High-resolution three-dimensional scanning optical image system for intrinsic and extrinsic contrast agents in tissue

Transcription

1 REVIEW OF SCIENTIFIC INSTRUMENTS VOLUME 73, NUMBER 1 JANUARY 2002 High-resolution three-dimensional scanning optical image system for intrinsic and extrinsic contrast agents in tissue Yueqing Gu, a) Zhiyu Qian, b) Jinxian Chen, Dana Blessington, Nimmi Ramanujam, and Britton Chance Department of Biochemistry/Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania Received 7 March 2001; accepted for publication 15 October 2001 This article presents the theory and development of a three-dimensional 3D imaging instrument capable of determining the biochemical properties of tissue by measuring the absorption or fluorescence of different intrinsic and extrinsic agents simultaneously. A bifurcated optical fiber bundle, serving to deliver the excitation light and collect the emission or reflection light, scans over the flat tissue surface retrieving optical signals in each pixel. Two-dimensional 2D images of a series of subsequent sections are obtained after signal conversion and processing to yield a 3D image. Manipulation of the scanning step and diameter size of the fibers within the bundle, the spatial resolution of the instrument attains a maximum of m 3. The wavelength range is extended from ultraviolet to the near infrared NIR through specialized optical design, typically employed for the NIR extrinsic contrast agents study. The instrument is most applicable in situations involving the measurement of fluorescence or absorption at any specific wavelength within the spectrum range. Flavoprotein and nicotinamide adeine dinucleotide are the two typical intrinsic agents indicating the oxidization and reduction status of the tissue sample, with their fluorescence detected at wavelengths of 540 and 440 nm, respectively. Oxy and deoxy hemoglobin are two other significant intrinsic agents for evaluating the blood oxygenation saturation by recording their absorptions at two different wavelengths of 577 and 546 nm. These intrinsic agents were measured in this study for comparison of biochemical properties of rat liver in different gas inhalation treatments. Indocyanine green, a NIR extrinsic contrast agent measured at wavelengths of 780 nm/830 nm as excitation/emission can indicate blood pooling by displaying the distribution of blood vessels within a9ltumor. The advantage of high sensitivity, spatial resolution, and broad applied potentiality were demonstrated by the instrument during these experiments American Institute of Physics. DOI: / I. INTRODUCTION a Electronic mail: guyueqing_99@yahoo.com b Also at: Department of Testing and Measurement Engineering, Nanjing University of Aeronautics and Astronautics, Nanjing , People s Republic of China. One of the current goals in biomedical research is to design sophisticated diagnostic tools that can provide a direct link between the biochemical properties of tissues and patient care. Diagnostic tools based on the optical spectroscopy have the potential to link crucial tissue characteristics to individual patient care. 1 5 In particular, fluorescence techniques have capability to provide high sensitivity/specificity and quantitative profiles of tissue biochemical and morphological features. 6,7 Metabolic states of tissues directly related to the diseases can provide important parameters for the diagnosis, prognosis, and treatment of diseases, like tumors Maintenance of physiological oxygenation of normal tissue depends on the controlled balance between supply and demand. The vascular system supplies oxygen (O 2 ) and the mitochondria in the cells consume O 2 to generate biological energy for tissue function in the form of adenosine triphosphate ATP. Generally, under physical conditions, the supply of oxygen meets and usually exceeds the tissue metabolic demand. In unusual or abnormal conditions, the supply of oxygen may be inadequate to meet the metabolic demands of the tissue. On the demand side, abnormalities in energy metabolism are known to be related to defects in O 2 delivery or regulation of respiratory enzymes, combined with a high demand for energy caused by cell proliferation. On the supply side, deficiencies in nutrient supply, such as low vascular density and uneven vascular distribution, appear to play an important role. The disparity in supply and demand of oxygen results in hypoxia which leads to anaerobic