NIH Public Access

Author Manuscript
J Biomed Opt. Author manuscript; available in PMC 2009 September 17.
Published in final edited form as:
NIH-PA Author Manuscript

J Biomed Opt. 2008 ; 13(2): 021113. doi:10.1117/1.2907968.

Optical clearing of unsectioned specimens for three-dimensional

imaging via optical transmission and emission tomography

Mark Oldham and Harshad Sakhalkar

Duke University Medical Center Department of Radiation Oncology Durham, North Carolina 27710
Tim Oliver
Duke University Medical Center Department of Cell Biology Durham, North Carolina 27710
G. Allan Johnson
Duke University Medical Center Department of Radiology Durham, North Carolina 27710
Mark Dewhirst
Duke University Medical Center Department of Radiation Oncology Durham, North Carolina 27710
NIH-PA Author Manuscript

Abstract
Optical computed tomography (optical-CT) and optical emission computed tomography (optical-
ECT) are new techniques that enable unprecedented high-resolution 3-D multimodal imaging of
tissue structure and function. Applications include imaging macroscopic gene expression and
microvasculature structure in unsectioned biological specimens up to 8 cm3. A key requisite for these
imaging techniques is effective sample preparation including optical clearing, which enables light
transport through the sample while preserving the signal (either light absorbing stain or fluorescent
proteins) in representative form. We review recent developments in optical-CT and optical-ECT, and
compatible “fluorescence-friendly” optical clearing protocols.

Keywords
optical; clearing; imaging; 3D; emission; tomography; gene; expression; vasculature; HIF1;
xenograft
NIH-PA Author Manuscript

1 Introduction
The ability to image co-registered biological structure and function in three dimensions in
whole unsectioned tissue specimens is of significant present interest. In cancer research, for
example, new antiangiogenic agents have potential to enhance the therapeutic effects of
principal cancer treatments such as radiation and chemotherapy.1,2 There is a need for greater
understanding of the complex relationships governing the response of the global
microvasculature to these agents to help development of more effective therapies.3-5 These
efforts have been hampered by the lack of a truly 3-D imaging modality with sufficiently high
spatial resolution and contrast to determine subtle microvasculature detail. Such a modality
would facilitate study of the vascular response to new therapeutic agents, to variations in
fractionation of application,6,7 the restructuring of vasculature networks,8 and the distribution
of functional response to hypoxia.9,10 Imaging fine microvascular structure presents a

© 2008 Society of Photo-Optical Instrumentation Engineers.

Address all correspondence to Mark Oldham, Radiation Oncology and Biomedical Engineering, Duke University Medical Center, Room
04216, Red Zone - Duke South Clinics, Durham, NC 27710; Tel: 919 668 0349; Fax: 919 681 7183; [email protected].
 Oldham et al. Page 2

challenge to existing imaging techniques, including micro-CT (micro-computed tomography)

and micro-MRI (micro-magnetic resonance imaging) due to the combined requirement for high
contrast and high spatial resolution. Similarly, there is great interest in techniques that can
NIH-PA Author Manuscript

image the efficacy of drug and gene delivery techniques, gene expression, and the genetic
response of tumors to therapy.11-14 Optical responses to these challenges are now feasible with
the development of fluorescent reporter genes [e.g., red and green fluorescent proteins (RFP/
GFP), respectively] and targeted fluorophors15,16 [e.g., FITC fluorescin conjugated lectin].
Confocal and two-photon imaging methods have been applied to obtain some 3-D data, but
these methods are limited to a few hundred micrometers depth from the imaging surface.17,18

Three-dimensional optical-computed-tomography (optical-CT) and optical-emission-

computed-tomography (optical-ECT), when combined with optical clearing techniques, can
yield high-contrast and high-resolution 3-D images of microvasculature and/or any fluorescent
moiety. They represent powerful new techniques for investigating structural, functional, and
genetic therapeutic responses and relationships.19

In previous work optical-CT has been developed in the context of high resolution 3-D
dosimetry for verification of radiation therapy treatments.20-23 Later, the technique was
extended to include emission tomography, and applications were explored in developmental
embryology and embryonic gene expression.24,25 Recently, a novel implementation was
presented26 along with initial applications imaging xenograft tumors27 and whole unsectioned
NIH-PA Author Manuscript

rodent organs. In this paper, we review recent developments in optical-CT and optical-ECT
techniques and compatible clearing protocols28 and illustrate their application with an in-house
benchtop imaging system (Fig. 1).

