The revolution of in vivo cancer biology enabled by fluorescent proteins is described. The high extinction coefficients, quantum yields, and unique spectral properties of fluorescent proteins have been taken advantage of in order to visualize, in real time, the important aspects of cancer in living animals, including tumor cell trafficking, invasion, metastasis, and angiogenesis. Fluorescent proteins enable whole-body imaging of tumors on internal organs. These multicolored proteins have allowed the color-coding of cancer cells growing in vivo with distinction of different cell types, including host from tumor, with single-cell resolution.

To improve the labeling efficiency of a low-density lipoprotein (LDL)-based photosensitizer (PS) for achieving high probe to protein payload, a tetra-t-butyl silicon phthalocyanine bearing two oleate moieties at its axial positions, SiPcBOA, is designed and synthesized. Using this novel strategy, SiPcBOA reconstituted LDL (r-SiPcBOA-LDL) with a very high payload (SiPcBOA to LDL molar ratio >3000 to 35001:1) is obtained. Using electron microscopy, we find reconstituted LDL (rLDL) with such a high payload essentially retains the mean particle size of native LDL. Since acetylated LDL binds to scavenger receptors of endothelial and microglial cells instead of LDLR, SiPcBOA reconstituted acetylated LDL (r-SiPcBOA-AcLDL) is also prepared to serve as a negative control to validate the LDL receptor (LDLR) targeting specificity. Confocal microscopy studies demonstrate that the internalization of r-SiPcBOA-LDL by human hepatoblastoma G2 (HepG2) tumor cells is mediated by LDLR pathway. The in vitro photodynamic therapy (PDT) response of HepG2 cells to r-SiPcBOA-LDL is compared to SiPcBOA (free drug control) using a clonogenic assay. The slopes of the linear regression fit to the logarithmic data for these two plots are significantly different from each other (p=0.0007), indicating greatly enhanced efficacy of LDLR-targeted PDT.

The rat represents an excellent mammalian model for broadening medical knowledge, and a wealth of information on its physiology has been obtained from its use as an experimental organism. Furthermore, its ample body size allows various surgical manipulations that cannot be performed on a mouse. Many rat models mimic human diseases and have therefore been used in a variety of biomedical studies, including physiology, pharmacology, and transplantation. In an effort to create specifically designed rats for new biomedical research and the field of regenerative medicine, we develop an engineered rat system on the basis of transgenic technology and succeed in establishing unique rats that possess genetically encoded color probes: green fluorescent protein (GFP), DsRed2 (red liver), Cre/LoxP (red to green), and LacZ (blue and luminescence). In this work, we highlight their characteristics and describe recent applications for tissue engineering and regeneration. Coupled with recent progress in modern imaging systems, these transgenic rats are providing powerful tools for the elucidation of many cellular processes in biomedical science, and may lead to innovative medical treatments.

We develop a highly specific antibody-dye conjugate for optical imaging of peripheral lymph nodes. The contrast agent consists of the monoclonal antibody recognizing endothelial ligands for the lymphocyte homing receptor L-selectin, MECA-79, and a near-infrared (near-IR) fluorescent indotricarbocyanine dye. The targeting and biodistribution behavior of MECA-79 is studied after radio-iodination and intravenous injection into mice demonstrating specific uptake in lymph nodes and accumulation in high endothelial venules (HEV). After conjugation of MECA-79 with indotricarbocyanine dye, the fluorescence imaging properties of the MECA-79 dye conjugate are examined by intravenous injection in nude mice and laser-induced fluorescence whole-body imaging in vivo. The MECA-79 antibody-dye conjugate accumulates in peripheral lymph nodes, whereas an isotype antibody-dye conjugate does not. Specific lymph node near-IR fluorescent signals become detectable within minutes after injection, and stable imaging persists for more than 24 h. The results demonstrate that vascular targeting of endothelial expression of glyocproteins is feasible to visualize the accumulation of near-IR fluorescent MECA-79 in lymph nodes, making this technology potentially useful to characterize processes of inflammation.

