
specimen integration mode. For example, detector system 118 can
include one or more imaging detectors such as CCD cameras for
capturing multiple views of specimen fluorescence. The spatially
resolved fluorescence images captured by the detector system may
each be integrated to provide a single integrated fluorescence
intensity measurement for each captured view of the specimen.
Alternatively, detector system 118 can include non-imaging
photodetectors such as photodiodes and/or photomultiplier tubes for
measuring integrated fluorescence from multiple views of a
specimen. Light collecting optics 114 can include one or more
lenses positioned to direct emitted fluorescence onto the active
areas of such photodetectors.
[0247] In some embodiments, measurement system 100 can be
configured to monitor a single view of a specimen and record a
measurement of the total emitted fluorescence intensity measured in
the monitored view. In other embodiments, measurement system 100
can be configured to monitor multiple views of the specimen.
Integrated fluorescence intensity measurements from the multiple
views can be combined to give a measurement of the total integrated
flux of fluorescence from the entire surface of the specimen. This
total integrated flux measurement can be used to determine, for
example, a mass of a fluorescent entity internal to the specimen
and giving rise to the measured fluorescence. In many cases, the
mass of the entity determined using multiple views of the specimen
is more accurate than a mass determined using only a single view of
the specimen.
[0248] In some embodiments, multiple viewing mode can be used to
collect multiple views of a fluorescing specimen, and the
fluorescence intensity in each of the views can be integrated. For
example, pyramid 200 in FIG. 4 can be used to collect fluorescence
emission from multiple sides of a specimen. The images of the
specimen can be directed to an array detector such as a CCD,
imaged, and integrated electronically, for example. Alternatively,
the light from each of the multiple views can be collected using
mirrors, lenses, and other similar optical elements, and be
directed onto a non-imaging detector such as a photodiode or a
photomultiplier tube that produces a single measurement of the
integrated fluorescence intensity.
[0249] Another embodiment of a measurement system 340 for
performing integrated fluorescence measurements by collecting
fluorescence emissions from multiple sides of a specimen is shown
in FIG. 10. System 340 includes a reflecting surface 342 that
directs illumination light 108 onto the surface of a specimen 2. A
labeled entity 3 within specimen 2 emits fluorescence in all
directions in response to illumination light 108. Some fluorescence
rays, such as ray 346, are emitted in a direction that is
substantially opposite to the direction of incidence of
illumination light 108, and are directed by surface 342 to a
detector system (not shown). Other rays, such as ray 348, are
directed by reflecting surface 344 toward surface 342, and
thenceforth to a detector system. In this embodiment, individual
views of the specimen are not resolved, and all of the collected
fluorescence emission is directed to a non-imaging detector. In
general, system 340 can be a two dimensional system as shown in
FIG. 10, or system 340 can be a three dimensional system in which
surface 342 is one angled surface of a reflective pyramid such as
pyramid 200, and surface 344 is a reflective surface of a concave
mirror having a spherical, paraboloidal, or other suitable shape
for collecting emitted fluorescence radiation. Either
two-dimensional or three-dimensional systems can be configured to
collect fluorescence radiation from multiple sides of a
specimen.
[0250] As discussed previously, the measurements of integrated
fluorescence intensity for each of the monitored views can be
corrected using data obtained from measurement system 100 operating
in another measurement mode. For example, depth/thickness
measurements obtained in multiple wavelength illumination mode or
in multiple wavelength emission mode can be used to calculate a
correction factor to apply to integrated fluorescence intensity
measurements in order to compensate for absorption of fluorescence
radiation by specimen tissues.