2 Basic Principles of the Optical-CT and Optical-ECT Techniques

Optical-CT can be conceived as the optical analog of x-ray CT. Three-dimensional images of
the distribution of optical attenuation throughout a sample are reconstructed from optical
projection images of light transmitted through the sample. Optical-ECT is the optical analog
of SPECT (single-photon emission tomography). In optical-ECT, 3D images of the distribution
of emitting light sources (e.g., fluorochromes) are reconstructed from emission images of light
emitted from the sample. Some of the established benefits associated with combining image
data from complimentary modalities, such as x-ray-CT/SPECT or x-ray-CT/PET (positron
emission tomography), translate over to the optical-CT/optical-ECT combination. These
include the facility for accurate registration of transmission and emission image data and the
potential for relating tissue structure and function, as demonstrated here in relation to xenograft
tumor imaging.
NIH-PA Author Manuscript

2.1 Imaging Hardware and Acquisition

Optical projection/emission images are acquired of a sample suspended in (for example) an
agarose gel, mounted vertically on a rotating platform inside a small aquarium made from
antireflection-coated glass. The gel rotates inside a high-refractive-index matched fluid, which
minimizes refraction, enabling straight-line projection/emission images to be acquired. In
optical-CT (transmission mode) light from a uniform back-light traverses through the sample
to image formation at the CCD. Projections are acquired in a “step-and-image” manner at small
angular increments (e.g., 0.3 deg) through a complete 360-deg revolution. In optical-ECT
(emission mode) excitation light causes longer wavelength fluorescent light to be emitted
within the sample, which is then also imaged on the CCD, as illustrated. The use of telecentric
lenses in the optical imaging system facilitates accurate optical-CT and optical-ECT images,
as image formation occurs from light traveling parallel to the optic axis. Acquisition proceeds
in the same step-and-image procedure, with the same settings and constraints as already
described. A key difference is that narrow-bandwidth optical filters are used to select both the

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 3

excitation and emitting wavelengths. High-quality filters greatly enhance effective image
quality by reducing any contaminant autofluorescence. A DSRed2 filter set was used to select
for the excitation and emission wavelengths corresponding to RFP (558 and 583 nm,
NIH-PA Author Manuscript

respectively). Exposure of the sample to excitation light was minimized by utilization of a

shutter, which permitted exposure only during image acquisition. Consistent interprojection
normalization was achieved by keeping the exposure time constant for each projection.

Once a complete set of projection or emission images has been acquired, 3-D reconstructions
of the sample were created using the commercial COBRA Feldkamp filtered back-projection
algorithm (Exxim Computing Corp, Pleasantown, California). Input parameters were adjusted
to reflect the geometries of the image acquisition. In principle, the spatial resolution can
approach the resolution of the CCD camera as the resolution of the lens is usually far higher.
In practice, however, resolution is lost through the tomographic reconstruction and artifacts
arising from motion point spread function and projection noise. Present prototype systems have
been reported to have a modulation transfer function (MTF) ∼10% at 20 lp/mm, but substantial
improvements are expected. The microoptical-CT scanner presented in Fig. 1 represents a
second-generation and very fast scanning system when compared with the first-generation
optical-CT devices described elsewhere.20-22,29,30 The time for a typical acquisition of 360
projections was about 5 min.

