Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-μm step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

The peripheral lymphatic vascular system is a part of the immune body system comprising a complex network of lymph vessels and nodes that are flowing lymph toward the heart. Traditionally the imaging of lymphatic vessels is based on the conventional imaging modalities utilizing contrast fluorescence materials. Given the important role of the lymphatic system there is a critical need for the development of noninvasive imaging technologies for functional quantitative diagnosis of the lymph vessels and lymph flow without using foreign chemicals. We report a label free methodology for noninvasive in vivo imaging of blood and lymph vessels, using long-exposure laser speckle imaging approach. This approach entails great promise in the noninvasive studies of tissues blood and lymph vessels distribution in vivo.

The author presents a graphics processing unit (GPU) programming for real-time Fourier domain optical coherence tomography (FD-OCT) with fixed-pattern noise removal by subtracting mean and median. In general, the fixed-pattern noise can be removed by the averaged spectrum from the many spectra of an actual measurement. However, a mean-spectrum results in artifacts as residual lateral lines caused by a small number of high-reflective points on a sample surface. These artifacts can be eliminated from OCT images by using medians instead of means. However, median calculations that are based on a sorting algorithm can generate a large amount of computation time. With the developed GPU programming, highly reflective surface regions were obtained by calculating the standard deviation of the Fourier transformed data in the lateral direction. The medians and means were then subtracted at the observed regions and other regions, such as backgrounds. When the median calculation was less than 256 positions out of a total 512 depths in an OCT image with 1024 A-lines, the GPU processing rate was faster than that of the line scan camera (46.9 kHz). Therefore, processed OCT images can be displayed in real-time using partial medians.

The dynamic behavior of phase singularities, or optical vortices, in the pseudo-phase representation of dynamic speckle patterns is investigated. Sequences of band-limited, dynamic speckle patterns with predetermined Gaussian decorrelation behavior were generated, and the pseudo-phase realizations of the individual speckle patterns were calculated via a two-dimensional Hilbert transform algorithm. Singular points in the pseudo-phase representation are identified by calculating the local topological charge as determined by convolution of the pseudo-phase representations with a series of 2×2 nabla filters. The spatial locations of the phase singularities are tracked over all frames of the speckle sequences, and recorded in three-dimensional space (x,y,f), where f is frame number in the sequence. The behavior of the phase singularities traces 'vortex trails' which are representative of the speckle dynamics. Slowly decorrelating speckle patterns results in long, relatively straight vortex trails, while rapidly decorrelating speckle patterns results in tortuous, relatively short vortex trails. Optical vortex analysis such as described herein can be used as a descriptor of biological activity, flow, and motion.

Traditional white-light and fluorescent imaging techniques provide powerful methods to extract high-resolution information from two-dimensional (2-D) sections, but to retrieve information from a three-dimensional (3-D) volume they require relatively slow scanning methods that result in increased acquisition time. Using an ultra-high speed liquid lens, we circumvent this problem by simultaneously acquiring images from multiple focal planes. We demonstrate this method by imaging microparticles and cells flowing in 3-D microfluidic channels.

We report a novel small-animal whole-body imaging system called ring-shaped confocal photoacoustic computed tomography (RC-PACT). RC-PACT is based on a confocal design of free-space ring-shaped light illumination and 512-element full-ring ultrasonic array signal detection. The free-space light illumination maximizes the light delivery efficiency, and the full-ring signal detection ensures a full two-dimensional view aperture for accurate image reconstruction. Using cylindrically focused array elements, RC-PACT can image a thin cross section with 0.10 to 0.25 mm in-plane resolutions and 1.6 s/frame acquisition time. By translating the mouse along the elevational direction, RC-PACT provides a series of cross-sectional images of the brain, liver, kidneys, and bladder.