[0251] In many applications, a sufficiently accurate estimate of
tumor mass (or, more generally, the mass of a structural entity)
can be obtained by measuring a representative portion of the
emitted flux of fluorescence photons from a labeled specimen. It is
generally not necessary to capture every emitted photon. Steps for
estimating the mass of a labeled tumor in a specimen such as a
mouse are shown on flow chart 350 in FIG. 11. Steps 352, 354, 356
and 358 include measuring the total emitted fluorescence intensity
in four different views of the specimen. The integrated
fluorescence intensities from each of the views are combined in
step 360 to produce a representative fluorescence emission index
for the specimen. The fluorescence emission index is then used to
estimate the mass of the labeled tumor. The combination of the
fluorescence intensities from each of the views can be performed
according to a formula or computer algorithm. For example, the
total fluorescence intensities from each of the views can simply be
added together to produce an overall total fluorescence intensity
index. In some embodiments, scaling factors based on depth
measurements performed using other operating modes of the
measurement system can be used to produce a scaled linear
combination of the integrated fluorescence intensities from each
view of the specimen. Similarly, the determination of tumor mass
from the index can be performed using a look-up table or a
mathematical algorithm, for example.
[0252] One or more calibration steps can be performed initially and
at selected intervals in order to correlate the fluorescence index
with tumor mass. For example, in an initial calibration step, a
fluorescence index can be determined for a particular specimen, and
the specimen can subsequently be sacrificed in order to measure the
mass of a labeled tumor directly. Further calibration steps can be
performed periodically to validate and/or enhance the relationship
between the predictive measured optical signal and the biological
mass under study.
Structured Illumination Mode
[0253] Measurement system 100 can also provide an illumination
light intensity profile that is either uniform or structured (e.g.,
a spatially varying illumination intensity profile and/or an
illumination profile that is controlled in time sequence) at the
specimen position. This provides different means to obtain
structural and optical information from the specimen, the
information serving as input to 3D reconstruction algorithms. The
light source used for structured fluorescence excitation can
include one or more light source elements such as conventional
lamps, LEDs, or lasers, for example. The illumination light can be
delivered to the specimen directly or via fiber optics, dichroic
beamsplitters, light pipes, diffusers, or any other optical device
to transport and/or condition the light.
[0254] For example, in some structured illumination modes of
operation, different sides of a specimen can be illuminated in a
chosen sequence. Moreover, each side of the specimen can be
illuminated with a patterned light intensity profile, or with a
sequence of light intensity patterns. Even a single light intensity
pattern, used to illuminate one or more sides, can be used to
realize the benefits of structured illumination. In general,
structured illumination mode provides for either simultaneous
illumination of a specimen with a structured light source, or
direction-sequential illumination with a structured light
source.
[0255] In a first aspect, structured illumination of a specimen can
be provided by multiple light source elements arranged in a desired
configuration around the specimen. For example, FIG. 12 shows an
embodiment of a measurement system 100 that includes a source 102
configured to direct light 104 to a lens 431, which directs the
light to reflect from a dichroic beamsplitter 430 and to pass
through an imaging lens 433. The light is divided into two
counter-propagating portions by mirror 440. One of the portions is
directed to reflect from mirror 442, pass through lens 434, reflect
from a first surface 200a of pyramid 200, and impinge upon specimen
2 from above in the plane of the figure. The second portion of the
illumination light is directed to reflect from mirror 442, pass
through lens 439, reflect from a second surface 200b of pyramid
200, and impinge on the specimen from below in the plane of the
figure. The structured illumination provided by the two beams
induces fluorescence in the specimen. Portions of the emitted
fluorescence retrace the optical paths of the excitation beams to
beamsplitter 430. Due the red-shift of the emitted fluorescence,
the fluorescence radiation is transmitted through dichroic
beamsplitter 430 and is imaged by lens 432 as two different views
116 of specimen 2. The two views are captured by detector system
118, which includes a CCD array. The embodiment shown in FIG. 12 is
an epi-fluorescence measurement system, and the incident light and
the emitted fluorescence encounter several optical elements common
to the optical path of each.