2.2 Optical Clearing for Optical-CT and Optical-ECT

NIH-PA Author Manuscript

A significant difference between the optical and x-ray imaging analogs is that the poor optical
transmission of biological tissue necessitates ex vivo sample preparation to improve optical
transmission (the optical clearing process). Despite this limitation, accurate “in vivo” functional
information is entirely feasible because optical stains and fluorescing labels can be applied in
vivo, such that representative staining/labeling is achieved for subsequent imaging. Meaningful
imaging of any functional parameter therefore requires preservation of the condition of that
stain/label through the excision and sample preparation procedures. Optical clearing can be
achieved by dehydration and reperfusion of the tissue with a transparent solution of high
refractive index close to that of the cell nuclear and organelle membranes. In general, better-
reconstructed image quality (both optical-CT and optical-ECT) is directly associated with
better optical clearing, provided the signal of interest (here isotonic-ink in the vasculature, and
fluorescent proteins) are not affected by the optical clearing process. Illustrative projection
images through various unsectioned tissue samples that were cleared and labeled using the
processes described later are shown in Fig. 2(a). The images were acquired using the system
shown in Fig. 1(a), with a backlight source of wavelength 633 nm. Corresponding transmission
characteristics of the samples relative to methyl-salicylate are shown in Fig. 2(b). All samples
were imaged immersed in methyl salicylate. The lowest transmission was observed for the rat
NIH-PA Author Manuscript

heart because of the high concentration of ink taken up in the vasculature and ventricle
chambers. The highest transmission is observed in the brain, where ink uptake was greatly
reduced due to the blood brain barrier.

2.2.1 General sample preparation—All samples were whole (i.e., unsectioned) at the
time of imaging and optically cleared to enable visible light penetration through the sample.
The principle of optical clearing is to replace the water-based cellular fluid with a solution of
high refractive index to match that of the cell, nuclear, and organelle membranes.31,32
Achieving quality optical clearing is a key step that enables the feasibility of both optical-CT
and optical-ECT. All tissue samples imaged in this paper were first set in 0.75% agarose gel
by weight. The tissue samples were positioned centrally and ∼1 cm up from the bottom of the
gel. Staining and fixing procedures varied between samples, and details are discussed in the
corresponding following sections. The purpose of the agarose gel was to stabilize the sample
during rotation incurred in the optical-CT/optical-ECT acquisition. Each sample (agarose and

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 4

imbedded tissue) was then immersed in a succession of graded ethanol/water solutions, until
the tissue was completely dehydrated. Once the sample was fully permeated with 100%
ethanol, they were then immersed in a succession of graded ethanol with methylsalicylate or
NIH-PA Author Manuscript

benzyl-alcohol benzyl benzoate (BABBs), until the ethanol was completely replaced by the
higher refractive index liquid. The large pore size of agarose gel facilitates efficient fluid
exchange. Methyl salicylate was used for clearing any tissue sample containing fluorescent
proteins, as better fluorescent preservation has been observed. For nonfluorescent samples,
optical clearing was achieved with BABBs solution, which leads to slightly better optical
clarity. These are the only clearing solutions we have tested so far but, in principle, any
transparent solution with high refractive index (∼1.54) that can be perfused into tissue can be
a viable clearing agent. The solutions have negligible influence on the physical size and shape
of organ. Furthermore, hematoxylin-and-eosin (H&E) histological evaluation of cleared
specimens are virtually indistinguishable from control noncleared specimens. However, we
have been unsuccessful so far in achieving immunohistological staining on tissue samples that
have been optically cleared in this way.

2.2.2 “Fluorescence-friendly” optical clearing—Oldham et al.27 investigated the

feasibility of 3-D imaging the viable tumor burden in HCT116 tumor xenografts. The HCT116
cell line had been transfected with a gene coding for constitutively expressed33 RFP. Viable
tumor cells therefore express red fluorescence when exposed to the excitation wavelength. The
initial challenge was to develop a tissue optical-clearing procedure that would preserve RFP
NIH-PA Author Manuscript

