Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

A microscope-based imaging system has been developed that records single photon emissions from luminescent samples. The time and position of each detected photon is recorded so that integration times for image reconstruction can be selected and adjusted during data analysis. The system performs automated acquisition of brightfield and epifluorescence images that are correlated with the photon images. The features of this system will be demonstrated in the context of studying the dynamics of living cells using luminescent markers such as luciferase and aequorin, which can be introduced by microinjection or transfection. The value of using this approach to study the effects of pharmacological agents on gene expression in living cells will be presented.

We have performed separation of bacterial and cultured cancer cells from peripheral human blood in microfabricated electronic chips by dielectrophoresis. The isolated cells were examined by staining the nuclei with fluorescent dye followed by laser induced fluorescence imaging. We have also related DNA and RNA from the isolated cells electrically and detected specific marker sequences by DNA amplification followed by electronic hybridization to immobilized capture probes. Efforts toward the construction of a 'laboratory-on- a-chip' system will be presented which involves the selection of DNA probes, dyes, reagents and prototyping of the fully integrated portable instrument.

Red light is transmitted through live tissue more efficiently than other wavelengths of visible light, thus by red-shifting the emission of bioluminescent reporters, we may enhance their utility for in vivo monitoring of biological processes. Codon changes at positions that may shift the yellow-green emission to red, based on studies of a related luciferase, were introduced into a variant of the North American firefly luciferase. Clones containing the desired mutation were selected based on the introduction of unique restriction enzyme sites and transfected into NIH 3T3 cells. Expression levels were evaluated using an intensified charge coupled device camera. Upon spectral analysis, all mutant luciferases demonstrated red-orange emission. Two emission peaks were detected in each spectrum, each clone with different peak heights at 560 nm and 610 nm. Sequence analyses of the compete coding region of several clones confirmed the presence of the target mutations, although sequence variation was observed at several secondary sites, likely resulting from the infidelity of Taq polymerase used in the mutagenesis protocol. A clone that demonstrated a strong 610 nm peak with a minimum shoulder at 560 nm was selected for use in animals. In summary, a red-shifted mutant of a well-characterized luciferase reporter gene was generated. Red light from this enzyme may both penetrate mammalian tissues to a greater extent and provide a tool for multicolor biological assays.

Fluorescence imaging is a useful tool to study the sequence an dynamics of the spread of excitability in biological tissues. Gastrointestinal muscles are particularly amenable to imaging using standard video rates because the frequency of events i slow and propagation velocities are slow. Calcium-sensitive fluorescent indicators such as fluo-3 provide effective markers of excitability because optically they exhibit high quantum yields and calcium plays important biological roles including regulating intracellular signaling and muscle contraction. Video sequences of gastrointestinal tissues demonstrate the existence of multiple preferred locations to indicate excitability. The spatial and temporal resolution of microscope-based imagin system allows pacing sites to be identified within single muscle bundles. Anisotropic conduction velocities result in spatially complex patterns of excitability where the range of propagation appears to be limited by 'collisions' with neighboring excitable events or recently activated regions. Although standard video rates are generally not sufficient to monitor more rapid excitable event such as nerve action potentials, fluorescence imaging can be used to investigate excitability mechanisms in tissues such as smooth muscles where event frequencies and propagation velocities are low.

Changes in pHⁱⁿ and (Ca²⁺)^cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca²⁺ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca²⁺ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca²⁺ regulatory systems: plasmalemmal vacuolar- type H⁺-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca²⁺ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca²⁺ regulation via H⁺-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHⁱⁿ regulation in microvascular endothelial cells.

Quantum efficiency, (Phi) , and molar absorption coefficients, (epsilon) , are determined for Terbium and Europium polyazamacrocylic, phosophonic acid chelating agents that have been shown to be used in biomedical imaging applications.