[0256] FIG. 12 shows a two-dimensional projection of a
three-dimensional measurement system. Therefore, optical elements
that are not positioned in the plane of the figure are not
depicted. For example, other surfaces of pyramid 200 are not shown
in figure - these are used to capture other views of the specimen.
In general, other light source elements, mirrors, beamsplitters,
and other optical elements can also be present. For example, a
second set of light conditioning and collecting optics can be used
to capture two additional views of the specimen propagating into
and out of the plane of FIG. 12. In some embodiments, the
measurement system can be only two-dimensional, however, as
depicted. Further, in some embodiments, surfaces 200a and 200b can
be two surfaces of a mirror, or they can be the surfaces of two
separate mirrors. In general, many combinations of optical elements
can be provided in order to capture a two- or three-dimensional set
of views of specimen 2. The foregoing discussion applies as well to
the embodiments of FIGS. 13-16, any of which may be configured to
operate in a two-dimensional or three-dimensional imaging
modality.
[0257] Another embodiment of a measurement system that provides a
structured illumination source is shown in FIG. 13. In this
embodiment, light provided by optical fiber bundles 510 arranged
around specimen 2 is used to illuminate the specimen and induce
fluorescence. The emitted fluorescence is collected by a series of
optical elements that are similar to those of FIG. 12. Since the
illumination and fluorescence radiation do not share a common
optical path, the embodiment of FIG. 13 is an example of a
non-epi-fluorescence measurement system. In this embodiment, for
example, the fiber bundle source elements 510 can be positioned to
illuminate different sides of specimen 2 via the reflective
surfaces of pyramid 200. Further, each of the source elements 510
can be selectively enabled to provide direction-sequential
illumination when desired, or the sources may all be simultaneously
enabled.
[0258] FIG. 14 shows another embodiment of a measurement system in
which optical fiber bundles 510 are used together with dichroic
beamsplitters 610 to direct structured illumination light onto
specimen 2 from selected directions. Fluorescence radiation emitted
by specimen 2 in response to the illumination light is red-shifted
and is therefore transmitted by beamsplitters 610 and eventually
detected by detector system 118. The optical fiber bundle source
elements 510 may all provide illumination light simultaneously, or
source elements 510 may be turned on and off to provide
direction-sequential illumination of specimen 2.
[0259] Yet another embodiment is shown in FIG. 15, wherein light
from fiber bundles 510 is transmitted through dichroic
beamsplitters 710 and directed by surfaces 200a and 200b of pyramid
200 to impinge upon specimen 2. Fluorescence emitted by specimen 2
is reflected by dichroic beamsplitters 710 and captured as multiple
views 116 of specimen 2 by detector system 118.
[0260] In a second aspect, structured illumination may be provided
by combining some or all of the light source elements in an
illumination system with one or more additional optical elements
configured to modify the spatial intensity distribution of the
illumination light source elements. For example, in FIG. 13, each
of the fiber bundle source elements 510 may be used in combination
with image-forming optics and one or more optical elements such as
diffractive elements, spatial masks, or spatial light modulators
(e.g., MEMS digital light processors, liquid crystal modulators),
or the like in order to modulate the spatial distribution of light
emanating from each of the fiber bundles. Source element
interference effects can also be used to produce a composite
illumination source having a modulated spatial intensity profile.
Further, this aspect can be combined with the previous structured
illumination and direction-sequential aspects to produce an
illumination source that provides structured and
direction-sequential illumination, wherein individual source
elements in a structured and/or direction-sequential source can
have an output intensity profile with a chosen modulation.
[0261] In some embodiments where a passive or active optical device
is used to modulate the intensity profile of a light source
element, the surface of the optical device can be imaged by one or
more imaging lenses onto the surface of a specimen. For example,
FIG. 16 is a schematic diagram of a system that provides for
structured illumination of specimen. Many of the elements of FIG.
16 are similar to those of FIG. 12. In addition, FIG. 16 includes a
spatial light modulator 532 positioned and configured to modify the
spatial intensity profile of light 104 provided by source 102.