fluorescence in the cleared tissue. A series of plating experiments were performed, where the
transfected HCT116 tumor cells were exposed to a variety of tissue-fixing and clearing agents
representing different potential clearing processes. Full details of the clearing procedures are
given in Ref. 28. The effect on RFP and GFP fluorescence is shown in Fig. 3. The significant
result from these experiments is that substantial fluorescence preservation was achieved when
the initial cell fixation was in ethanol. Fixation in either PFA or methanol resulted in almost
complete loss of fluorescence [Figs. 3(A) and 3(B)]. Furthermore, after ethanol fixation, the
cells proved robust to subsequent exposure to either clearing agent BABBs or MetSal. To
successfully image RFP in whole xeneograft tumors, the results of the plating experiments
must be transferred to whole tumor specimens. This was achieved by first performing
perfusion-fixation of the tumors in situ, by aortic cannulation and gravitational drip feed of
ethanol. Tumors were then placed in ethanol at 4°C overnight, and set in 0.75% agarose the
next day. The clearing procedure involved perfusion graded solutions of water:ethanol, and
then ethanol:methyl-salicylate. The total clearing process for tissue samples of order 1 cm3
typically takes from 1 to 3 weeks, depending on size of sample, under gentle motion-assisted
diffusion processes at room temperature. A similar procedure was used irrespective of the
particular agent used for dehydration and clearing steps. The sample is initially placed in a
75:25 ethanol:water solution, which is changed to 100% ethanol after 12 h, and then refreshed
NIH-PA Author Manuscript

on a daily basis for 4 days. Complete dehydration is necessary to avoid precipitation of opaque
regions of agarose, which may occur if water and clearing agent react with agarose gel. The
sample is then placed in a 50:50 solution of ethanol:clearing agent. After 24 h, the solution is
changed to 100% clearing agent, which is also replenished on a daily basis.

3 Application of Optical-CT and Optical-ECT

3.1 Imaging Xenograft Tumors
Oldham et al.27 presented preliminary data imaging HCT116RFP xenograft colon cancer
tumors, containing constitutive RFP labeling, grown on the hind legs of nude mice, following
the procedures of an Institutional Animal Care and Use Committee (IACUC) approved
protocol. When the tumors had grown to a ∼1 cm length, labeling/staining of the tumor
microvasculature was achieved by tail vein injection and subsequent natural circulation around

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 5

the body of a double bolus of isotonic india ink and fluorescent probe (lectin conjugated with
FITC). The carbon-based ink particles circulate in the blood stream and are phagocytosed inside
endothelial cells of the vessels, thereby marking patent microvasculature.34 At 5 to 10 min
NIH-PA Author Manuscript

postinfusion, the mouse was sacrificed and the tumor removed for sample preparation. Lectin
actively binds to endothelial cells of the microvasculature, providing independent labeling of
the microvasculature amenable for optical-ECT imaging. The implementation of both passive
and active labeling of microvasculature enables cross-validation and comparison of both
techniques. A single optical transmission or projection image of the whole HCT116 tumor is
shown in Fig. 4(A), and contrasted with micro-x-ray-CT and micro-MRI images acquired with
state of the art small animal imaging systems35,36 [Figs. 4(B) and 4(C)] to highlight the
excellent contrast and resolution of optical-CT. Exquisite visualization of the microvasculature
was observed in the optical projection, although the 3-D nature of the vascular network is lost.
In this instance, the vasculature is primarily seen on the periphery of the tumor, with a few
larger vessels penetrating to the tumor core. As these tumors were implanted subcutaneously,
especially dense and intensive vasculature is seen along one side of the tumor where it was
attached to the underlying fascia of the animal.

Quantitative information is available from CT reconstructions of the projections. Figures 5

(A)–5(C) show such a reconstruction from 240 projections acquired at 1.5-deg increments. The
original projection images (1040×1388 pixels) were downsized to 748×998 (to match present
software restrictions) and reconstructed on a 512×512×512 grid. The pixel dimensions in the
NIH-PA Author Manuscript