The effect of sampling region size and tissue heterogeneity is examined using fluorescence histogram assessment in a rat prostate tumor model with benzoporphyrin derivative fluorophore. Spatial heterogeneity in the fluorescence signal occurs on both macroscopic and microscopic scales. The periphery of the tumor is more fluorescent than the center. Fluorescence is also highest nearest the blood vessels immediately after injection, but over time this fluorescence becomes uniform through the tumor tissue. Using microscopy analysis, the fluorescence intensity histogram distributions follow a normal distribution, yet as the sampling area is increased from the micron scale to the millimeter scale, the variance of the distribution decreases. The mean fluorescence intensity is accurately measured with a millimeter size scale, but this cannot provide accurate measurements of the microscopic variance of drug in tissue. Fiber probe measurements taken in vivo are used to confirm that the variance observed is smaller than would be expected with microscopic sampling, but that the average fluorescence can be measured with fibers. Sampling tissue with fibers smaller than the intercapillary spacing could provide a way to estimate the spatial variance more accurately. In summary, sampling fiber size affects the fluorescence intensities detected and use of multiple region microscopic sampling could provide better information about the distribution of values that occur.

The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.

Optics has played a key role in the rapidly developing field of molecular imaging. The spectroscopic nature and high-resolution imaging capabilities of light provide a means for probing biological morphology and function at the cellular and molecular levels. While the use of bioluminescent and fluorescent probes has become a mainstay in optical molecular imaging, a large number of other optical imaging modalities exist that can be included in this emerging field. In vivo imaging technologies such as optical coherence tomography and reflectance confocal microscopy have had limited use of molecular probes. In the last few years, novel nonfluorescent and nonbioluminescent molecular imaging probes have been developed that will initiate new directions in coherent optical molecular imaging. Classes of probes reviewed in this work include those that alter the local optical scattering or absorption properties of the tissue, those that modulate these local optical properties in a predictable manner, and those that are detected utilizing spectroscopic optical coherence tomography (OCT) principles. In addition to spectroscopic OCT, novel nonlinear interferometric imaging techniques have recently been developed to detect endogenous molecules. Probes and techniques designed for coherent molecular imaging are likely to improve the detection and diagnostic capabilities of OCT.

Heat stress responses are analyzed in cancer cells by applying different microscopy techniques for targeting various fluorescently labeled or native structures. Thermotreatments are performed at 40, 45, 50, and 56 °C, respectively, for 30 min each, while controls were kept at 37 °C. Actin cytoskeletons labeled with Alexa Fluor® 488-conjugated phalloidin are imaged by wide-field fluorescence microscopy (WFFM). Structural plasma membrane stabilities are labeled with fluorescent quantum dots and analyzed by laser scanning microscopy (LSM). High-resolution atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to study morphological features and surface structures. Fluorescence images reveal F-actin to be a comparatively thermolabile cell component showing distinctive alteration after heat treatment at 40 °C. Destabilization of actin cytoskeletons proceed with increasing stress temperatures. Active reorganization of plasma membranes coincidental to heat-induced shrinkage and rounding of cell shapes, and loosening of monolayered tissue are observed after treatment at 45 or 50 °C. Active stress response is inhibited by stress at 56 °C, because actin cytoskeletons as well as plasma membranes are destroyed, resulting in necrotic cell phenotypes. Comparing data measured with the same microscopic technique and comparing the different datasets with each other reveal that heat stress response in MX1 cells results from the overlap of different heat-induced subcellular defects.

In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25°C, the in vitro max of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37°C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be

We investigate the intersubject signal variability of near-infrared spectroscopy (NIRS), which is commonly used for noninvasive measurement of the product of the optical path length and the concentration change in oxygenated hemoglobin (C) and deoxygenated hemoglobin (C) and their sum (C) related to human cortical activation. We do this by measuring sensorimotor cortex activation in 31 healthy adults using 24-measurement-position near-infrared (NIR) topography. A finger-tapping task is used to activate the sensorimotor cortex, and significant changes in the hemisphere contralateral to the tapping hand are assessed as being due to the activation. Of the possible patterns of signal changes, 90% include a positive C, 76% included a negative C, and 73% included a positive C. The C and C are less consistent because of a large intersubject variability in C; in some cases there is a positive C. In the cases with no positive C in the contralateral hemisphere, there are cases of other possible changes for either or both hemispheres and no cases of no change in any hemoglobin species in either hemisphere. These results suggest that NIR topography is useful for observing brain activity in most cases, although intersubject signal variability still needs to be resolved.