The investigation of cyanine dyes as contrast agents in optical tumor imagin has been a focus of our recent work. We have shown that i.v. injected hydrophilic indotricarbocyanine derivatives enable tumor detection by fluorescence imagin and by frequency-domain absorption spectroscopy. Our current objective is to extend this approach by conjugating these dyes with specific biomolecules in order to enhance targetability and to introduce acid-cleavable links that enable dye release in acidic cell compartments. Accordingly, we have synthesized cyanine dyes which contain different acid-cleavable hydrazone links and which were coupled to peptides, proteins and antibodies. We have studied the release of the dyes under various pH conditions. Our results show that dye release from transferrin increased under acidic conditions, while at neutral pH the stability was higher. Additionally, we observed pH-dependent fluorescence enhancement during cleavage. Cellular fluorescence microscopy experiments indicated that intracellular trapping is possible. In conclusion, cyanine dyes bound to biomolecules by acid- cleavable bonds could act as promising optical contrast agents. Further work will include optimization of release rates by chemical modification and in vivo imaging studies.

It was assumed that each cell is a homogeneous suspension may have a slightly different pH and membrane potential. A wide range of pH-sensitive fluorescent dyes BCECF, SNARF, FITC, carboxyfluorescein, fluorescein and pyranine have been carefully tested for the accuracy and reliability of their pH-response inside living cells. The intracellular milieu was simulated by a series of mineral buffers with addition of proteins. The pH values have been determined from the excitation ratios 490/435 nm for BCECF, FITC, carboxyfluorescein and fluorescein, and 450/400 nm for pyranine, emission ratios 518/529 nm for BCECF and 635/590 nm for SNARF. The spectrally determined values were then compared with the pH values of buffers measured by a glass electrode. Using the data from the calibration procedure, we evaluated individual intracellular pH values of a large number of cells within one cell population. The confocal ratio fluorescence microscopy revealed pH maps from which both cytoplasmic and vacuolar pH values could be determine, flow cytometry gave enormous amount of average intracellular pH values of individual cells of a whole cell population. Each cell population exhibited significant differences in both cytoplasmic pH values among individual cells. The pH distribution of a typical cell population appeared to fit a Gaussian curve. In yeast it was a Gaussian curve with half- width values around 0.4 pH unit. The men pH values depended on the growth phase, H-ATPase activity and external pH values. The preliminary result with the new membrane potential dye tetramethylrhodamine methyl ester indicated that similarly to pH values, there is a heterogeneity in membrane potential values among cell sin one cell population. The data presented above suggest that each ell behaves as an individual with an individual set up of its metabolism. This 'fine tuning' of the metabolism result in slightly higher or lower pH or membrane potential values that can be detected by fluorescence techniques.

Radiometallated peptides that exhibit high specificity for cognate receptors over expressed on cancer cells offer important potential as site-directed diagnostic and therapeutic radiopharmaceutical. The formation of effective radioactive drugs for specific in vivo targeting of cancerous tumors is being facilitated by the integration of novel chelation strategies and receptor-avid derivatives. Significant efforts are being made to design Technetium-99m labeled for diagnostic imaging of cancerous tumors for use in conjunction with Single Photon Emission Tomography instrumentation in nuclear medicine. Receptor avid radiopharmaceutical are also being developed that utilize other radionuclides for imaging and therapeutic applications. Despite the technological challenges that must be overcome, radiolabeled receptor avid peptide conjugates are providing promising site-directed targeting agents for the assessment and treatment of cancerous tumors in humans.

The ligand, 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrakis(methylenephosphonic acid, ethyl ester) (DOTPME) was made membrane permeable by preparing its acetoxymethyl (AM) derivative (DOTPME-AM). The synthetic approach was to prepare the AM ester of the phosphonate side-chain prior to attachment to the macrocyclic ring. P NMR was used to demonstrate that DOTPME-AM can penetrate cell membranes, get hydrolyzed by cellular esterases to regenerate charged DOTPME, and hence become trapped inside cells. This technology offers the potential of designing Ca²⁺ and Mg²⁺ specific ligands for analytical, noninvasive measurement of these ions by ³¹P NMR.