image are thus ∼30 μm, although a greater number of projections must be acquired to meet the
Nyquist criteria for this resolution limit. Significant vascular penetration is observed to be
limited to the lower part of the tumor [Fig. 5(B)], indicating this region was relatively well
perfused. The corresponding reconstructions of the emitting FITC distribution within the
tumor, acquired with the FITC filters, are shown in Figs. 5(D)–5(F). In general, a clear
correlation and agreement is observed between the optical-CT and optical-ECT images. Well-
perfused regions appear bright in the optical-CT images (corresponding to regions high ink
absorption) and also as bright regions in the optical-ECT images (where the scale is inverted
such that light pixel values correspond to high emission of light and hence high concentration
of FITC). The HCT116 tumor was also imaged in optical-ECT mode with DSRed2 filter set
[Figs. 5(G) to 5(H)]. This image is significant as it represents the 3-D distribution of RFP
emitted by viable tumor cells. The correlations between the corresponding views in Fig. 5 are
striking, and clearly show that regions of high RFP expression correlate closely with the well-
perfused regions. This makes intuitive sense, as one would expect the more viable regions of
the tumor to correlate with perfusion. A precise interpretation is complex due to the novelty
of these techniques, and requires reference to more established imaging modalities. Further
interpretation of the optical-ECT images would require an attenuation correction, similar to
that routinely encountered in SPECT imaging. Comparison with conventional histological
NIH-PA Author Manuscript

sections [Fig. 5(J)] provided strong supporting evidence for the conclusions derived above
from the optical imaging modalities. The peripheral band of well-perfused viable cells, inferred
from all three optical reconstructions [Figs. 5(B), 5(E), and 5(H)] exhibits strong H&E staining
in histological section (Fig. 5(J)) and therefore viable cells in this region. The large central
areas devoid of vasculature and viable cells, as determined from the optical modalities, are
indeed found to be devoid of H&E stain, indicating regions of necrosis.

The images shown in Fig. 5 were acquired with a prototype microscope-based optical system
that incorporated nontelecentric optical components with limited suitability for tomographic
imaging due primarily to limited depth of field. More recent images of similar tumors acquired
with the bench system of Fig. 1 are shown in Fig. 6. A significant improvement in image quality
is observed, through the removal of artifacts associated with the limited depth of field of the
microscope system. Figure 6 also presents the first reconstructions of endogenous gene
expression in three dimensions in xenograft tumors using optical-ECT. This was achieved

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 6

using FITC filter sets and an HCT116 cell line that was double labeled with reporter proteins.
Constitutive RFP-labeled viable tumor (as in the preceding), but secondary GFP labeling was
incorporated to report the expression of the HIF1 gene.33 While further work (for example,
NIH-PA Author Manuscript

development of an attenuation correction for optical-ECT) and independent validation are

required to fully understand the interpretation of these images, the potential utility is evident.

3.2 Whole Rodent Organ Imaging

Vascular staining in rodent organs was achieved by tail vein injection of isotonic ink as
described in Sec. 3.1. Organs were prepared for optical-CT imaging using the procedure
outlined in Sec. 2.1. Reconstructed images of a rat heart and lung are shown in Figs. 7 and 8,
respectively. Postimaging histological study of these samples confirmed that the lighter regions
in these images correspond to tissues with higher optical attenuation (greater ink staining), and
higher perfusion.26 In the heart images, the most prominent features are the coronary arteries,
ventricles, and other areas of high blood content. Excellent detail of general heart structure is
also observed. Similar observations are made in the lung images of Fig. 8. The major and minor
bronchial airways are clearly visible, as are interesting variations in perfusion around the lung.

4 Discussion and Conclusions

Optical-CT and optical-ECT are relatively new imaging modalities that, when combined with
optical clearing techniques, can provide unique 3-D information in high resolution and high
NIH-PA Author Manuscript

contrast on the structure and function (including gene expression) of tissue. The optical clearing
techniques reviewed here were developed from earlier techniques developed to clear thin
sections of tissue for optical microscopy. A significant step forward was the development of
clearing protocols that preserve the fluorescence output of unsectioned bulk tissue samples.
28 As with any new imaging modality, accurate interpretation of image content is gradually
established by reference and correlation to alternative more established methodologies. The
bulk of this effort has yet to be accomplished. Here, we reviewed the physical basis and
demonstrated preliminary application to imaging xenograft tumor microvasculature and, for
the first time, preliminary 3-D images showing endogenous gene expression in xenograft
tumors (HIF1 expression), and whole rodent organs. The true potential of these techniques may
be even wider, as it should be feasible to image a wide range of other tumor and normal tissue
structures and functions, depending on the development of corresponding optical probes.