A new class of gradient refractive index (GRIN) lens is introduced and analyzed. The interior iso-indicial contours mimic the external shape of the lens, which leads to an invariant geometry of the GRIN structure. The lens model employs a conventional surface representation using a coincoid of revolution with a higher-order aspheric term. This model has a unique feature, namely, it allows analytical paraxial ray tracing. The height and the angle of an arbitrary incident ray can be found inside the lens in a closed-form expression, which is used to calculate the main optical characteristics of the lens, including the optical power and third-order monochromatic aberration coefficients. Moreover, due to strong coupling of the external surface shape to the GRIN structure, the proposed GRIN lens is well suited for studying accommodation mechanism in the eye. To show the power of the model, several examples are given emphasizing the usefulness of the analytical solution. The presented geometry-invariant GRIN lens can be used for modeling and reconstructing the crystalline lens of the human eye and other types of eyes featuring a GRIN lens.

A theoretical model investigating the dependence of optoacoustic (OA) signal on blood oxygen saturation (SO₂) is discussed. The derivations for the nonbandlimited and bandlimited OA signals from many red blood cells (RBCs) are presented. The OA field generated by many RBCs was obtained by summing the OA field emitted by each RBC approximated as a fluid sphere. A Monte Carlo technique was employed generating the spatial organizations of RBCs in two-dimensional. The RBCs were assumed to have the same SO₂ level in a simulated configuration. The fractional number of oxyhemoglobin molecules, confined in a cell, determined the cellular SO₂ and also defined the blood SO₂. For the nonbandlimited case, the OA signal amplitude decreased and increased linearly with blood SO₂ when illuminated by 700 and 1000 nm radiations, respectively. The power spectra exhibited similar trends over the entire frequency range (MHz to GHz). For the bandlimited case, three acoustic receivers with 2, 10, and 50 MHz as the center frequencies were considered. The linear variations of the OA amplitude with blood SO₂ were also observed for each receiver at those laser sources. The good agreement between simulated and published experimental results validates the model qualitatively.

Activatable fluorescent molecular probes are predominantly nonfluorescent in their inactivated state due to intramolecular quenching, but increase fluorescence yield significantly after enzyme-mediated hydrolysis of peptides. Continuous wave in vivo detection of these protease-activatable fluorophores in the heart, however, is limited by the inability to differentiate between activated and nonactivated fractions of the probe and is frequently complicated by large background signal from probe accumulation in the liver. Using a cathepsin-activatable near-infrared probe (PGC-800), we demonstrate here that fluorescence lifetime (FL) significantly increases in infarcted murine myocardial tissue (0.67 ns) when compared with healthy myocardium (0.59 ns) after 24 h. Furthermore, we show that lifetime contrast can be used to distinguish in vivo cardiac fluorescence from background nonspecific liver signal. The results of this study show that lifetime contrast is a helpful addition to preclinical imaging of activatable fluorophores in the myocardium by reporting molecular activity in vivo due to changes in intramolecular quenching. This characterization of FL from activatable molecular probes will be helpful for advancing in vivo imaging of enzyme activity.

Diffuse, optical near infrared imaging is increasingly being used in various neurocognitive contexts where changes in optical signals are interpreted through activation maps. Statistical population comparison of different age or clinical groups rely on the relative homogeneous distribution of measurements across subjects in order to infer changes in brain function. In the context of an increasing use of diffuse optical imaging with older adult populations, changes in tissue properties and anatomy with age adds additional confounds. Few studies investigated these changes with age. Duncan et al. measured the so-called diffusion path length factor (DPF) in a large population but did not explore beyond the age of 51 after which physiological and anatomical changes are expected to occur [Pediatr. Res. 39(5), 889-894 (1996)]. With increasing interest in studying the geriatric population with optical imaging, we studied changes in tissue properties in young and old subjects using both magnetic resonance imaging (MRI)-guided Monte-Carlo simulations and time-domain diffuse optical imaging. Our results, measured in the frontal cortex, show changes in DPF that are smaller than previously measured by Duncan et al. in a younger population. The origin of these changes are studied using simulations and experimental measures.