We use near-infrared dynamic multiple scattering of light [diffusing-wave spectroscopy (DWS)] to detect the activation of the somato-motor cortex in 11 right-handed volunteers performing a finger opposition task separately with their right and left hands. Temporal autocorrelation functions g(1)(r,) of the scattered light field are measured during 100-s periods of motor task alternating with 100-s resting baseline periods. From an analysis of the experimental data with an analytical theory for g(1)(r,) from a three-layer geometry with optical and dynamical heterogeneity representing scalp, skull, and cortex, we obtain quantitative estimates of the diffusion coefficient in cortical regions. Consistent with earlier results, the measured cortical diffusion coefficient is found to be increased during the motor task, with a strong contralateral and a weaker ipsilateral increase consistent with the known brain hemispheric asymmetry for right-handed subjects. Our results support the interpretation of the increase of the cortical diffusion coefficient during finger opposition being due to the functional increase in cortical blood flow rate related to vasodilation.

The influence of skin on the bias and reproducibility of regional cerebral oxygenation measurements is investigated using cw near-infrared spectroscopy (NIRS). Receiving optodes are placed over the left and right hemispheres of a piglet (C3, C4 EEG placement code) and one transmitting optode centrally (Cz position). Optical densities (OD) are measured during stable normo, mild, and deep hypoxemia. This is done for skin condition 1: all optodes on the skin; skin condition 2: transmitting optode on the skin and one receiving optode on the skull; and skin condition 3: all optodes on the skull. Absolute changes of oxy- (cO2Hb), deoxyhemoglobin (cHHb), and total hemoglobin (ctHb) concentrations [µmol/L] are calculated from the ODs. These absolute changes are calculated for each skin condition with respect to normoxic condition. Additionally, for skin condition 2, the difference of concentration changes between receiver 1 (skull) and receiver 2 (skin) is calculated. The effect of skin removal is an average increase of attenuation changes by a factor of 1.66 (=0.51 OD) and of the concentration changes due to the arterial oxygen saturation steps by 23%. We conclude that skin significantly influences regional oxygenation measurements. Nevertheless, it is hypothesized that the estimated concentration changes are dominated by changes of the oxygenation in the brain.

Tumor hypoxia has been shown to have prognostic value in clinical trials involving radiation, chemotherapy, and surgery. Tumor oxygenation studies at microvascular levels can provide understanding of oxygen transport on scales at which oxygen transfer to tissue occurs. To fully grasp the significance of blood oxygen delivery and hypoxia at microvascular levels during tumor growth and angiogenesis, the spatial and temporal relationship of the data must be preserved and mapped. Using tumors grown in window chamber models, hyperspectral imaging can provide serial spatial maps of blood oxygenation in terms of hemoglobin saturation at the microvascular level. We describe our application of hyperspectral imaging for in vivo microvascular tumor oxygen transport studies using red fluorescent protein (RFP) to identify all tumor cells, and hypoxia-driven green fluorescent protein (GFP) to identify the hypoxic fraction. 4T1 mouse mammary carcinoma cells, stably transfected with both reporter genes, are grown in dorsal skin-fold window chambers. Hyperspectral imaging is used to create image maps of hemoglobin saturation, and classify image pixels where RFP alone is present (tumor cells), or both RFP and GFP are present (hypoxic tumor cells). In this work, in vivo calibration of the imaging system is described and in vivo results are shown.