Lenses 530, 431, 433, 434, and 439 are positioned to image the
surface of spatial light modulator 532 onto specimen 2, providing
for configurable, patterned illumination on multiple sides of the
specimen. In other embodiments, spatial light modulator 532 can be
positioned in another imaging relationship to the specimen, such as
in a conjugate image plane.
[0262] Generally, two- or three-dimensional implementations of the
embodiments shown in FIGS. 13-15 can also include one or more
spatial light modulators, masks, diffractive optics, or other
devices that modify the spatial intensity profile of one or more
light source elements. The surfaces of these devices can also be
imaged by a set of imaging lenses onto the surface of a specimen,
as described above for FIG. 16, or the devices can be positioned in
another imaging relationship to the specimen, such as in a
conjugate image plane.
[0263] In general, structured illumination modes of operation can
be used to acquire additional information about a specimen that is
not available using a standard unmodulated illumination source.
Structured illumination can be used to illuminate a specimen from a
single direction or from any number of multiple directions,
simultaneously or in sequential fashion. Structured illumination
can further be used in combination with any of the measurement
modes discussed previously in order to extract additional
information about the specimen under study. For example, structured
illumination can be used to provide profile or topographic
information about a specimen by illuminating selected regions of
the surface of the specimen. In some embodiments, illumination
light is focused by light conditioning optics 106 to a spot having
a small diameter (relative to the size of the specimen) in a focal
plane positioned to coincide with the surface of the specimen. The
position of the focal plane can then be adjusted by reconfiguring
light conditioning optics 106 or by translating the specimen using
illumination stage 110. By capturing multiple images of the
illumination spot on the specimen surface for different focal plane
positions, the position of "best focus" can be measured since the
depth of focus of light conditioning optics 106 is limited. As an
example, the position of best focus may correspond to a focal plane
position that produces a smallest measured spot diameter on an
image of the specimen surface. By performing similar measurements
at other positions on the surface of the specimen, a series of
"best focus" focal plane positions is determined, and these
correspond to a surface topographic map or height profile of the
specimen.
[0264] In addition, single or multiple views that include radiation
emitted from the specimen, such as fluorescence radiation, in
response to structured illumination can be captured by detector
system 118.
[0265] Structured illumination patterns can include, for example,
arrays of illumination points overlaying the surface of a specimen,
a single illumination point scanned over the surface of a specimen,
an illumination grid, and in general, any other desired structured
illumination pattern. In general, an operator of measurement system
100 can selectively illuminate the entire surface of specimen 2 or
any portion thereof, in simultaneous fashion or in a
direction-sequential modality.
[0266] Depth and/or tissue thickness information for labeled
sub-surface structural entities internal to a specimen can be
obtained via structured illumination in the same manner as for the
multiple wavelength illumination and multiple wavelength emission
measurement modes discussed previously. For example, illumination
of a specimen in a selected region, followed by measurement of
specimen fluorescence by capturing multiple specimen views 116
using light collecting optics 114 and detector system 118, can be
used in order to determine the amount of tissue through which
emitted fluorescence radiation propagates in each of the views,
thereby establishing the internal position of the illuminated
portion of the structural entity of interest.
[0267] Alternatively, or in addition, direction-sequential
structured illumination in selected regions of the specimen surface
and imaging of the emitted fluorescence or scattered light can be
used together with turbid media scattering models to determine
surface topographic features of the emitting or scattering
entity.