metabolism and tissue acidosis. In general, the metabolic pathway in mitochondria consists of glycolysis, the citric acid cycle, and electron transport. Food molecules to be consumed for energy are first converted into acetyl CoA glycolysis. The citric acid cycle metabolizes acetyl CoA into the reduced electron carriers, nicotinamide adenine dinucleotide NADH, and reduced flavin adenine nucleotide (FADH 2 ). NADH is the reduced form of the electron carriers called pyridine nucleotide PN while FADH 2 is the oxidized form of the electron carriers called flavoprotein FP. The reduced carriers transfer their electrons through the electron transport chain to O 2, the ultimate /2002/73(1)/172/7/$ American Institute of Physics

2 Rev. Sci. Instrum., Vol. 73, No. 1, January D optical scanning in tissue 173 FIG. 1. Flavoprotein FP and pyridine nucleotide PN equilibrium in CAC. electron acceptor that oxidizes them into NAD and FAD. Subsequently, water is produced which drives the synthesis of ATP. The ratio of oxidized and reduced electron carriers gives a measure of steady-state metabolism. If metabolism were elevated, e.g., during exercise or tumor growth, there would be a shift towards an increased concentration of oxidized electron carriers. If the converse were true, the equilibrium would shift towards an increased concentration of reduced electron carriers. Oxygen can also perturb the metabolic equilibrium. A deficiency in the oxygen supply will reduce the number of reduced electron carriers that are oxidized and hence increase the concentration of the reduced form of these carriers. Figure 1 shows the oxidized electron carriers/reduced electron carriers FP/PN equilibrium in a citric acid cycle. In summary, the metabolism of cells can be characterized by the redox ratio, i.e., the ratio of concentration of FP and PN. Blood oxygenation is also a very important clinical parameter, which can be used to assess the vital state of organ and physiological conditions of patients. 16,17 Absorption of hemoglobin has the great potential of quantitatively measuring hemoglobin concentration and blood oxygenation saturation in tissue based on the fact of a substantial absorption difference between oxy and deoxy hemoglobin, which is the basis of most near infrared NIR instruments for in vivo measurement. The relationship between the cellular metabolism and intravascular hemoglobin saturation defines the oxygen flux from the capillary to cell. The oxygen flux in turn provides insight into the understanding of the supply versus demand of oxygen in tissue. This supports redox state and oxygenation saturation as being the most crucial parameters related to the tissue properties. On the other hand, the feasibility of diffuse optical tomography in NIR windows nm to image tissue in vivo and noninvasion promotes an elevated prospective for tissue function. Previous studies based on the intrinsic contrast agents oxy and deoxy hemoglobin have illustrated the progress achieved for tissue, especially for tumor tissue measurements However, the contrast ratio of the intrinsic agents on tumor and normal tissue is limited. In order to increase the sensitivity and specificity for tumor tissue detection, NIR extrinsic contrast agents are used to enhance the tumor: background ratio Except for indocyanine green ICG approved by the Food and Drug Administration of the United States, other NIR contrast agents remain in the trial process. Many researchers present a collaboration of their work as a contribution to this continuous study. The Weissleder group is developing the NIR molecular beacon which is activated by a tumor-associated enzyme. 30 The Glickson group is compounding a low density lipoprotein receptor specified NIR contrast agent. 34 The Achilefu and Kai Licha groups are modifying the ICG in an effort to enhance the qualities of its properties Prior to using these contrast agents, it is necessary to distinctively evaluate their biochemical characteristics such as biocompatibility, toxicity, pharmacokinetics, and specificity. The delivery of these extrinsic contrast agents to different organs is the most important parameter for the study. Using the high-resolution threedimensional 3D image system discussed in this article, we can localize the contrast agents in m 3 voxel. Meanwhile, we can study the effect of extrinsic contrast agents on the metabolic state of the tissue by measuring the fluorescence of FP, PN, and NIR contrast agents simultaneously. A snap freeze-clamping technique should be used to get the metabolic state of the tissue at the expected time. When performing the measurement, the tissue sample was immersed in the liquid nitrogen, which can preserve the metabolic state on tissue and also improve the fluorescent quantum yields, thus further improving the signal-to-noise S/N ratio. Our purpose is to design an instrument for studying the intrinsic and extrinsic contrast agents simultaneously. 