We develop a clinical visible-light spectroscopy (VLS) tissue oximeter. Unlike currently approved near-infrared spectroscopy (NIRS) or pulse oximetry (SpO2%), VLS relies on locally absorbed, shallow-penetrating visible light (475 to 625 nm) for the monitoring of microvascular hemoglobin oxygen saturation (StO2%), allowing incorporation into therapeutic catheters and probes. A range of probes is developed, including noncontact wands, invasive catheters, and penetrating needles with injection ports. Data are collected from: 1. probes, standards, and reference solutions to optimize each component; 2. ex vivo hemoglobin solutions analyzed for StO2% and pO2 during deoxygenation; and 3. human subject skin and mucosal tissue surfaces. Results show that differential VLS allows extraction of features and minimization of scattering effects, in vitro VLS oximetry reproduces the expected sigmoid hemoglobin binding curve, and in vivo VLS spectroscopy of human tissue allows for real-time monitoring (e.g., gastrointestinal mucosal saturation 69±4%, n=804; gastrointestinal tumor saturation 45±23%, n=14; and p<0.0001), with reproducible values and small standard deviations (SDs) in normal tissues. FDA approved VLS systems began shipping earlier this year. We conclude that VLS is suitable for the real-time collection of spectroscopic and oximetric data from human tissues, and that a VLS oximeter has application to the monitoring of localized subsurface hemoglobin oxygen saturation in the microvascular tissue spaces of human subjects.

Raman spectroscopy is an optical technique able to interrogate biological tissues, giving us an understanding of the changes in molecular structure that are associated with disease development. The Kerr-gated Raman spectroscopy technique uses a picosecond pulsed laser as well as fast temporal gating of collected Raman scattered light. Prostate samples for this study were obtained by taking a chip at the transurethral resection of the prostate (TURP), and bladder samples from a biopsy taken at transurethral resection of bladder tumor (TURBT) and TURP. Spectra obtained through the bladder and prostate gland tissue, at different time delays after the laser pulse, clearly show change in the spectra as depth profiling occurs, eventually showing signals from the uric acid cell and urea cell, respectively. We show for the first time, using this novel technique, that we are able to obtain spectra from different depths through both the prostate gland and the bladder. This has major implications in the future of Raman spectroscopy as a tool for diagnosis. With the help of Raman spectroscopy and Kerr gating, it may be possible to pick up the spectral differences from a small focus of adenocarcinoma of the prostate gland in an otherwise benign gland, and also stage the bladder cancers by assessing the base of the tumor post resection.

To evaluate the potential of a new in vivo confocal Raman microprobe, we undertake a pilot study in human skin. A fiber optic probe is operated with a 633-nm laser and trials are conducted in healthy volunteers. We examine changes in molecular composition and structure of the stratum corneum, from different volunteers, from different anatomical sites and skin layers. Main spectral variations are detected in the following regions: 800 to 900 cm–1 (amino acids); 1200 to 1290 cm–1 (proteins); and 1030 to 1130 cm–1, 1300 to 1450 cm–1, and 2800 to 2900 cm–1 (lipids). Curve fitting of the amide 1 region performs in detail protein secondary structural variations of the amide 1 band. Protein conformation is also found to vary depending on the anatomical site and volunteer. Similar analysis of the 730- to 1170-cm–1 spectral window reveals a different organization of lamellar lipids: gel for forearm and palm, and liquid-crystalline phase for fingertips. All these variations result from changes in the stratum corneum components such as natural moisturizing factor (NMF), lipids (namely ceramides), and water. Hierarchical clustering classification is also performed to sort out Raman data obtained from different subjects. Further improvement of the confocal probe would be to adapt a 360-deg configuration enabling access to other anatomical sites.

There is a lack of systematic investigations comparing optical coherence tomography (OCT) with histology. OCT assessments were performed on the upper back of 16 healthy subjects. Epidermis thickness (ET) was assessed using three methods: first, peak-to-valley analysis of the A-scan (ET-OCT-V); second, manual measurements in the OCT images (ET-OCT-M); third, light microscopic determination using routine histology (ET-Histo). The relationship between the different methods was assessed by means of the Pearson correlation procedure and Bland and Altman plots. We observed a strong correlation between ET-Histo (79.4±21.9 µm) and ET-OCT-V (79.2±15.5 µm, r=0.77) and ET-OCT-M (82.9±15.8 µm, r=0.75), respectively. Bland and Altman plots revealed a bias of –0.19 µm (95% limits of agreement: –27.94 µm to 27.56 µm) for ET-OCT-V versus ET-Histo and a bias of 3.44 µm (95% limits of agreement: –24.9 µm to 31.78 µm) for ET-OCT-M versus ET-Histo. Despite the strong correlation and low bias observed, the 95% limits of agreement demonstrated an unsatisfactory numerical agreement between the two OCT methods and routine histology indicating that these methods cannot be employed interchangeably. Regarding practicability, precision, and indication spectrum, ET-OCT-V and ET-OCT-M are of different clinical value.