The relative opacity of mammalian tissue permits the transmission of light from internal biological light sources in small laboratory animals. As such internally expressed bioluminescence can be detected externally revealing spatiotemporal information about tagged biological functions. Enzymes that emit light, photoproteins, have been characterized photoproteins have been used as reporters in a variety of in vitro and ex vivo assays and are now being employed as sources of internal biological light that can be eternally monitored in living animals. Using this approach, spatiotemporal changes in patterns of gene expression, infectious disease and tumor cell growth can be revealed in real time. Monitoring light emissions from internal sources provides a powerful method for cellular and molecular analyses in living animals. This approach is particularly well suited for the evaluation of potential therapeutics including the efficacy of novel DNA-based therapies and vaccines.

The combination of a very high gain image intensifier, a CCD camera and an image processor which eliminates read out noise of the CCD camera, enables one to detect single photon events by photon counting imaging. The detected photons are then counted in the image processor to generate an image. The image intensifier is also designed to minimize dark noise that determines the sensitivity limit. The photon counting imaging system can be used as a standard intensified CCD camera by lowering the gain of the intensifier. This is useful for focusing and getting an image of the entire object in brightfield, which is used as the reference for the luminescent image. This is a very flexible imagin system used to monitor luminescence; such as gene expression, genetically modified luminescent microorganisms, ATP from microorganisms and luminescence from aequorin stimulated by calcium ions. This system also makes it possible to see luminescence generated inside of small animals through their skin without harming them and it is becoming an important tool for In-Vivo monitoring of small animals. In this paper, we discuss recent technique of photon counting imaging.

Green fluorescent protein (GFP)-expressing cell-lines have been established by our laboratory that permit the visualization and imaging of primary tumors and micrometastases in live tissue and live animals. Hamster and human cancer cell-lines were transfected with vectors containing the humanized GFP cDNA. Stable high-level expression of GFP was maintained in subcutaneously and orthotopically growing tumors in nude or SCID mice. Subsequent micro-metastases were visualized by GFP fluorescence in live tissue of systematic organs down to the single-cell level. GFP-expressing lung and prostate cancer were visualized to metastasize widely throughout the skeleton when implanted orthotopically in nude mice. With these GFP-cell lines, we have developed models that closely mimic the clinic situation. We have now developed a mean to visualize the onset and progression of angiogenesis of growing and spreading tumors by injecting a fluorescent rhodamine dye to the GFP-tumor-bearing mice indicate that the onset and extent of tumor angiogenesis depends on the site and type of tumor growing in the animal. These models are ideal for studying the mechanisms of cancer metastasis and for discovery of angiogenesis and metastasis inhibitors.

A recently developed bioluminescent assay was used to study the influence of age and inoculum size on the acute susceptibility of newborn and juvenile BALB/c mice to Salmonella gastrointestinal infection. Three strains of Salmonella were tagged by expression of the lux operon from Photohabdus luminescenes. Using a range of inoculum sizes varied over 6 orders of magnitude, mice aged 0-6 weeks were infected by oral inoculation. LIght emitted from the tagged bacteria and transmitted through mouse tissues was used to noninvasively monitor disease progression over 7 days. In neonatal mice there was evidence of gastrointestinal infection at 24 hours even with small inocular, and at 4-7 days, the patterns of photon emission and remained and healthy throughout the study period. Inoculation of neonates with tagged LB5000 and BJ66 resulted in severe gastrointestinal infections with low and intermediate sizes of inocula respectively. These strains are known to be of reduced virulence in adult mice. These age-related differences in susceptibility emphasize the need to define virulence in the context of age of the host, and implicate maturation of innate resistance factors in determining disease patterns. Identifying these host-factors and further defining the bacterial determinants of virulence in the neonatal host will be facilitated by this noninvasive study of infection using bioluminenscent methods.

The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

Evaluation of antineoplastic therapies would be enhanced by sensitive methods that noninvasively asses both tumor location and neoplastic growth kinetics in living animals. Since light is transmitted through mammalian tissues, it was possible to externally monitor growth and regression of luciferase labeled murine tumor cells engrafted into immunodeficient mice. External quantification of tumor burden revealed the biological impact of the chemotherapeutic agent cyclophosphamide on the kinetics of tumor growth in living animals. Therapeutic activity was apparent but this drug did not eliminate the NIH 3T3 cell signal over the 28 d time course. This novel, noninvasive system allowed sensitive, real time spatiotemporal analyses of neoplastic cell growth and may facilitate rapid optimization of effective therapeutic treatment regimes.