20 years ago the chance group developed a redox scanner for measuring the intrinsic contrast agents Based on this instrument, we extended the wavelength range to the NIR window, and modified the circuit to improve the sensitivity and S/N ratio. Software can give rise to a 3D image. In this article, we present the theory and development of the instrument that can measure the redox state, blood oxygenation saturation based on intrinsic contrast agents, and NIR extrinsic contrast agents simultaneously. Two applications are presented to demonstrate the instrument. II. DESIGN OF THE INSTRUMENT A. Optical design of the instrument The instrument is designed mainly based on the fluorescence of PN, FP, NIR extrinsic contrast agents, and the absorption of oxy and deoxy hemoglobin. It can be easily extended over the wavelength range for various contrast agents. So, the optical design is the crux of the matter. 1. Measurement of the redox state The reduced form of PN NADH and the oxidized form of FP FAD emit fluorescence when excited with ultraviolet and bluelight, respectively. Figure 2 I displays the excitation and emission spectra of PN and FP in reduced and oxidized form. 11,12 Panels a and b display the oxidized and reduced FP excitation and emission spectra, respectively. Panels c and d show the oxidized and reduced PN excitation and emission spectra, respectively. Clearly, the excitation and

3 174 Rev. Sci. Instrum., Vol. 73, No. 1, January 2002 Gu et al. emission of FP is maximal when it is oxidized and minimal when it is reduced. The converse is true for PN. Hence, FP fluorescence indicates the concentration of the oxidized electron carrier at the terminal point of the electron transport chain, while PN fluorescence reflects the concentration of reduced electron carrier entering the chain. The ratio of fluorescence of FP PN, which is very slightly affected by hemoglobin and protein concentration, gives the measure of the metabolism. So, by monitoring PN and FP fluorescence simultaneously, one can monitor the metabolic changes of cells and tissues. In order to obtain the maximum fluorescence, the excitation wavelength should be selected within the range of nm for PN and nm for FP. The emission wavelength should be within the range of nm for PN and nm for FP see Fig. 2 I. However, oxy and deoxy hemoglobin have the maximum absorption within the range of nm as shown in Fig. 2 III, which will slightly absorb the excitation light for FP and the fluorescence of PN. In our design we can take advantage of the light source to make compensation. In this instrument, mercury arc is used as the source to provide the wavelength from nm. The emission spectrum is shown in Fig. 2 II. We select the peak energy at 365 nm as the excitation for PN and 436 nm for FP. Moreover, this wavelength avoids the excitation of collagen fluorescence which has the absorption between the range of nm with the absorption maximum at 325 nm. Considering all factors and compromising, we use optical filters to select 366 nm U-360, HBW40 nm, 65%T Edmund Scientific Company and 436 nm 440DF20, 50%T, Omega Optical Inc. as the excitation, 460 nm 455DF20, o.d. 5.5, Omega Optical Inc. and 530 nm 525DF20, o.d. 5.5, Omega Optical Inc. as the emission for PN and FP, respectively, which can reach a higher sensitivity and good S/N ratio. The optical density o.d. value of the emission filters outside the bandpass range are larger than 5.5, which can avoid the light leakage between the corresponding excitation and emission filters and also decrease the environment noise, which can further improve the S/N ratio. 2. Measurement of blood oxygenation The absorption of light in tissue is solely dependent on the oxy and deoxy hemoglobin. The concentration ratio of oxy and deoxy hemoglobin can reflect the hemoglobin saturation and thus reflect the oxygen delivery. In general, we can quantify the hemoglobin with dualwavelength, 2. 