Carotid angioplasty and stenting is a minimally invasive endovascular procedure that may benefit from in vivo high resolution imaging for monitoring the physical placement of the stent and potential complications. The purpose of this pilot study was to evaluate the ability of optical coherence tomography to construct high resolution 2D and 3D images of stenting in porcine carotid artery. Four Yorkshire pigs were anaesthetized and catheterized. A state-of-the-art optical coherence tomography (OCT) system and an automated injector were used to obtain both healthy and stented porcine carotid artery images. Data obtained were then processed for visualization. The state-of-the-art OCT system was able to capture high resolution images of both healthy and stented carotid arteries. High quality 3D images of healthy and stented carotid arteries were constructed, clearly depicting vessel wall morphological features, stent apposition and thrombus formation over the inserted stent. The results demonstrate that OCT can be used to generate high quality 3D images of carotid arterial stents for accurate diagnosis of stent apposition and complications under appropriate imaging conditions.

In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.

We evaluated frontal brain activation during a mixed attentional/working memory task with graded levels of difficulty in a group of 19 healthy subjects, by means of time-domain functional near-infrared spectroscopy (fNIRS). Brain activation was assessed, and load-related oxy- and deoxy-hemoglobin changes were studied. Generalized linear model (GLM) was applied to the data to explore the metabolic processes occurring during the mental effort and, possibly, their involvement in short-term memorization. GLM was applied to the data twice: for modeling the task as a whole and for specifically investigating brain activation at each cognitive load. This twofold employment of GLM allowed (1) the extraction and isolation of different information from the same signals, obtained through the modeling of different cognitive categories (sustained attention and working memory), and (2) the evaluation of model fitness, by inspection and comparison of residuals (i.e., unmodeled part of the signal) obtained in the two different cases. Results attest to the presence of a persistent attentional-related metabolic activity, superimposed to a task-related mnemonic contribution. Some hemispherical differences have also been highlighted frontally: deoxy-hemoglobin changes manifested a strong right lateralization, whereas modifications in oxy- and total hemoglobin showed a medial localization. The present work successfully explored the capability of fNIRS to detect the two neurophysiological categories under investigation and distinguish their activation patterns.

The parameters of an off-axis cylindrical mirror-focused line-scanning system were studied to optimize the flatness of the 2 mm scan field. The scanning system parameters included the beam size, the distance between the scanning and the focusing mirror, the angle between the incident beam and the reflected beam, the optical scan angle, and the effective focal length of the cylindrical mirror. Because of the off-axis line-scanning system configuration, the scanning could be carried out either in the tangential (Y-scan) or in the sagittal (X-scan) plane. A 53 nm spectral bandwidth light source was used to evaluate the imaging performance of the scanning system. Since reflective optics is employed in this work for focusing, the scanning system could be used with a higher spectral bandwidth light source for optical coherence tomography applications. The effect of the angle between of the incident and reflected beams, the distance between the mirrors, the focal length of the cylindrical mirror and the scanning directions, on the flatness of the scan field were studied. It was proved that the sagittal scanning is least sensitive to variations in scanning system parameters and thus provides maximum flexibility in design.

It is challenging to image fluorescence objects with high spatial resolution in a highly scattering medium. Recently reported temperature-sensitive indocyanine green-loaded pluronic nanocapsules can potentially alleviate this problem. Here we demonstrate a frequency-domain temperature-modulated fluorescence tomography system that could acquire images at high intensity-focused ultrasound resolution with use of these nanocapsules. The system is experimentally verified with a phantom study, where a 3-mm fluorescence object embedded 2 cm deep in a turbid medium is successfully recovered based on both intensity and lifetime contrast.

Multifrequency (0 to 0.3  mm−1), multiwavelength (633, 680, 720, 800, and 820 nm) spatial frequency domain imaging (SFDI) of 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) was used to recover absorption, scattering, and fluorescence properties of glioblastoma multiforme spheroids in tissue-simulating phantoms and in vivo in a mouse model. Three-dimensional tomographic reconstructions of the frequency-dependent remitted light localized the depths of the spheroids within 500 μm, and the total amount of PpIX in the reconstructed images was constant to within 30% when spheroid depth was varied. In vivo tumor-to-normal contrast was greater than ∼ 1.5 in reduced scattering coefficient for all wavelengths and was ∼ 1.3 for the tissue concentration of deoxyhemoglobin (ctHb). The study demonstrates the feasibility of SFDI for providing enhanced image guidance during surgical resection of brain tumors.