Many tumor cells are characterized by the overexpression of highly specific transferrin receptors. Transferrin is bound to its receptor, which is followed by rapid internalization into intracellular compartments; it can thus be used for the specific delivery of coupled contrast agents in to tumor cells. The aim of our study was to investigate the tumor- specificity of transferrin-modified contrast agents for optical imaging and magnetic resonance imaging (MRI). Indotricarbocyanine, a near-IR absorbing dye, and ultrasmall superparamagnetic iron oxide particles, a contrast material for MRI, were covalently coupled to transferrin and examined in vitro and in vivo for their potential as tumor-specific contrast agents. CEll culture experiments using HT29 and A431 tumor cells showed specific uptake of transferrin- modified contrast agents by the cells at 37 degrees C, while uptake was inhibited at 4 degrees C or in the presence of an excess of unlabeled transferrin. Optical imaging of tumor- bearing nude mice after intravenous injection of transferrin-ICC resulted in pronounced tumor fluorescence. Tumor specificity was also observe in MRI, where transferrin-USPIO accumulated in SMT2A tumor-bearing rats. We conclude that transferrin-modified contrast agents for optical imaging and MRI can be specifically delivered to tumor tissue by use of transferrin receptor pathways.

We have measured the spectroscopic properties of upconverting phosphors relevant to their use of in-vitro diagnostics and other application. These novel reporters have a number of useful properties for biological and medical applications, including no autofluorescent background, no photobleaching, excitation with inexpensive and compact cw diode laser sources, and narrow emission bands. Properties we have measured include the intensity dependence of the absolute phosphorescence power per phosphor particle, the intensity-dependent temporal response, and histograms of phosphorescence intensities on a single particle basis.

A nanosecond gating technique applied to conventional, 'off the shelf', photomultiplier tubes (PMTs) is described. This technique is most suitable for detection and analysis of fluorescence, induced by pulsed lasers. A high voltage square wave generator is applied to 3 dynodes of the PMT. The result is a fast time-gated optical detector with a high dynamic range of approximately 80 dB. The active state of the gate, i.e. the pulsewidth of the applied square wave, sets the integration time in accordance to the decay time of the fluorescence signal. It can be selected by the user from less than 2 ns to several hundred nanoseconds. The pulse generator gets its energy from a transmission line, which is charged by a DC voltage of 650 Volts. This determines the magnitude of the pulsed voltage, which is applied to the 3 PMT stages. Twice the electrical length of the transmission line gives the pulse width. The pulse is initiated by a fast electrical switch with switching times of approximately 700 ps and a low time jitter of only 200 ps related to the trigger input. A distinct separation of the fluorescence and superposing background, e.g. scattered laser light, is achieve due to fast pulse transitions. Output signals from the PMT are processed by a fast integration. This can reduce the effects of electrical cross talk of the gating pulse into the signal line to sufficiently low values.

To establish optical in situ detection of mitochondrial malfunction, non-radiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. An experimental setup was developed, where these signals are detected simultaneously using a time-gated technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the apparatus are described, and preliminary result of cultivated endothelial cells are reported. Results are compared with those obtained from a previously established microscopic system and discussed in view of potential applications.

Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

This study investigates the spectro-temporal fluorescence emission of normal and diseased coronary arteries with graded levels of atherosclerosis. Fluorescence emission of 58 excised human coronary artery samples was induced with N₂ laser pulses and detected with a MCP-PMT connected to a digital oscilloscope. The samples were H and E and Movat stained and histologically classified in accordance with AHA classification. An algorithm based on Laguerre expansion of kernels was used to deconvolve the intrinsic fluorescence impulse response function from the measured transient pulse. A biexponential function depicted the fluorescence decay characteristics. We noticed 1) in spectral domain: peak fluorescence intensity was at 380 nm for normal and initial lesions samples and blue-shifted for advanced lesions; intensity at 450-480 nm decreased from approximately 65 percent peak intensity for normal samples to approximately 30 percent for Type V lesions; 2) in time domain: longer lasting emission for the advanced lesions. The decay constants varied as a function emission wavelength and lesion type. For instance the time constants for Type V lesions measured at 390 nm were significantly larger that those measured on normal arterial wall. The fast term decay contributed to a higher degree to the impulse response function for normal tissue. These results reveal that the analysis of the temporal characteristics of fluorescence can be used to differentiate between coronary lesion and normal coronary wall. The time domain information complements the spectral domain intensity data for improved differentiation between graded levels of coronary lesions.