[0268] FIG. 17 is a flow chart 800 showing steps involved in a
structured illumination measurement, where the information gained
is used to as input to a 3D reconstruction model. In a first step
802, a specimen having one or more fluorescence-labeled internal
structural entities of interest is illuminated on a chosen first
side using a structured illumination source. In step 804,
fluorescence radiation emitted by the labeled specimen is collected
from multiple sides of the specimen using light collecting optics
114 and detector system 118. The light collecting optics can
include, for example, a multi-faceted pyramid such as pyramid 200,
along with lenses, mirrors, and other optical elements. The light
from multiple sides of the specimen can be imaged as a set of views
of the specimen on a CCD detector, for example. The information
contained in the multiple fluorescence images is used as input to a
3D reconstruction algorithm in step 806, where the algorithm
produces a 3D model of the internal structure of the specimen. In
order to further refine the model, in step 808, a second side
different from the first side is selected and illuminated, and
fluorescence radiation emitted by the specimen due to illumination
on the second side is collected on multiple sides of the specimen.
The information in the new set of fluorescence images is extracted
and used as input to the 3D reconstruction algorithm in step 810 to
generate corrections to the 3D model of the specimen's structure.
These corrections are used in step 806 to generate an improved 3D
specimen model. Steps 808, 810, and 806 then continue in cyclic
fashion in order to self-consistently improve the calculated 3D
structure model, on each iteration choosing a different side for
specimen illumination. The algorithm can be interrupted when the
model is sufficiently detailed or accurate, or when successive
iterations no longer produce significant changes in structure or
accuracy.
[0269] Direction-sequential and structured illumination of a
specimen and multiple captured views of the specimen undergoing
fluorescence or light scattering can also provide complementary
information for 3D reconstruction algorithms. For example,
direction-sequential illumination can be used to perform
time-of-flight fluorescence measurements on a specimen. In
specimens where an internal fluorescing entity such as a tumor is
asymmetrically positioned with respect to a nominal axis of the
specimen, the temporal dependence of the fluorescence emitted by
the specimen in multiple views of the specimen may vary, providing
information about absorption and scattering of illumination light
by specimen tissues.
[0270] In another aspect, structured illumination can be used to
prevent illumination light from being directed to the eyes of a
biological specimen. For example, mice are commonly used specimens
in fluorescence imaging studies, and when anaesthetized, the
eyelids of mice often do not fully close. The relatively intense
light sources used to induce fluorescence in specimens such as mice
could conceivably exceed permissible exposure levels, or induce
involuntary nervous responses in mice such as twitching or other
movements that reduce the reproducibility and accuracy of measured
data. Structured illumination provides a means for avoiding these
consequences. In some embodiments, for example, one or more
modulating elements such as masks, spatial light modulators (e.g.,
MEMS digital light processors, liquid crystal modulators), and the
like may be used in conjunction with light source 102 to reduce the
intensity of illumination light that is incident on the surface of
the specimen in the vicinity of the specimen's eyes in order to
reduce the likelihood of causing a nervous response .
[0271] In some embodiments, for example, illumination stage 110 can
be provided with a shield to prevent exposure of the specimen's
eyes to illumination light. FIG. 18 shows an embodiment of
illumination stage 110 that includes a support platform 390 and a
specimen holder 392. Light shield 394 includes an optically opaque
light block affixed to a rotatable and extendible arm so that the
light block can be configurably positioned over the eyes of a
specimen such as a mouse in order to prevent illumination light
from reaching the specimen's eyes. Other embodiments of light
shield 394 are possible. For example, light shield 394 can be
implemented as a movable shield affixed to specimen holder 392.
Alternatively, for example, light shield 394 can be incorporated
into support platform 390, with the specimen in specimen holder 392
positioned relative to platform 390 such that light shield 394
prevents illumination light from entering the eyes of the
specimen.
General Measurement System Components
[0272] Each of the operating modes of measurement system 100 have
been discussed with reference to specific embodiments and
configurations of the measurement system. However, it is important
to recognize that in general, many different configurations and
operating modes of the measurement system are possible, and
different operating modes can be used in combination or in
complementary fashion.