The increased sensitivity of spectral domain optical coherence tomography (OCT) has driven the development of a new generation of technologies in OCT, including rapidly tunable, broad bandwidth swept laser sources and spectral domain OCT interferometer topologies. In this work, the operation of a turnkey 1300-nm swept laser source is demonstrated. This source has a fiber ring cavity with a semiconductor optical amplifier gain medium. Intracavity mode selection is achieved with an in-fiber tunable fiber Fabry-Perot filter. A novel optoelectronic technique that allows for even sampling of the swept source OCT signal in k space also is described. A differential swept source OCT system is presented, and images of in vivo human cornea and skin are presented. Lastly, the effects of analog-to-digital converter aliasing on image quality in swept source OCT are discussed.

We evaluate the performance of our novel hybrid optical coherence tomography (OCT) and scintillating probe, demonstrate simultaneous OCT imaging and scintillating detection, and validate the system using an atherosclerotic rabbit model. Preliminary data obtained from the rabbit model suggest that our prototype positron probe detects local uptake of fluorodeoxyglucose (FDG) labeled with 18F positron (beta) radionuclide emitter, and the high-uptake regions correlate with sites of injury and extensive atherosclerosis areas. Preliminary data also suggest that coregistered high-resolution OCT images provide imaging of detailed plaque microstructures, which cannot be resolved by positron detection.

This study evaluates the capability of a photothermal (PT) assay to monitor the impact of nicotine on pancreatic cancer cells (AR42J). The specific PT response is closely proportional to nicotine concentrations at concentration range 1nM to 100 µM, while at high concentrations of nicotine ranging from 1 mM to 50 mM, PT response shows dramatic decrease. According to the theoretical model, the mechanism of the PT assay is associated with metabolic and apoptotic-related shrinking of local cellular absorbing nanoscale zones caused by increased local absorption at low nicotine doses, while high doses of nicotine lead to apoptotic release of absorbing component (cytochrome c) into the intracellular space, and necrotic swelling of organelles, thereby causing a decrease in local absorption. This model is verified with conventional imaging and with Annexin-V Propidium iodide kits. The PT assay, in addition to its high sensitivity (3 orders of magnitude better than conventional assay), shows the potential to distinguish between various functional states of cells that are associated with changes in metabolism, early and late stages of apoptosis, and necrosis. Comparison of PT responses of pancreatic tumor cells AR42J with isolated primary pancreatic acinar cells and HepG2 cells shows a universal nature of PT assay.

The mitotic spindle is a subcellular protein structure that facilitates chromosome segregation and is crucial to cell division. We describe an image processing approach to quantitatively characterize and compare mitotic spindles that have been imaged three dimensionally using confocal microscopy with fixed-cell preparations. The proposed approach is based on a set of features that are computed from each image stack representing a spindle. We compare several spindle datasets of varying biological (genotype) and/or environmental (drug treatment) conditions. The goal of this effort is to aid biologists in detecting differences between spindles that may not be apparent under subjective visual inspection, and furthermore, to eventually automate such analysis in high-throughput scenarios (thousands of images) where manual inspection would be unreasonable. Experimental results on positive- and negative-control data indicate that the proposed approach is indeed effective. Differences are detected when it is known they do exist (positive control) and no differences are detected when there are none (negative control). In two other experimental comparisons, results indicate structural spindle differences that biologists had not observed previously.

Refractive index of tissue is an essential parameter in many bio-optical experiments, yet little data can be found in literature. Several methods have been proposed to measure refractive index in tissue samples, but all have specific limitations, such as low accuracy, the need for large amounts of tissue, or the complexity of the measurement setup. We propose a new method using a standard confocal microscope and requiring only small tissue samples. A thin slice of tissue is put next to a layer of immersion fluid of exactly the same thickness. The actual thickness of the fluid layer is directly measured with the microscope, as there is no refractive index mismatch. A difference between index of refraction of the tissue and of the immersion medium causes an axial scaling factor. The optical thickness of the specimen is thus measured with the microscope, and as its actual thickness equals the known thickness of the fluid layer, the axial scaling factor is readily determined. From this factor, we calculate the refractive index of the tissue. We use a diffraction model to take the point spread function (PSF) of the microscope into account, so we can determine the index of refraction to a very high accuracy. We demonstrate the method on bovine muscle tissue and find a value of n=1.382±0.004, at 592 nm.

Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.

Optimization of device-tissue interface parameters may lead to an improvement in the efficacy of fluorescence spectroscopy for minimally invasive disease detection. Although illumination-collection geometry has been shown to have a strong influence on the spatial origin of detected fluorescence, devices that deliver and/or collect light at oblique incidence are not well understood. Simulations are performed using a Monte Carlo model of light propagation in homogeneous tissue to characterize general trends in the intensity and spatial origin of fluorescence detected by angled geometries. Specifically, the influence of illumination angle, collection angle, and illumination-collection spot separation distance are investigated for low and high attenuation tissue cases. Results indicate that oblique-incidence geometries have the potential to enhance the selective interrogation of superficial or subsurface fluorophores at user-selectable depths up to about 0.5 mm. Detected fluorescence intensity is shown to increase significantly with illumination and collection angle. Improved selectivity and signal intensity over normal-incidence geometries result from the overlap of illumination and collection cones within the tissue. Cases involving highly attenuating tissue produce a moderate reduction in the depth of signal origin. While Monte Carlo modeling indicates that oblique-incidence designs can facilitate depth-selective fluorescence spectroscopy, optimization of device performance will require application-specific consideration of optical and biological parameters.

Computer simulation is used to facilitate the design of fiber-probe geometries that enable enhanced detection of optical signals arising from specific tissue depths. Obtaining understanding of the relationship between fiber-probe design and tissue interrogation is critical when developing strategies for optical detection of epithelial precancers that originate at known depths from the tissue surface. The accuracy of spectroscopic diagnostics may be enhanced by discretely probing the optical properties of epithelium and underlying stroma, within which the morphological and biochemical features vary as a function of depth. While previous studies have investigated controlling tissue-probing depth for fluorescence-based modalities, in this study we focus on the detection of reflected light scattered by tissue. We investigate how the depth of optical interrogation may be controlled through combinations of collection angles, source-detector separations, and numerical apertures. We find that increasing the obliquity of collection fibers at a given source-detector separation can effectively enhance the detection of superficially scattered signals. Fiber numerical aperture provides additional depth selectivity; however, the perturbations in sampling depth achieved through this means are modest relative to the changes generated by modifying the angle of collection and source-detection separation.

Potentially transplantable kidneys experience warm ischemia, and this injury is difficult to quantify. We investigate optical spectroscopic methods for evaluating, in real time, warm ischemic kidney injury and reperfusion. Vascular pedicles of rat kidneys are clamped unilaterally for 18 or 85 min, followed by 18 or 35 min of reperfusion, respectively. Contralateral, uninjured kidneys serve as controls. Autofluorescence and cross-polarized light scattering images are acquired every 15 s using 335-nm laser excitation (autofluorescence) and 650±20-nm linearly polarized illumination (light scattering). We analyze changes of injured-to-normal kidney autofluorescence intensity ratios during ischemia and reperfusion phases. The effect of excitation with 260 nm is also explored. Average injured-to-normal intensity ratios under 335-nm excitation decrease from 1.0 to 0.78 at 18 min of ischemia, with a return to baseline during 18 min of reperfusion. However, during 85 min of warm ischemia, average intensity ratios level off at 0.65 after 50 min, with no significant change during 35 min of reperfusion. 260-nm excitation results in no autofluorescence changes with ischemia. Cross-polarized light scattering images at 650 nm suggest that changes in hemoglobin absorption are not related to observed temporal behavior of the autofluorescence signal. Real-time detection of kidney tissue changes associated with warm ischemia and reperfusion using laser spectroscopy is feasible. Normalizing autofluorescence changes under 335 nm using the autofluorescence measured under 260-nm excitation may eliminate the need for a control kidney.