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

We developed a novel trimodality system for human breast imaging by integrating photoacoustic (PA) and thermoacoustic (TA) imaging techniques into a modified commercial ultrasound scanner. Because light was delivered with an optical assembly placed within the microwave antenna, no mechanical switching between the microwave and laser sources was needed. Laser and microwave excitation pulses were interleaved to enable PA and TA data acquisition in parallel at a rate of 10 frames per second. A tube (7 mm inner diameter) filled with oxygenated bovine blood or 30 mM methylene blue dye was successfully detected in PA images in chicken breast tissue at depths of 6.6 and 8.4 cm, respectively, for the first time. The SNRs at these depths reached ∼ 24 and ∼ 15  dB, respectively, by averaging 200 signal acquisitions. Similarly, a tube (13 mm inner diameter) filled with saline solution (0.9%) at a depth of 4.4 cm in porcine fat tissue was successfully detected in TA images. The PA axial, lateral, and elevational resolutions were 640 μm, 720 μm, and 3.5 mm, respectively, suitable for breast cancer imaging. A PA noise-equivalent sensitivity to methylene blue solution of 260 nM was achieved in chicken tissue at a depth of 3.4 cm.

We present a dynamic laser speckle method to easily discriminate filamentous fungi from motile bacteria in soft surfaces, such as agar plate. The method allows the detection and discrimination between fungi and bacteria faster than with conventional techniques. The new procedure could be straightforwardly extended to different micro-organisms, as well as applied to biological and biomedical research, infected tissues analysis, and hospital water and wastewaters studies.

We present spectral domain polarization-sensitive optical coherence tomography (SD PS-OCT) imaging of peripheral nerves. Structural and polarization-sensitive OCT imaging of uninjured rat sciatic nerves was evaluated both qualitatively and quantitatively. OCT and its functional extension, PS-OCT, were used to image sciatic nerve structure with clear delineation of the nerve boundaries to muscle and adipose tissues. A long-known optical effect, bands of Fontana, was also observed. Postprocessing analysis of these images provided significant quantitative information, such as epineurium thickness, estimates of extinction coefficient and birefringence of nerve and muscle tissue, frequency of bands of Fontana at different stretch levels of nerve, and change in average birefringence of nerve under stretched condition. We demonstrate that PS-OCT combined with regular-intensity OCT (compared with OCT alone) allows for a clearer determination of the inner and outer boundaries of the epineurium and distinction of nerve and muscle based on their birefringence pattern. PS-OCT measurements on normal nerves show that the technique is promising for studies on peripheral nerve injury.

Hematoxylin and eosin (H&E) stain is currently the most popular for routine histopathology staining. Special and/or immuno-histochemical (IHC) staining is often requested to further corroborate the initial diagnosis on H&E stained tissue sections. Digital simulation of staining (or digital staining) can be a very valuable tool to produce the desired stained images from the H&E stained tissue sections instantaneously. We present an approach to digital staining of histopathology multispectral images by combining the effects of spectral enhancement and spectral transformation. Spectral enhancement is accomplished by shifting the N-band original spectrum of the multispectral pixel with the weighted difference between the pixel's original and estimated spectrum; the spectrum is estimated using M < N principal component (PC) vectors. The pixel's enhanced spectrum is transformed to the spectral configuration associated to its reaction to a specific stain by utilizing an N × N transformation matrix, which is derived through application of least mean squares method to the enhanced and target spectral transmittance samples of the different tissue components found in the image. Results of our experiments on the digital conversion of an H&E stained multispectral image to its Masson's trichrome stained equivalent show the viability of the method.

Myocardial infarction often leads to an increase in deposition of fibrillar collagen. Detection and characterization of this cardiac fibrosis is of great interest to investigators and clinicians. Motivated by the significant limitations of conventional staining techniques to visualize collagen deposition in cardiac tissue sections, we have developed a Fourier transform infrared imaging spectroscopy (FT-IRIS) methodology for collagen assessment. The infrared absorbance band centered at 1338  cm−1, which arises from collagen amino acid side chain vibrations, was used to map collagen deposition across heart tissue sections of a rat model of myocardial infarction, and was compared to conventional staining techniques. Comparison of the size of the collagen scar in heart tissue sections as measured with this methodology and that of trichrome staining showed a strong correlation (R = 0.93). A Pearson correlation model between local intensity values in FT-IRIS and immuno-histochemical staining of collagen type I also showed a strong correlation (R = 0.86). We demonstrate that FT-IRIS methodology can be utilized to visualize cardiac collagen deposition. In addition, given that vibrational spectroscopic data on proteins reflect molecular features, it also has the potential to provide additional information about the molecular structure of cardiac extracellular matrix proteins and their alterations.