Acknowledgments
This work has arisen out of work funded by National Institutes of Health (NIH) Grant No. R01 CA 100835. The MR
and microCT were performed at the Duke Center for In Vivo Microscopy an NCRR/NCI National Resource (P41
RR005959/R24 CA092656).
NIH-PA Author Manuscript

References
1. Dewhirst MW, Richardson R, Cardenas-Navia I, Cao Y. The relationship between the tumor
physiologic microenvironment and angiogenesis. Hematol. Oncol. Clin. North Am 2004;18:973–990.
[PubMed: 15474330]
2. Jain RK. Antiangiogenic therapy for cancer: current and emerging concepts. Oncology 2005;19:7–16.
[PubMed: 15934498]
3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature Sep;2000 407:249–257.
[PubMed: 11001068]
4. Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK. Tumour biology: herceptin acts as an anti-
angiogenic cocktail. Nature Mar;2002 416:279–280. [PubMed: 11907566]

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 7

5. Wachsberger P, Burd R, Dicker AP. Improving tumor response to radiotherapy by targeting

angiogenesis signaling pathways. Hematol. Oncol. Clin. North Am 2004;18:1039–1057. [PubMed:
15474334]
NIH-PA Author Manuscript

6. Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, Fuks Z,

Kolesnick R. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science May;
2003 300:1155–1159. [PubMed: 12750523]
7. Shan S, Robson ND, Cao Y, Qiao T, Li CY, Kontos CD, Garcia-Blanco M, Dewhirst MW. Responses
of vascular endothelial cells to angiogenic signaling are important for tumor cell survival. FASEB J
2004;18:326–328. [PubMed: 14688196]
8. Jain RK. Antiangiogenic therapy for cancer: current and emerging concepts. Oncology 2005;19:7–16.
[PubMed: 15934498]
9. Moeller BJ, Cao Y, Vujaskovic Z, Li CY, Haroon ZA, Dewhirst MW. The relationship between hypoxia
and angiogenesis. Semin. Radiat. Oncol 2004;14:215–221. [PubMed: 15254864]
10. Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular
radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cells
2004;5:429–441.
11. Barker SD, Dmitriev IP, Nettelbeck DM, Liu B, Rivera AA, Alvarez RD, Curiel DT, Hemminki A.
Combined transcriptional and transductional targeting improves the specificity and efficacy of
adenoviral gene delivery to ovarian carcinoma. Gene Ther 2003;10:1198–1204. [PubMed:
12833129]
12. Brown EB, Campbell RB, Tsuzuki Y, Xu L, Carmeliet P, Fukumura D, Jain RK. In vivo measurement
NIH-PA Author Manuscript

of gene expression, angiogenesis and physiological function in tumors using multiphoton laser
scanning microscopy. Nat. Med 2001;7:864–868. [PubMed: 11433354]
13. Wilke M, Fortunati E, van den Broek M, Hoogeveen AT, Scholte BJ. Efficacy of a peptide-based
gene delivery system depends on mitotic activity. Gene Ther 1996;3:1133–1142. [PubMed: 8986440]
14. Relph KL, Harrington KJ, Pandha H. Adenoviral strategies for the gene therapy of cancer. Semin.
Oncol 2005;32:573–582. [PubMed: 16338423]
15. Contag CH, Jenkins D, Contag PR, Negrin RS. Use of reporter genes for optical measurements of
neoplastic disease in vivo. Aquacultural Eng 2000;2:41–52.
16. Hoffman R. Green fluorescent protein imaging of tumour growth, metastasis, and angiogenesis in
mouse models. Lancet Oncol 2002;3:546–556. [PubMed: 12217792]
17. Alexandrakis G, Brown EB, Tong RT, McKee TD, Campbell RB, Boucher Y, Jain RK. Two-photon
fluorescence correlation microscopy reveals the two-phase nature of transport in tumors. Nat. Med
2004;10:203–207. [PubMed: 14716306]
18. Secomb TW, Hsu R, Park EY, Dewhirst MW. Green's function methods for analysis of oxygen
delivery to tissue by microvascular networks. Ann. Biomed. Eng 2004;32:1519–1529. [PubMed:
15636112]
19. Oldham M, Oliver T, Dewhirst M. A novel method of imaging unperturbed tumor vasculature in 3D.
Int. J. Radiat. Oncol., Biol., Phys., Suppl 2005;63:S134.
NIH-PA Author Manuscript