In this study, we examine the possibility of differentiating between classes of atherosclerotic lesions based on time- resolved fluorescence spectroscopy and we compare the performance of classification schemes that use either the time-resolved spectra or only the intensity spectra. Transient fluorescence emissions induced by pulsed nitrogen laser excitation was measured on 87 excised samples of human aorta. The samples were classified histologically using the AHA classification Predictor variables derived from the time-resolved spectra included the spectral intensities at 360-510 nm and parameters of a biexponential fit of the fluorescence impulse response function. Stepwise discriminant analysis using these predict variables showed that a few predictor variables sufficed to correctly classify 89 percent of the samples. Excluding the time- dependent decay and using only the spectral intensities, the percentage of correctly classified cases was significantly lower: 51 percent. These results establish that time- resolved fluorescence spectroscopy markedly improved on the performance of steady-state fluorescence spectroscopy for fine classification of atherosclerotic lesions.

Near-IR biomedical optical imaging consists of imaging interior volumes on the basis of optical property contrast from measurement conducted at the air-tissue interface. However, the ability to optically image or detect diseased tissue volumes located deep within tissues depends upon the contrast provided by differences in absorption and scattering. The exogenous contrast offered by fluorescent contrast agents may be superior to that provided by nonfluorescing, light-absorbing compounds, when the optical measurement are conducted with frequency-domain techniques. However, the reconstruction of internal fluorescent properties of quantum efficiency and lifetime has been difficult, especially when the finite partitioning of fluorescent compounds takes place between normal and diseased tissues. Also, the correct absorption coefficient map is required for the successful reconstruction of lifetime. Herein we present a novel fluorescence-enhanced imaging algorithm for frequency-domain photon migration measurements imaging differs in that it utilizes measurements of generated fluorescent wave instead of scattered excitation wave. Using synthetic data sets, we demonstrate fluorescence-enhanced imaging using FDA approved fluorescent agent. Indocyanine Green. Our results show the fluorescence-enhanced imaging algorithm works well up to 10:1 dye uptake ratio, and it is relatively insensitive to measurement noise. In addition, we present the lifetime reconstruction with a modified fluorescence-enhanced imaging technique.

Two time-domain fluorescence imaging techniques have been developed and tested for the detection of malignancies after administration of a marker having a fluorescence lifetime longer than that of the tissue natural fluorescence. The first technique, based on the time-gated approach, relies on the acquisition of fluorescence images after a suitable delay with respect to the excitation pulses in order to discriminate the long living exogenous fluorescence from the short living endogenous one. The second technique, called lifetime imaging, measures the spatial map of the fluorescence decay time of the sample, allowing an indirect detection of the regions where the concentration of the marker is higher. The first method is simpler, does not require any image processing, leading to a true real time video, but it works better when a rather strong signal comes from the sample. The second method requires two or more images to be acquired and processed sequentially; therefore, it is slower, but proved to be more sensitive in low signal conditions. The two techniques have been applied for the detection of skin tumors in humans after the topical application of (delta) -aminolevulinic acid ointment, which promotes the accumulation of the endogenous porphyrin Protoporphyrin IX preferentially in proliferative tissues. Preliminary results are encouraging.