16,17 The absorption coefficient ( a )in units of cm can be written in terms of the Beer Lambert relationship FIG. 2. I The excitation and emission spectra of FP and PN in the oxidized and reduced state. II The emission spectra of the mercury arc II. III The absorption spectra of oxy and deoxy hemoglobin III. a Hb a 2 Hb Hb HbO2 2 2 Hb HbO2 HbO 2, HbO 2 2, where is the extinction coefficient (cm M ), HbO 2 and Hb are the tissue concentration of the oxy and deoxygenated form of hemoglobin, and is the background absorption on tissue. Generally, the background absorbers include water and lipids. 35,36 In the range of nm, the absorption coefficient for these background absorbs is very low, below cm. Compared with hemoglobin absorption about 10 5 cm in the visible range, 37 the background parameter can be ignored in the visible range. Here we just select visible wavelengths for hemoglobin measurement although NIR wavelengths are always used for in vivo measurements. If the hemoglobin saturation is defined as Y HbO 2 / Hb HbO 2 ), the one can combine the equations for tissue absorption: a Hb Y 2 HbO2 Hb 2 2 a. 2 Hb Y HbO2 Hb Furthermore, if 2 is the isosbestic wavelength of hemoglobin, then the equation can be rewritten as a Hb 2 a HbO2 Hb Y Hb Hb Thus, inspection of Eqs. 2 and 3 show that the ratio of absorption due to the hemoglobin measured at two wavelengths is a unique function of tissue hemoglobin saturation and independent of total hemoglobin concentration Hb HbO 2, the blood volume. As shown in Fig. 2 III, 546 nm is the isosbestic wavelength and 577 nm has the maximum different absorption between the oxy and deoxy hemoglobin. To take advantage of mercury lamp, 577 nm 580DF27, 55T%, Omega Optical 2

4 Rev. Sci. Instrum., Vol. 73, No. 1, January D optical scanning in tissue 175 FIG. 3. Optical design of the instrument. FIG. 4. Transmission spectra of the cold mirror. Inc. was selected as, and 546 nm 540DF37, 50T%, Omega Optical Inc. was for 2 to measure the hemoglobin saturation. 3. Optical design The simultaneous measurement of redox state and blood oxygenation is accomplished by using two optical filter wheels. One wheel contains the excitation filters and the other contains emission filters. The two wheels are synchronized for symmetry of the four excitation holes aligned to the four emission holes, shown in Fig. 3. Each of the filter pairs responds to one parameter allowing four parameters to be measured simultaneously. When performing measurements, the synchronized filter wheels are driven at a speed of 60 Hz with optical signals from the four emission filters detected by a photomultiplier tube PMT R928, Hamamatsu at 240 Hz frequency. In order to balance the intensity of signals in the four channels, the placement of neutral density filters o.d. value is between 0.1 and 2 is sometimes necessary. This optical design is illustrated in Fig. 3. Filtered illumination from the mercury arc lamp is coupled into one branch of a bifurcated optical fiber bundle 7 quartz fibers, 70 m core diameter for each, 0.34 numerical aperture, 1 fiber for emission in center, 6 fibers for excitation around the emission fiber and projected onto the surface of the tissue. The fluorescence of FP, PN, and reflected light of, 2 from the tissue surface can be collected through another branch of the bifurcated fiber bundle and propagated to the PMT after the emission filters. Signals from the PMT are amplified and converted into the digital data through an analog-to-digital converter 2210 series, 12-bit, Real Time Devices, Inc. and imaged as four pictures for each parameter. Image analysis of collected signals results in the redox ratio FP/ FP PN and blood oxygenation HbO 2 /(Hb HbO 2 ) on tissue. Extrinsic contrast agents can enhance the tumor:background ratio and thus improve the sensitivity/specificity in assessment of the tumor tissue. The NIR extrinsic contrast agents are especially applied to tumor diagnosis and therapy because the NIR windows nm maintaining the maximum penetrating depth in living tissue, present the advantage of noninvasion in vivo measurement. Extension of the wavelength of the system to the NIR range would significantly enhance the application potential of the instrument. In this system, a specialized cold mirror 650DRXRU, R: 300/600 nm T: nm, Chroma Technology Corp. is positioned between the light source and focus lens. The cold mirror reflects wavelengths shorter than 600 nm and transport wavelengths longer than 600 nm. Figure 