Faulty postures, scoliosis and sagittal plane deformities should be detected as early as possible to apply preventive and treatment measures against major clinical consequences. To support documentation of the severity of deformity and diminish x-ray exposures, several solutions utilizing analysis of back surface topography data were introduced. A novel approach to automatic recognition and localization of anatomical landmarks of the human back is presented that may provide more repeatable results and speed up the whole procedure. The algorithm was designed as a two-step process involving a statistical model built upon expert knowledge and analysis of three-dimensional back surface shape data. Voronoi diagram is used to connect mean geometric relations, which provide a first approximation of the positions, with surface curvature distribution, which further guides the recognition process and gives final locations of landmarks. Positions obtained using the developed algorithms are validated with respect to accuracy of manual landmark indication by experts. Preliminary validation proved that the landmarks were localized correctly, with accuracy depending mostly on the characteristics of a given structure. It was concluded that recognition should mainly take into account the shape of the back surface, putting as little emphasis on the statistical approximation as possible.

The use of a novel all-optical photoacoustic scanner for imaging the development of tumor vasculature and its response to a therapeutic vascular disrupting agent is described. The scanner employs a Fabry-Perot polymer film ultrasound sensor for mapping the photoacoustic waves and an image reconstruction algorithm based upon attenuation-compensated acoustic time reversal. The system was used to noninvasively image human colorectal tumor xenografts implanted subcutaneously in mice. Label-free three-dimensional in vivo images of whole tumors to depths of almost 10 mm with sub-100-micron spatial resolution were acquired in a longitudinal manner. This enabled the development of tumor-related vascular features, such as vessel tortuosity, feeding vessel recruitment, and necrosis to be visualized over time. The system was also used to study the temporal evolution of the response of the tumor vasculature following the administration of a therapeutic vascular disrupting agent (OXi4503). This revealed the well-known destruction and recovery phases associated with this agent. These studies illustrate the broader potential of this technology as an imaging tool for the preclinical and clinical study of tumors and other pathologies characterized by changes in the vasculature.

Diffuse optical spectroscopy (DOS) provides a powerful tool for fast and noninvasive disease diagnosis. The ability to leverage DOS to accurately quantify tissue optical parameters hinges on the model used to estimate light-tissue interaction. We describe the accuracy of a lookup table (LUT)-based inverse model for measuring optical properties under different conditions relevant to biological tissue. The LUT is a matrix of reflectance values acquired experimentally from calibration standards of varying scattering and absorption properties. Because it is based on experimental values, the LUT inherently accounts for system response and probe geometry. We tested our approach in tissue phantoms containing multiple absorbers, different sizes of scatterers, and varying oxygen saturation of hemoglobin. The LUT-based model was able to extract scattering and absorption properties under most conditions with errors of less than 5 percent. We demonstrate the validity of the lookup table over a range of source-detector separations from 0.25 to 1.48 mm. Finally, we describe the rapid fabrication of a lookup table using only six calibration standards. This optimized LUT was able to extract scattering and absorption properties with average RMS errors of 2.5 and 4 percent, respectively.

The combined use of surface acoustic wave (SAW) and phase-sensitive optical coherence tomography (PhS-OCT) is useful to evaluate the elasticity of layered biological tissues, such as normal skin. However, the pathological tissue is often originated locally, leading to the alternation of mechanical properties along both axial and lateral directions. We present a feasibility study on whether the SAW technique is sensitive to detect the alternation of mechanical property along the lateral direction within tissue, which is important for clinical utility of this technique to localize diseased tissue. Experiments are carried out on purposely designed tissue phantoms and ex vivo chicken breast samples, simulating the localized change of elasticity. A PhS-OCT system is employed not only to provide the ultra-high sensitive measurement of the generated surface waves on the tissue surface, but also to provide the real time imaging of the tissue to assist the elasticity evaluation of the heterogeneous tissue. The experimental results demonstrate that with PhS-OCT used as a pressure sensor, the SAW is highly sensitive to the elasticity change of the specimen in both vertical and lateral directions with a sensing depth of ∼ 5  mm with our current system setup, thus promising its useful clinical applications where the quantitative elasticity of localized skin diseases is needed to aid in diagnosis and treatment.