The objective of this work is to identify an imaging modality which can best locate heterogeneous tissue volumes when designer contrast agents are used. We used a multi- pixel, homodyne, frequency-domain photon migration detection system to acquire images of 830 nm fluorescent heterogeneities immersed within a tissue-simulating phantom that contained 0.5 percent Intralipid solution. An expanded beam of 25 mW, 778 nm light modulated at 100 MHz illuminated the phantom surface. Specifically, we monitor fluorescence average intensity, modulation amplitude, phase, and modulation ratio resulting from micromolar concentrations of indocyanine green and DTTCI embedded within tissue- mimicking, highly scattering media. The results indicate that under conditions of perfect uptake, only phase and modulation distinguish dye solutions that possess equivalent fluorescence yield but unequal lifetime when both heterogeneities are located 0.5 cm from the illumination surface. Enhanced phase contrast was observed for fluorescent solutions with short lifetimes located within a surrounding of longer lived fluorophore and visa versa. These results have important implications for the development of contrast agents whose lifetimes depend on the local biochemical environment.

In this paper we demonstrate the ability to detect the frequency-domain fluorescent signal from the contrast agent indocyanine green within the mammary chain of dogs with spontaneous mammary tumors. We use a gain-modulated image intensifier to rapidly capture multi-pixel images of the fluorescent modulation amplitude, modulation phase, and average intensity signals. Excitation is provided by a 100 MHz amplitude-modulated, 780 nm laser diode. Time series images of the uptake and clearance of the contrast agent in the diseased tissue are also presented.

Temporal emission profiles of fluorescein dye-labeled polymers of molecular weight range 4 K to 500 K at two polarization directions parallel and perpendicular to that of the linearly polarized exciting light were measured. The polarization decay time of dye-polymer conjugates increases with molecule weight. Images of an object containing varying molecular weight fluorescein dye-labeled polymers embedded inside turbid media were investigated. The difference image at high polymer molecular weight was found much brighter and clearer than that at low molecular weight. This observation demonstrates that high molecular weight dye-polymer conjugates can be used to enhance the imaging depth and visibility of objects hidden inside scattering media and tissues.

Stochastic methods originally devised for geophysical tomography are adapted to the biomedical optical tomography problem. Frequency domain measurements of modulated NIR light are inverted using a Bayesian approximate extended Kalman filter. Minimum variance updates for the linearized problem are calculated from explicit models of the parameters error covariance, the covariance of the system noise, and the measurement error covariance. The method is not iterative per se, but may be applied iteratively to account for strong nonlinearities. Data-driven zonation is used to dynamically reduce the parameterization for improved efficiency, sensitivity, and stability of the inversion. By modeling the parameters as beta distributed random variables, estimates are kept within feasible limits without ad hoc adjustments. In preliminary studies using synthetic domains we have successfully resolved spatially heterogeneous parameters such as absorption, fluorescence lifetime, and quantum efficiency. The method is shown to be much more accurate and computationally efficient than a more traditional Newton-Raphson method. On a 33 by 33 grid, distributed values of a single unknown parameter can be accurately identified in under 2 minutes on a 350 MHz Pentium.

In this contribution, the inverse optical imaging problem is formulated as both simply bound-constrained and unconstrained minimization problems in order to illustrate the reduction in computational time and storage. The Galerkin finite element method is used for the numerical solution of the excitation and emission diffusion equations. The inverse approach employs the truncated Newton method with trust region, and modified automatic reverse differentiation method is used to calculate the gradients thus sped up the computation of the optimization problem.

In order for photon migration imaging to be clinically feasible, data acquisition must be fast and efficient. Rapid data acquisition minimizes error due to patient movement and improves patient acceptance. Multi-pixel imaging techniques offer the fastest method of data acquisition, however difficulties may exist in coupling the data with inversion algorithms. Single pixel measurements, while easier to couple to an inversion algorithm, require longer data acquisition times. Herein a heterodyne procedure for speeding the data acquisition time using single pixel techniques is presented. This simple extension of the heterodyne method enables the use of multiple sources simultaneously with the signal from various source-detector pairs discriminated by FFT analysis.

Ischemia-reperfusion injury is the leading cause of early dysfunction following transplantation. Currently, there are no techniques available to accurately measure ischemic changes during organ storage. Therefore, the interest exists in developing non-invasive monitoring techniques. We used NADH and tryptophan as fluorescent markers, since both are intrinsic fluorophores and excellent indicators for levels of hypoxia and protein denaturation, respectively.