Fluorescence molecular tomography (FMT) has emerged as a means of quantitatively imaging fluorescent molecular probes in three dimensions in living systems. To assess the accuracy of FMT in vivo, translucent plastic tubes containing a turbid solution with a known concentration of Cy 5.5 fluorescent dye are constructed and implanted subcutaneously in nude mice, simulating the presence of a tumor accumulating a fluorescent molecular reporter. Comparisons between measurements of fluorescent tubes made before and after implantation demonstrate that the accuracy of FMT reported for homogeneous phantoms extends to the in vivo situation. The sensitivity of FMT to background fluorescence is tested by imaging fluorescent tubes in mice injected with Cy 5.5-labeled Annexin V. For small tube fluorochrome concentrations, the presence of background fluorescence results in increases in the reconstructed concentration. This phenomenon is counteracted by applying a simple subtraction correction to the measured fluorescence data. The effects of varying tumor photon absorption are simulated by imaging fluorescent tubes with varying ink concentrations, and are found to be minor. These findings demonstrate the in vivo quantitative accuracy of fluorescence tomography, and encourage further development of this imaging modality as well as application of FMT in molecular imaging studies using fluorescent reporters.

We suitably adapt the design of a tissue-equivalent phantom used for photoacoustic imaging to construct phantoms for optical elastography. The elastography phantom we consider should have optical properties such as scattering coefficient, scattering anisotropy factor, and refractive index; mechanical properties such as storage and loss modulus; and acoustic properties such as ultrasound velocity, attenuation coefficient, and acoustic impedance to match healthy and diseased tissues. The phantom is made of poly (vinyl alcohol) (PVA) and its mechanical, optical, and acoustic properties are tailored by physical cross-linking effected through subjecting a suitable mix of PVA stock and water to a number of freeze-thaw cycles and by varying the degree of hydrolysis in the PVA stock. The optical, mechanical, and acoustic properties of the samples prepared are measured by employing different techniques. The measured variations in the values of optical scattering coefficient, scattering anisotropy factor, and refractive index and storage modulus are found to be comparable to those in normal and diseased breast tissues. The acoustic properties such as sound speed, acoustic attenuation coefficient, and density are found to be close to the average values reported in the literature for normal breast tissue.

Irradiation of the ocular lens of numerous species by near-UV or short-visible wavelengths induces a blue-green fluorescence, which can be a source of intraocular veiling glare. Wavelengths longer than the ~365-nm lens absorption peak induce progressively weaker but also progressively more red-shifted fluorescence emission. The more red-shifted emission has a higher luminous efficiency and, in fact, earlier studies in this laboratory have demonstrated that the lens fluorescence in the nonhuman primate yields an approximately constant luminous efficiency when excited by equal radiant exposures over the wavelength range from 350 to 430 nm. Now, with the recent development and projected widespread use of "blue" diode lasers, a further study extending the measurements of the induced fluorescence efficiency and of the consequent veiling glare to the human lens seemed timely. The current study quantifies the fluorescence intensity induced in the human lens, both in terms of radiance and luminance, as a function of exciting light intensity, excitation wavelength, and subject age. The spatial distribution of the emitted fluorescence is also examined. These data are shown to imply that exposure to near-UV/blue wavelength sources at "safe" exposure levels (according to existing laser safety standards) can induce a veiling glare intense enough to degrade visual performance, and that the fluorescence intensity and consequent glare disruption show little dependence on subject age.

The purpose of the study is to analyze and compare differences in the optical properties between normal and adenomatous human colon tissues in vitro at 630-, 680-, 720-, 780-, 850-, and 890-nm wavelengths using a Ti:sapphire laser. The optical parameters of tissue samples are determined using a double integrating sphere setup at seven different laser wavelengths. The inverse Monte Carlo simulation is used to determine the optical properties from the measurements. The results of measurement show that the optical properties and their differences vary with a change of laser wavelength for normal and adenomatous colon mucosa/submucosa and normal and adenomatous colon muscle layer/chorion. The maximum absorption coefficients for normal and adenomatous human colon mucosa/submucosa are 680 nm, and the minimum absorption coefficients for both are 890 nm. The maximum difference of the absorption coefficients between both is 56.8% at 780 nm. The maximum scattering coefficients for normal and adenomatous colon mucosa/submucosa are 890 nm, and the minimum scattering coefficients for both are 780 nm. The maximum difference of the scattering coefficients between both is 10.6% at 780 nm. The maximum absorption coefficients for normal and adenomatous colon muscle layer/chorion are 680 nm, and the minimum absorption coefficients for both are 890 nm. The maximum difference of the absorption coefficients between both is 47.9% at 780 nm. The maximum scattering coefficients for normal and adenomatous colon muscle layer/chorion are 890 nm, and the minimum scattering coefficients for both are 680 nm. The maximum difference of the scattering coefficients between both is 9.61% at 850 nm. The differences in absorption coefficients between normal and adenomatous tissues are more significant than those in scattering coefficients.