4 shows the transmission response curve. This demonstrates the optical ability of two light sources functioning simultaneously with set positions described in Fig. 3. The mercury arc provides the wavelength range from ultraviolet to visible. Suitable NIR laser diodes, light emitting diodes LEDs, or a xenon lamp can be selected as the second source to provide NIR light. The wavelength range from ultraviolet to the infrared can be easily broadened, which obviously widens the application of the system for measuring absorption or fluorescence of various intrinsic and extrinsic contrast agents. In our study, the 780 nm laser diode LD 30 MW, Sharp Corp., 750 nm LD 0 MW, Sharp Corp., 760 nm 30 MW, Hamamatsu, 750 nm LED 40 MW, Hamamatsu and 660 nm LD 40 MW, Lasermate Corp. are typically assigned as the second source to provide the NIR light in different experiments. When measuring extrinsic contrast agents, FP and PN are preferable as intrinsic agents for simultaneous measurement. B. Description of the instruments The 3D image system incorporates two light sources, a cold mirror, two synchronized optical filter wheels set with bandpass filters symmetrically, a bifurcated light guide, two PMTs, a sample chamber, and seven motors with driven circuits. Figure 5 shows the diagram of the instrument. Lights from the two light sources mercury arc and NIR light source are simultaneously coupled into the excitation arm of the bifurcated light guide and propagated to the tissue sample. The fluorescence and reflection of various intrinsic and extrinsic agents are collected by the PMT1 through the emission arm of the fiber bundle after emission filters. Due to the high optical density o.d. 5.5 outside the bandpass windows of the emission filters, the fluorescence is effectively collected without reflection. The signals from the PMT1 are amplified and converted into digital signals after 2210 analog-to-digital converter ADC for computer processing. The PMT2 is used here to monitor and then eliminate the fluctuation effects of the light sources on detected signals. The two synchronized filter wheels make the four cor-

5 176 Rev. Sci. Instrum., Vol. 73, No. 1, January 2002 Gu et al. FIG. 5. Diagram of the 3D image instrument. responding filter pairs plug into the optical path in timeshare at 240 Hz frequency. Two step motors SM1, SM2 drive the optical fiber bundles to scan over the tissue surface 70 m from the fused end of the fiber bundle to tissue surface in the X, Y plane for projection of a 2D image. The resolution for each pixel is m 2. Step motors 3 and 4 SM3, SM4 control movement of the sample chamber in the X, Z plane, delivering the tissue sample to its designated position for milling and for measurements. The resolution in the Z direction is 10 m, allowing the tissue to be cut in 10 m thickness. After continual 2D images are recorded for subsequent sections, a 3D image is rendered. The spatial resolution for the instrument can reach to m 3. Figure 6 depicts the mechanical design of the sample chamber with the system. 15 The tissue sample is fixed in a liquid nitrogen (LN 2 ) filled chamber with the depth of LN 2 monitored by a thermal resistor. The low temperature preserves the metabolic state of the tissue and increase the fluorescent quantum yield. The metal cutter driven by a high speed rotating motor shaves the tissue sample and gives it a flat surface necessary for proper measurement. This cutter can be set to automatically divide the tissue into subsequent sections based on the initial input parameters. FIG. 6. Mechanical design of the sample chamber in the instrument from Quistorff and Chance. The systematic operation of the apparatus, including milling and measurement, is simplified by preprogramming a computer associated to the instrument. III. APPLICATION This image system, initially designed to measure the intrinsic and extrinsic contrast agents simultaneously, can now be widely used in the measurements of fluorescence and absorption over the entire wavelength range. The following present two of the applications for evaluating the instrument. A. Comparison of rat liver in different gas inhalations Redox ratio and hemoglobin saturation are the most important parameters in evaluating tissue s properties. Here the metabolic states of the rat livers in different gas inhalations are characterized by using the image system. Male rats weighing from 200 to 300 g were used in compliance with