20. Oldham M, Siewerdsen JH, Kumar S, Wong J, Jaffray DA. Optical-CT gel-dosimetry I: basic
investigations. Med. Phys 2003;30:623–634. [PubMed: 12722814]
21. Oldham M, Siewerdsen JH, Shetty A, Jaffray DA. High resolution gel-dosimetry by optical-CT and
MR scanning. Med. Phys 2001;28:1436–1445. [PubMed: 11488576]
22. Gore JC, Ranade M, Maryanski MJ, Schulz RJ. Radiation dose distributions in three dimensions from
tomographic optical density scanning of polymer gels: I. Development of an optical scanner. Phys.
Med. Biol 1996;41:2695–2704. [PubMed: 8971963]
23. Doran SJ, Koerkamp KK, Bero MA, Jenneson P, Morton EJ, Gilboy WB. A CCD-based optical-CT
scanner for high-resolution 3D imaging of radiation dose distributions: equipment specifications,
optical simulations and preliminary results. Phys. Med. Biol 2001;46:3191–3213. [PubMed:
11768500]
24. Sharpe J. Optical projection tomography as a new tool for studying embryo anatomy. J. Anat
2003;202:175–181. [PubMed: 12647867]

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 8

25. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sorensen J, Baldock R, Davidson D. Optical
projection tomography as a tool for 3D microscopy and gene expression studies. Science Apr;2002
296:541–545. [PubMed: 11964482]
NIH-PA Author Manuscript

26. Oldham M, Sakhalkar H, Wang YM, Guo P, Oliver T, Bentley R, Vujaskovic Z, Dewhirst M. Three-
dimensional imaging of whole rodent organs using optical computed and emission tomography. J.
Biomed. Opt 2007;12:014009–1. 014009–10. [PubMed: 17343484]
27. Oldham M, Sakhalkar H, Oliver T, Wang YM, Kirpatrick J, Cao Y, Badea C, Johnson GA, Dewhirst
M. Three-dimensional imaging of xenograft tumors using optical computed and emission
tomography. Med. Phys 2006;33:3193–3202. [PubMed: 17022212]
28. Sakhalkar HS, Dewhirst M, Oliver T, Cao Y, Oldham M. Functional imaging in bulk tissue specimens
using optical emission tomography: fluorescence preservation during optical clearing. Phys. Med.
Biol Apr;2007 52:2035–2054. [PubMed: 17404454]
29. Kelly RG, Jordan KJ, Battista JJ. Optical-CT reconstruction of 3D dose distributions using the ferrous-
benzoic-xylenol (FBX) gel dosimeter. Med. Phys 1998;25:1741–1750. [PubMed: 9775382]
30. Oldham M, Kim L. Optical-CT gel-dosimetry. II: Optical artifacts and geometrical distortion. Med.
Phys 2004;31:1093–1104. [PubMed: 15191297]
31. Tuchin VV. Optical clearing of tissues and blood using the immersion method. J. Phys. D
2005;38:2497–2518.
32. Zhou F, He Y, Wang RK. A theoretical model on optical clearing of biological tissue with chemical
active agents. Progress in Biomedical Optics and Imaging, Proc. SPIE 2004;5486:173–179.
33. Cao Y, Li CY, Moeller BJ, Yu D, Zhao Y, Dreher MR, Shan S, Dewhirst MW. Observation of incipient
NIH-PA Author Manuscript

tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation. Cancer
Res July;2005 65:5498–5505. [PubMed: 15994919]
34. Hilmas DE, Gillette EL. Tumor microvasculature following fractionated x irradiation. Radiology
1975;116:165–169. [PubMed: 806094]
35. Badea C, Hedlund LW, Johnson GA. Micro-CT with respiratory and cardiac gating. Med. Phys
2004;31:3324–3329. [PubMed: 15651615]
36. Chen BT, Yordanov AT, Johnson GA. Ventilation-synchronous magnetic resonance microscopy of
pulmonary structure and ventilation in mice. Magn. Reson. Med 2005;53:69–75. [PubMed:
15690504]
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 9
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 1.
Schematic of light paths through a prototype optical-CT (a) and optical-ECT (b) imaging
system. In optical-CT, light from a uniform backlight traverses through the sample to form
projection images captured by a CCD camera. In optical-ECT, incident light orthogonal to the
imaging axis stimulates fluorescence in the sample. In both modes, a telecentric lens is used
to form an image dominated by light parallel to the optic axis, thereby best approximating the
parallel ray geometry and minimizing scatter contamination. (Based on Oldham et al.26)