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of ~100 pN/s, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25±1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

The intracellular localization and specific organelle association of mRNA may reflect essential functions, stages, and stability of mRNA. We report the direct visualization of subcellular localization of K-ras and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs in live HDF cells using molecular beacons together with membrane-permeabilization and peptide-based delivery. Unexpectedly, we found that both K-ras and GAPDH mRNAs colocalize with mitochondria. Extensive control studies are performed, including the use of fluorescence in-situ hybridization (FISH), negative-control beacons, and the detection of colocalization of 28S ribosomal RNA with the rough endoplasmic reticulum (ER), suggesting that the mRNA localization and colocalization patterns observed in our study are true and specific. Our observation reveals intriguing subcellular associations of mRNA with organelles such as mitochondria, which may provide new insight into the transport, dynamics, and functions of mRNA and mRNA-protein interactions.

UV extinction spectra of effluent dialyzate in the process of hemodialysis and peritoneal dialysis are studied. It is found that they have distinctive individual features unique to each patient. The shape of the spectrum remains virtually invariable for a given subject during the whole period of the study (more than a year) and does not depend on the treatment modality or other factors like sex, age, the stage of chronic renal failure, and concomitant illnesses. Absorption bands in two spectral regions—255 to 265 nm and 288 to 298 nm—account for the unique character of dialyzate spectra. Spectra classification based on the ratio of integral extinctions in these regions are introduced. It is assumed that the shape of the spectrum depends on the relative contents of plasma components. Statistical analysis of individual features of spectra for the groups of patients undergoing hemodialysis (78 people) and peritoneal dialysis (42 people) is carried out. It is revealed that the distribution of patients by the ratio of integral extinctions in the specified spectral regions adheres to the normal law. Presumably individual features of spectra could be related to uric acid and a biochemically unidentified substance with an absorption peak near 260 nm.

We describe the protocol for an inexpensive and nondestructive optical reflectance assay for the measurement of biofilm formation. Reflectance data are obtained using an Ocean Optics (Dunedin, Florida) USB 2000 spectrometer with a polychromatic light source. A fiber optic cable is used both for illumination and collection, and Ocean Optics OOIBase32 Platinum software is used for preliminary processing of the data. Differences in reflectance data collected at times ranging from 2 to 24 h distinguish between cell attachment and volume growth for two strains of Enterococci. Confocal scanning laser microscopy imaging is used to confirm these results. Phase contrast microscopy images are also obtained in conjunction with reflectance measurements for several different biofilm specimens. The experiments consider biofilm formation on glass and polystyrene substrata, but the method can be used for many other abiotic substrata of interest, both opaque and nonopaque.

Real-time nonlinear optical Fourier filtering for medical image processing is demonstrated, exploiting light modulating characteristics of thin films of the biophotonic material bacteriorhodopsin (bR). The nonlinear transmission of bR films for a 442 nm probe beam with a 568 nm control beam and vice versa is experimentally studied in detail. The spatial frequency information carried by the blue probe beam is selectively manipulated in the bR film by changing the position and intensity of the yellow control beam. The feasibility of the technique is first established with different shapes and sizes of phantom objects. The technique is applied to filter out low spatial frequencies corresponding to soft dense breast tissue and displaying only high spatial frequencies corresponding to microcalcifications in clinical screen film mammograms. With the aid of an electrically addressed spatial light modulator (SLM), we successfully adapt the technique for processing digital phantoms and digital mammograms. Unlike conventional optical spatial filtering techniques that use masks, the technique proposed can easily accommodate the changes in size and shape of details in a mammogram.