the institution s guidelines. The rats were divided into three groups: group 1 consisted of controls air inhalation ; group 2 underwent carbogen inhalation 95% O 2 and 5% CO 2 for 120 s to induce hyperoxia; and group 3 underwent nitrogen inhalation for 60 s to induce hypoxia. Each rat was anesthetized by intraperitoneal injection of sodium pentobarbital at the dose of 0.1 ml/kg. A tracheal cannula was employed so that gas inhalation could be monitored. After the intended gas inhalation air, nitrogen, or carbogen the exposed liver lobe was freeze-clamped by an aluminum tong which was sufficiently precooled in liquid nitrogen. The liver samples were embedded in a compound mixture 60% sterile water, 30% glycerol, and 10% alcohol and fixed in the liquid nitrogen filled chamber for the measurement. The top m layers of the samples were ground off by the lowtemperature milling system preparing the flat-surfaced sample for redox scanning. Figure 7 displays redox ratio and Hb saturation images of rat liver acini in a normaxic air inhalation, A and B, hyperoxic carbogen inhalation, C and D, and hypoxic nitrogen inhalation state, respectively. The white point of the

6 Rev. Sci. Instrum., Vol. 73, No. 1, January D optical scanning in tissue 177 FIG. 7. Redox ratio and Hb saturation of rat liver after different gas inhalation. A, B: air inhalation; C. D: carbogen inhalation; E, F: nitrogen inhalation. color bar indicates maximum value, and the black point indicates the minimum value. The fine spatial resolution of the instrument 40 m 40 m per pixel allowed us to observe the liver acini, which is a microcirculatory unit of the liver parenchyma. Both redox ratio and relative Hb saturation were highest in the liver of carbogen inhalation and lowest in that of nitrogen inhalation. The values of the control sample were at an intermediate range. The changes of the redox ratio and relative Hb saturation images after different kinds of gas inhalations were accessed quantitatively by calculating the average and the standard deviation value of each profile, are shown in Fig. 8. In the liver acini, both redox ratio and Hb saturation increased after the carbogen inhalation, due to the increase of PO 2 in the breathing gas. Nitrogen induced hypoxia in the tissue and as a consequence caused an accumulation of reduced FP and PN, reflected by a decrease of average redox ratio. A decrease of relative Hb saturation was also observed as expected. FIG. 8. Average and standard deviation of the redox ratio and HB saturation after gas inhalation. A Redox ration; B relative saturation. In this study, sterile indocyanine green ICG, 7.5 nmol Akorn Inc. used as a NIR extrinsic contrast agent, was injected via the tail vein into the rat subject about 200 g bearing the 9 L glioma tumor. 7.5 nmol ICG average concentration in blood is about 0.5 M, assuming the blood volume of the rat is 15 ml was administered for 1 min into the bloodstream. The rat was then snap frozen immediately approximately 30 s after the injection. The tumor was excited from the frozen rat model along with peripheral normal tissue and embedded in the compound mixture described in Sec. III A for fluorescence measurement in the 3D image system. FP, PN, and ICG fluorescence signals were collected simultaneously at different excitation and emission wavelengths. ICG bounding to plasma proteins and lipoproteins is uptaken and washed out quickly through the blood circulation. At the beginning of the injection, ICG circulates in the vasculature of the rat model and is incapable of diffusion to the other tissues. This allows the ICG solution to reflect the distribution of the vasculature and therefore demonstrating the validity of ICG as the blood vessel indicator. Furthermore, it results in an enhanced tumor:background ratio due to the fact that the density of the vasculature in a tumor is B. Measurement of intrinsic and extrinsic contrast agents simultaneously FIG. 9. Images of FP, PN, PN/ FP PN, ICG in 9 L tumor at the depth 600 m below the surface.