[0273] Similarly, although some specific elements of measurement
system 100 have already been discussed, it should be recognized
that measurement system 100 can, in general, include any or all of
a wide variety of optical elements and components. The
determination of suitability of any particular component rests with
the operator of the measurement system, and is typically made based
on the nature, accuracy, and reproducibility of measurements made
with the system. In view of the foregoing, in this section we
summarize the different optical and other components that may be
included in measurement system 100.
[0274] In general, light source 102 can include one or more light
source elements configured to provide light 104. Light source 102
can include a single light-producing element such as a metal halide
lamp, a xenon arc lamp, a light-emitting diode, or a laser.
Alternatively, light source 102 can include any combination of
multiple light-producing elements suitable for a particular
measurement mode.
[0275] Light source 102 can provide illumination light in the
ultraviolet, visible, infrared, or another region of the
electromagnetic spectrum. In some embodiments, the light provided
by light source 102 can have a relatively wide spectral
distribution. For example, light source 102 can have a full-width
half-maximum (FWHM) spectral distribution of about 20 nm or
greater. In other embodiments, for example, light source 102 can
have a narrower spectral distribution, such as a spectral
distribution with FWHM less than about 20 nm (e.g., less than about
5 nm). In some embodiments, light source 102 can be a white light
source, and can have a very wide spectral distribution covering
substantially all of the visible region of the spectrum.
[0276] Light 104 can be provided in the form of a light beam, such
as a laser beam, or can have a more diffuse spatial intensity
profile, such as for a lamp.
[0277] In some embodiments, light source 102 can include two or
more light-producing elements providing light at the same or
different wavelengths. For example, embodiments may feature a light
source 102 that includes a first source element that produces
reference illumination light, the reference illumination light
including a broad distribution of wavelengths (i.e., white light),
and a second source element that produces measurement illumination
light having a relatively narrow distribution of wavelengths. The
reference light can be used to illuminate a specimen for purposes
of visualization during positioning, or surface profilometry, or 3D
modeling. The measurement source can be used to illuminate the
specimen to induce fluorescence emission, or to measure tissue
absorption, scattering, or transmission. Light source 102 may
further provide for selective illumination of a specimen using only
a subset of source elements. For example, if light source 102
includes both white light and fluorescence source elements, one
element can be used while the other is disabled.
[0278] In other embodiments, for example, light source 102 can
include two or more source elements having different central
wavelengths for use in multiple wavelength excitation modes of
measurement. A similar light source can be provided by using a
single, broadband light source element in combination with spectral
filters in either light source 102 or as part of light conditioning
optics 106.
[0279] In some embodiments, light source 102 can include multiple
source elements providing light of nominally the same wavelength.
For example, light source 102 can include multiple fiber optic
sources (e.g., 3 or more fiber optic sources, 4 or more fiber optic
sources, 5 or more fiber optic sources, 10 or more fiber optic
sources) arranged to illuminate a specimen. The number and spatial
distribution of light sources can be selected to provide a chosen
spatial illumination profile.
[0280] Light source 102 can also include one or more filters, such
as barrier filters, bandpass filters, or liquid crystal filters, in
order to produce light 104 having a selected distribution of
wavelength components.
[0281] Light conditioning optics 106, in general, include various
types of optical elements for modifying the properties of light 104
provided by light source 102. For example, light conditioning
optics 106 can include one or more lenses to focus and/or collimate
light, e.g., to focus illumination light to a position on the
surface of a specimen. Light conditioning optics 106 can also
include mirrors, beamsplitters, dichroic beamsplitters, and the
like. Dichroic beamsplitters can be particularly advantageous in
embodiments where illumination light 108 and emitted light 112
travel along optical paths that are substantially collinear, such
as in epi-fluorescence measurement systems. Dichroic beamsplitters
can be used, for example, to permit spatial separation of the
emitted and illumination light.
[0282] Light conditioning optics 106 can also include one or more
filters such as bandpass filters, barrier filters, graded filters,
epi-fluorescence filters, and/or liquid crystal filters. Filters
can be used in combination, and can be mounted in a filter wheel.