Non-invasive detection of fluorescence from the optical tracer indocyanine green is feasible in the adult human brain when employing a time-domain technique with picosecond resolution. A fluorescence-based assessment may offer higher signal-to-noise ratio when compared to bolus tracking relying on changes in time-resolved diffuse reflectance. The essential challenge is to discriminate the fluorescence originating from the brain from contamination by extracerebral fluorescence and hence to reconstruct the bolus kinetics; however, a method to reliably perform the necessary separation is missing. We present a novel approach for the decomposition of the fluorescence contributions from the two tissue compartments. The corresponding sensitivity functions pertaining to the brain and to the extracerebral compartment are directly derived from the in-vivo measurement. This is achieved by assuming that during the initial and the late phase of bolus transit the fluorescence signal originates largely from one of the compartments. Solving the system of linear equations allows one to approximate time courses of a bolus for each compartment. We applied this method to repetitive measurements on two healthy subjects with an overall 34 boluses. A reconstruction of the bolus kinetics was possible in 62% of all cases.

The determination of oxygen levels in blood and other tissues in vivo is critical for ensuring proper body functioning, for monitoring the status of many diseases, such as cancer, and for predicting the efficacy of therapy. Here we demonstrate, for the first time, a lifetime-based photoacoustic technique for the measurement of oxygen in vivo, using an oxygen sensitive dye, enabling real time quantification of blood oxygenation. The results from the main artery in the rat tail indicated that the lifetime of the dye, quantified by the photoacoustic technique, showed a linear relationship with the blood oxygenation levels in the targeted artery.

Time-resolved near-infrared spectroscopy allows for depth-selective determination of absorption changes in the adult human head that facilitates separation between cerebral and extra-cerebral responses to brain activation. The aim of the present work is to analyze which combinations of moments of measured distributions of times of flight (DTOF) of photons and source-detector separations are optimal for the reconstruction of absorption changes in a two-layered tissue model corresponding to extra- and intra-cerebral compartments. To this end we calculated the standard deviations of the derived absorption changes in both layers by considering photon noise and a linear relation between the absorption changes and the DTOF moments. The results show that the standard deviation of the absorption change in the deeper (superficial) layer increases (decreases) with the thickness of the superficial layer. It is confirmed that for the deeper layer the use of higher moments, in particular the variance of the DTOF, leads to an improvement. For example, when measurements at four different source-detector separations between 8 and 35 mm are available and a realistic thickness of the upper layer of 12 mm is assumed, the inclusion of the change in mean time of flight, in addition to the change in attenuation, leads to a reduction of the standard deviation of the absorption change in the deeper tissue layer by a factor of 2.5. A reduction by another 4% can be achieved by additionally including the change in variance.

We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 µm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 µm due to used laser wavelength of 0.66 µm.

Optical properties of flowing blood were analyzed using a photon-cell interactive Monte Carlo (pciMC) model with the physical properties of the flowing red blood cells (RBCs) such as cell size, shape, refractive index, distribution, and orientation as the parameters. The scattering of light by flowing blood at the He-Ne laser wavelength of 632.8 nm was significantly affected by the shear rate. The light was scattered more in the direction of flow as the flow rate increased. Therefore, the light intensity transmitted forward in the direction perpendicular to flow axis decreased. The pciMC model can duplicate the changes in the photon propagation due to moving RBCs with various orientations. The resulting RBC's orientation that best simulated the experimental results was with their long axis perpendicular to the direction of blood flow. Moreover, the scattering probability was dependent on the orientation of the RBCs. Finally, the pciMC code was used to predict the hematocrit of flowing blood with accuracy of approximately 1.0 HCT%. The photon-cell interactive Monte Carlo (pciMC) model can provide optical properties of flowing blood and will facilitate the development of the non-invasive monitoring of blood in extra corporeal circulatory systems.