7 178 Rev. Sci. Instrum., Vol. 73, No. 1, January 2002 Gu et al. ACKNOWLEDGMENTS The authors are grateful to H. Ma and Y. Chen for their helpful discussion on circuits. They thank M. Leonard for the excellent drafting. This article is supported by Grant No. CO FIG D image of ICG in 9 L tumor at different depths. usually higher than that found in normal tissue. Figure 9 is the 2D image for FP, PN, and ICG at the depth of 600 m below the skin covering the tumor. The histograms plotted for each image describe the distribution of the signals. The results indicate that FP and PN were found in high concentration along the edge of the tumor and low concentration in the tumor s center, an indication of mitochondria position along the edge. The redox ratio indicates the tumor s abundantly oxidized center and reduced edge. Conversely, ICG maintains a strong signal in the center of the tumor and a weak signal along the edge. The image clearly exhibits the distribution of the blood vessels found within the tumor and surrounding tissue. The tumor sample was sequentially scanned at depths of 0.6, 1.1, 1.6, 2.1, and 2.6 mm from the skin. A 3D image, compiled from all subsequent 2D images, specifies the location of the contrast agents in the tissue, as shown in Fig. 10. However, these images reveal the lack of a direct relationship between the redox state and ICG. In applying this instrument, several variables of extrinsic contrast agents were studied, especially focusing attention on their delivery into different organs. Numerous experiments have demonstrated the instrument s high sensitivity and signal-to-noise ratio. It has proven to be practical and feasible for simultaneous measurement of intrinsic and extrinsic agents. Also, it can be applied to assay requiring the measurement of fluorescence and absorption at specific wavelengths over the wavelength range from ultraviolet to NIR. The four channels recorded in this system allow for simultaneous measurement of the four different parameters. The high spatial resolution m admits the observation of the morphology of the tissue sample, depicting the delivery of the contrast agents or drugs for tumor diagnosis and therapy, especially for photodynamic therapy. The 3D optical image system expresses great potential for applications in the biochemistry and biomedical fields. 1 W. Cheong, S. A. Prahl, and A. J. Welch, IEEE J. Quantum Electron. 26, J. B. Fishin and E. Gratton, J. Opt. Soc. Am. A 10, G. Bishnoi, A. H. Hielscher, N. Ramanujam, S. Nioka, and B. Chance, Proc. SPIE 3597, H. Liu, D. A. Boas, Y. Zhang, A. G. Yodh, and B. Chance, Phys. Meas. Biol. 40, H. Y. Ma, Q. Xu, J. R. Ballesteros, V. Niziachristos, Q. Zhang, and B. Chance, Proc. SPIE 3597, N. Ramanujam, Neoplasia 2, K. Sokolov, R. Drezek, and K. Gossage, Opt. Express 5, A. Mayevsky, S. Lebourdais, and B. Chance, J. Neurosci. Res. 5, A. Mayevsky, H. Kaplan, and B. Chance, Brain Res. 367, J. C. Haselgrove, C. L. Bashford, and B. Chance, Brain Res. 506, B. Chance, B. Schoener, and R. Oshino, J. Biol. Chem. 254, B. Quistorff and B. Chance, Anal. Biochem. 108, B. Quistorff and B. Chance, Frontiers Biol. Energetics 2, B. Chance, J. Appl. Cardiol. 4, B. Chance, V. Legallais, J. Sorge, and N. Graham, Anal. Biochem. 66, E. M. Sevick, B. Chance, and J. Leigh, Anal. Biochem. 195, B. Chance, Proc. SPIE 1204, N. Ramanujam, C. Du, H. Y. Ma, and B. Chance, Rev. Sci. Instrum. 69, B. Chance, E. Anday, and S. Nioka, Opt. Express 2, S. Zhou, Y. Chen, Q. Zou, S. Nioka, X. Li, L. Pfaff, and B. Chance, Proc. SPIE 3597, A. M. Siege, J. J. A. Marota, and D. A. Boas, Opt. Express 4, B. W. Pogue, M. Testorf, and T. Mcbride, Opt. Express 1, V. Ntaiachristors, H. Ma, and B. Chance, Rev. Sci. Instrum. 70, B. Chance and K. A. Kang, Rev. Sci. Instrum. 67, H. Eda, I. Oda, Y. Ito, and Y. Wada, Rev. Sci. Instrum. 70, K. Licha, B. Riefke, V. Ntziachristos, A. Becker, B. Chance, and W. Semmler, Photochem. Photobiol. 72, S. Achilefu, R. B. Dorshow, J. E. Bugaj, and R. Rajagopalan, Invest. Radiol. 35, S. Achilefu, R. B. Dorshow, J. E. Bugaj, and R. Rajagopalan, Proc. SPIE 3917, K. Licha, V. Ntziachristos, B. Riefke, A. Becker, B. Chance, and W. Semmler, Proc. SPIE 3566, R. Weissleder, C. H. Tung, U. Mahmood, and A. Bogdanov, Nat. Biotechnol. 17, T. Desmettre, J. M. Devoisselle, and S. Mordon, Surv. Ophthalmol. 45, S. Mordon, J. M. Devoisselle, S. S. Begu, and T. Desmettre, Microvasc. Res. 55, J. S. Reynoilds, T. L. Troy, R. H. Mayer, A. B. Thompson, D. J. Waters, K. K. Cornell, P. W. Snyder, and E. M. Sevick, Photochem. Photobiol. 70, R. Zhou, K. Yang, H. Li, S. L. Katz, B. Chance, and J. D. Glickson, Ground report personal communication. 35 A. E. Cerussi, A. J. Berger, F. Bevilacqua, N. Shah, D. Jakubowski, J. Butler, R. F. Holcombe, and B. J. Tromberg, Acad. Radiol. 8, H. Q. Woodard and D. R. White, Br. J. Radiol. 59, Oregon Medical Laser Center