Filters are generally used to control the spectral properties of
illumination light 108. For example, one or more filters can be
used to eliminate from illumination light 108 spectral components
at one or more specimen fluorescence wavelengths.
[0283] In some embodiments, light conditioning optics 106 can
include one or more optical elements for controlling the spatial
intensity profile of illumination light 108. For example, light
conditioning optics 106 can include one or more spatial light
modulators, spatial aperture masks, diffractive optical elements,
or other elements configured to modulate the spatial intensity
distribution of illumination light 108. Examples of spatial light
modulators include MEMS digital light processors (Texas Instruments
DLP Products, 6550 Chase Oaks Blvd., Plano Tex. 75023) and liquid
crystal light modulators. In addition, other effects such as
multi-point source element interference can be used to induce
modulations in the spatial profile of illumination light 108.
[0284] For certain applications, the position of a specimen
relative to a focal plane of illumination light 108 may be
important. More particularly, it may be desirable in some
applications to ensure that the specimen is positioned in the focal
plane of the illumination light. One means of ensuring the correct
positioning of the specimen is provided by configuring light
conditioning optics 106 to produce structured illumination light
108 as a grid or other regular array pattern incident on a surface
of the specimen. The position of the specimen with respect to one
or more focusing optical elements can then be adjusted in order to
ensure that the spatial intensity profile of illumination light 108
is focused with sufficient sharpness at the surface of the
specimen. The spatial intensity profile of illumination light 108
can be imaged at a number of specimen positions, and the optimum
specimen position can be chosen from among the measurement
positions or interpolated.
[0285] Spatial light modulators and other optical devices and
elements for modifying the spatial intensity profile of
illumination light 108 can also be used to provide more spatially
uniform illumination of a specimen where desired in some
embodiments. For example, these modulating elements can be used to
provide a more uniform illumination profile from a single optical
source element by correcting for center-edge intensity fall-off.
Alternatively, or in addition, modulating elements can be used to
produce a composite light source 102 that includes multiple light
source elements, wherein the modulating elements are operated in
tandem in order to smooth the spatial intensity profile of
composite source 102.
[0286] In general, a specimen of interest is mounted on
illumination stage 110, and illumination light is directed to be
incident thereon. Illumination stage 110 can include a specimen
holder, for example, secured to a supporting platform. The
supporting platform can be affixed to a translation stage that
provides illumination stage 110 with multiple degrees of
translational freedom. The position of illumination stage 110 can
be changed in response to an automated signal from electronic
control system 122, for example, or in response to a manual signal
from an operator. In some embodiments, adjustment of the position
of the specimen relative to the imaging system can also be
accomplished by adjusting the positions of the light conditioning
optics and the light collecting optics while illumination stage 112
remains in the same position. In this way, the positions of the
focal plane of illumination light 108 and the object plane of the
light collecting optics 114 can be changed independently from one
another.
[0287] The specimen holder 392, illustrated schematically in FIG.
18, may in general be any type of support structure, holder, or
mount capable of supporting a specimen for study. The specimen
holder should generally permit illumination light 108 to be
incident on the surface of the specimen, and should also permit
emitted light 112 to emanate from specimen 2. The optical paths of
both the illumination and emitted light should be relatively
unobstructed by the specimen holder. Further, specimen holder 392
may be positioned in any orientation with respect to support
platform 390.
[0288] For example, in some embodiments, the specimen holder can
include several posts (e.g., 4 posts, 5 posts, 6 posts) arranged
with axes parallel to one another to form a substantially
regularly-shaped specimen area therebetween. For instance, a
specimen holder that includes 4 posts may have a substantially
rectangularly-shaped specimen area.