Osmotic disruption of the blood brain barrier (BBB) by intraarterial mannitol injection is sometimes the key step for the delivery of chemotherapeutic drugs to brain tissue. BBB disruption (BBBD) with mannitol, however, can be highly variable and could impact local drug deposition. We use optical pharmacokinetics, which is based on diffuse reflectance spectroscopy, to track in vivo brain tissue concentrations of indocyanine green (ICG), an optical reporter used to monitor BBBD, and mitoxantrone (MTX), a chemotherapy agent that does not deposit in brain tissue without BBBD, in anesthetized New Zealand white rabbits. Results show a significant increase in the tissue ICG concentrations with BBBD, and our method is able to track the animal-to-animal variation in tissue ICG and MTX concentrations after BBBD. The tissue concentrations of MTX increase with barrier disruption and are found to be correlated to the degree of disruption, as assessed by the ICG prior to the injection of the drug. These findings should encourage the development of tracers and optical methods capable of quantifying the degree of BBBD, with the goal of improving drug delivery.

We study the use of photochemical internalization (PCI) for enhancing chemotherapeutic response to malignant glioma cells in vitro. Two models are studied: monolayers consisting of F98 rat glioma cells and human glioma spheroids established from biopsy-derived glioma cells. In both cases, the cytotoxicity of aluminum phthalocyanine disulfonate (AlPcS2a)-based PCI of bleomycin was compared to AlPcS2a-photodynamic therapy (PDT) and chemotherapy alone. Monolayers and spheroids were incubated with AlPcS2a (PDT effect), bleomycin (chemotherapy effect), or AlPcS2a+bleomycin (PCI effect) and were illuminated (670 nm). Toxicity was evaluated using colony formation assays or spheroid growth kinetics. F98 cells in monolayer/spheroids were not particularly sensitive to the effects of low radiant exposure (1.5  J/cm² @ 5  mW/cm²) AlPcS2a-PDT. Bleomycin was moderately toxic to F98 cells in monolayer at relatively low concentrations-incubation of F98 cells in 0.1  μg/ml for 4 h resulted in 80% survival, but less toxic in human glioma spheroids respectively. In both in vitro systems investigated, a significant PCI effect is seen. PCI using 1.5  J/cm² together with 0.25  μg/ml bleomycin resulted in approximately 20% and 18% survival of F98 rat glioma cells and human glioma spheroids, respectively. These results show that AlPcS2a-mediated PCI can be used to enhance the efficacy of chemotherapeutic agents such as bleomycin in malignant gliomas.

Histological slices of skin samples with the subcutaneous adipose tissue after photothermal/photodynamic treatment are analyzed. In the case of subcutaneous indocyanine green injection and 808-nm diode laser exposure of the rat skin site in vivo, the greatest changes in tissue condition were observed. Processes were characterized by dystrophy, necrosis, and desquamation of the epithelial cells, swelling and necrosis of the connective tissue, and widespread necrosis of the subcutaneous adipose tissue. The obtained data are useful for safe layer-by-layer dosimetry of laser illumination of ICG-stained adipose tissue for treatment of obesity and cellulite.

When irradiated with nanosecond laser pulses, gold nanoparticles allow for manipulation or destruction of cells and proteins with high spatial and temporal precision. Gold nanorods are especially attractive, because they have an up-to-20-fold stronger absorption than a sphere of equal volume, which is shifted to the optical window of tissue. Thus, an increased efficiency of cell killing is expected with laser pulses tuned to the near infrared absorption peak of the nanorods. In contrast to the higher-absorption, experiments showed a reduced efficacy of cell killing. In order to explain this discrepancy, transient absorption of irradiated nanorods was measured and the observed change of particle absorption was theoretically analyzed. During pulsed irradiation a strong transient and permanent bleaching of the near-infrared absorption band occurred. Both effects limit the ability of nanorods to destroy cells by nanocavitation. The existence of nanocavitation and transient bleaching was corroborated by optoacoustic measurements.

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