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 10
NIH-PA Author Manuscript

Fig. 2.
(a) Light transmission projection images of a variety of optically cleared unsectioned tissue
samples, acquired using the imaging system illustrated in Fig. 1(a), with a backlight of
wavelength 633 nm and (b) light absorbtion characteristics of these samples acquired with a
spectrophotometer, relative to pure methyl salicylate.
NIH-PA Author Manuscript
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 11
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 3.
Fluorescent light intensity from cells labeled with RFP and GFP, after exposure to various
fixing and clearing solutions. Two cell lines were tested, human colon cancer (HCT116) and
human breast cancer (4T1). Comparisons are shown against wild-type (WT) controls, which
had no fluorescent labeling. (A) Effect of fixing agents on RFP intensity, (B) effect of fixing
agents on GFP intensity, (C) effect of clearing agents on RFP, and (D) effect of clearing agents
on GFP. (Based on Sakhalkar et al.28)
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 12
NIH-PA Author Manuscript

Fig. 4.
(A) Projection image of the HCT116 tumor after optical clearing, (B) In vivo micro-CT of the
same tumor on hind leg of mouse. The micro-CT image is reconstructed at 100-μm pixel
resolution and involved application of a new vascular contrast agent (fenestra). (C) in vivo T1-
weighted contrast-enhanced micro-MRI image of a similar tumor of same size acquired on the
2T system at the CIVM (Centre for Invivo Microscopy at Duke). MRI contrast was achieved
with a continuous infusion of magnevist. The location of the tumor is indicated by the dashed
line. (From Oldham et al.27)
NIH-PA Author Manuscript
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 13
NIH-PA Author Manuscript

Fig. 5.
(A) to (C) Orthogonal views of microvasculature of a HCT116 tumor stained with light-
absorbing ink and imaged by optical-CT incorporating a white light source. Light regions of
NIH-PA Author Manuscript

high perfusion are clearly visible as containing relatively high amounts of light attenuating ink.
(D) to (F) microvasculature of a HCT116 tumor stained with FITC-conjugated lectin and
imaged by optical-ECT incorporating the FITC excitation and emission filter set. (G) to (I) the
distribution of viable tumor cells in the HCT116 tumor obtained by optical-ECT using the
DSRed2 filter set to select for RFP excitation and emission. (J) conventional H&E stained
histological section corresponding to views (B), (E), and (H).
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 14
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 6.
Orthogonal views through 3-D reconstructions of HCT116 xenograft tumor imaged by optical-
CT and optical-ECT. The tumors were triply labeled, with RFP and GFP flourescent reporter
proteins, and light absorbing ink in the vasculature. Constitutive RFP labels the viable tumor
burden, and GFP labeled HIF1 expression. The upper row is optical-CT images of vasculature.
The middle row is optical-ECT images of the RFP (viable tumor) distribution. The lower row
is optical-ECT images of the GFP (HIF1) distribution.

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 15
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 7.
Orthogonal views (A) to (C) through a 3-D optical-CT reconstruction of a normal rat heart and
(D) a surface rendered image. Labels are LV/RV, right and left ventricles; AV, aortic valve;
RA, right atrium; PA, pulmonary arch; AA, ascending aorta; PV, pulmonary veins; and CA,
coronary arteries.
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.

 Oldham et al. Page 16
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 8.
(A) to (C) Orthogonal views through a 3-D optical-CT reconstruction of a normal rat lung and
(D) surface rendered image.
NIH-PA Author Manuscript

J Biomed Opt. Author manuscript; available in PMC 2009 September 17.