[0289] In other embodiments, the specimen holder may take the form
of a case made from a material that is substantially transparent to
the measurement light used. For example, the specimen holder can be
a glass case in the shape of a cylinder. The case can be airtight,
and may therefore be able to accommodate anaesthesia apparatus for
immobilizing a specimen. A sealed case also prevents contamination
of optical surfaces in the measurement system due to the presence
of the specimen. A glass case may also be autoclavable for
sterilization purposes and exhibit a low autofluorescence emission
signal.
[0290] In further embodiments, the specimen holder can simply
include one or more straps affixed to a base of the illumination
stage to secure the specimen in place.
[0291] Generally, it is desirable to construct the specimen holder
such that it is optically non-perturbative to both illumination and
emitted light. For example, the specimen holder can be constructed
from materials that are substantially transparent to radiation at
the wavelengths of the illumination and emitted light (i.e.,
fluorescence emission, scattered light). By filling air spaces
inside the specimen holder with an index-matching fluid, the
specimen holder can further be used to define the index of
refraction boundary conditions for one or more 3D reconstruction
algorithms, which may improve the accuracy of the reconstructed
specimen profile.
[0292] Light emitted from a specimen, e.g., fluorescence emission
from internal structural entities labeled with fluorescent
moieties, is captured by light collecting optics 114. In
particular, the optical elements of light collecting optics 114 are
configured to capture one or more views 116 of the specimen and
transmit the multiple views to detector system 118.
[0293] In some embodiments, such as epi-fluorescence measurement
systems for example, some optical elements may be common to both
light collecting optics 114 and light conditioning optics 106. For
example, light collecting optics 114 can include optical elements
such as lenses, mirrors, wavelength-neutral beamsplitters, dichroic
beamsplitters, and the like, some of which can be common to light
conditioning optics 106.
[0294] Light collecting optics 114 can also include filter elements
such as bandpass filters, barrier filters, liquid crystal filters,
and interference filters. The filters may, for example, be used in
some embodiments to spectrally resolve multiple views of the
specimen for applications such as autofluorescence removal. In
particular, filters can be used to separate spectral components in
one or more views that include multiple spectral components (e.g.,
2 or more spectral components, 3 or more spectral components, 10 or
more spectral components).
[0295] Generally, a wide variety of 3D models can be implemented in
computer programs constructed using standard programming techniques
and running on a processor within electronic control system 122.
Electronic control system 122 can include a processor or processing
unit, a user interface such as a keyboard and a monitor, and a
display device. Programs stored on computer readable media can be
transferred into electronic control system 122, and when executed,
may cause the processor to carry out the steps of analyzing the
measurement information provided to electronic control system 122
by detector system 118. Electronic control system 122 can further
be configured to display one or more views or images of the
specimen under study on the display device. Electronic control
system 122 can also implement algorithms for computing
goodness-of-fit metrics for use in positioning mode.
[0296] Electronic control system 122 can be configured to generate
one or more control signals either automatically or in response to
input from an operator. For example, electronic control system 122
can generate electronic signals for translating optical components
(e.g., light conditioning optics 106 and/or light collecting optics
114), for translating illumination stage 112, for capturing images
with detector system 118, for time-gating detector system 118, for
controlling light source elements in source 102, and for the
mechanical and electronic control of other measurement system
components and elements.
[0297] Although certain preferred embodiments described above
involve the measurement of multiple views of a specimen,
measurement systems configured to acquire only a single view or to
collect light emitted by a specimen from a single side can also
implement many of the described measurement techniques. For
example, techniques for positioning a specimen using a reference
image, for using structured illumination (including using a
structured illumination source that is configured to reduce
illumination in the vicinity of the specimen's eyes), and spectral
fluorescence measurements using multiple excitation wavelengths
and/or multiple different fluorescence labels, can all be
implemented with single view detection schemes. Detector systems
can be positioned to collect light emitted in any direction from a
specimen, such as in a reflected or transmitted direction relative
to the direction of incidence of illumination light, or in another
direction.
[0298] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *