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Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins.

Morphological changes of the adipose tissue at phototreatment are studied in vitro using optical coherence tomography. The 200 to 600 μm fat tissue slices are used in the experiments. The observed change in the tissue structure was associated with fat cell lipolysis and destruction caused by the photodynamic effect. It is found that overall heating of a sample from room to physiological temperature leads to deeper and faster morphology tissue changes if other processing conditions are kept constant. These data support the hypothesis that photodynamic/photothermal treatment induces fat cell lipolysis during some period after treatment., (© 2013 Society of Photo-Optical Instrumentation Engineers (SPIE))

Fractional laser ablation is one of the relatively safe and minimally invasive methods used to administer micro- and nanoparticles into the skin at sufficiently large depth. In this article, we present the results of delivery of TiO₂ nanoparticles and Al₂O₃ microparticles into skin. Fractional laser microablation of skin was provided by a system based on a pulsed Er:YAG laser with the following parameters: the wavelength 2940 nm, the pulse energy 3.0 J, and the pulse duration 20 ms. Ex vivo and in vivo human skin was used in the study. The suspensions of titanium dioxide and alumina powder in polyethylene glycol with particle size of about 100 nm and 27 μm, respectively, were used. In the ex vivo experiments, reflectance spectra of skin samples with administered particles were measured and histological sections of the samples were made. In the in vivo experiment, reflectance spectroscopy, optical coherence tomography, and clinical photography were used to monitor the skin status during one month after suspension administering. It is shown that particles can be delivered into dermis up to the depth 230 μm and distributed uniformly in the tissue. Spectral measurements confirm that the particles stay in the dermis longer than 1 month., (© 2013 Society of Photo-Optical Instrumentation Engineers (SPIE))

Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical-clearing agents into tissue, such as physical methods, chemical-penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced. [Formula: see text].

The high incidence and mortality of breast cancer requires an effective, rapid, and cost-effective method for its diagnosis. Here, visible and near-infrared spectroscopy in the wavelength range of 400 to 2200 nm is utilized for distinguishing the malignant tumor tissue from benign tumor and normal breast tissues. Based on the absorption and scattering spectra of fixed samples, three spectral analysis methods are proposed which include an absorption spectral analysis, a scattering spectral analysis, and a combined spectral analysis of the two. By comparison with the histopathological examination, the sensitivity, specificity, and accuracy of the three analysis methods are calculated. The results showed that the combined spectral analysis method can significantly enhance the effectiveness when compared with the sole absorption or scattering spectral analysis method. The sensitivity, specificity, and accuracy of the combined spectral analysis method are 100%, 87.82%, and 87.50% for the benign tumor tissue and 81.82%, 100%, and 87.5% for malignant tumor tissue, respectively. All of the three values are 100% for normal breast tissue. This study demonstrates that the combined spectral analysis method has better potential for in vitro optical diagnosis for breast lesions.

We fabricated composite nanoparticles consisting of a plasmonic core (gold nanorods or gold-silver nanocages) and a hematoporphyrin-doped silica shell. The dual photodynamic and photothermal activities of such nanoparticles against Staphylococcus aureus 209 P were studied and compared with the activities of reference solutions (hematoporphyrin or silica-coated plasmonic nanoparticles). Bacteria were incubated with nanocomposites or with the reference solutions for 15 min, which was followed by CW light irradiation with a few exposures of 5 to 30 min. To stimulate the photodynamic and photothermal activities of the nanocomposites, we used LEDs (405 and 625 nm) and a NIR laser (808 nm), respectively. We observed enhanced inactivation of S. aureus 209 P by nanocomposites in comparison with the reference solutions. By using fluorescence microscopy and spectroscopy, we explain the enhanced antimicrobial effect of hematoporphyrin-doped nanocomposites by their selective accumulation in the vicinity of the bacteria., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The main limitation of optical imaging techniques for studying biological tissues is light scattering leading to decreasing of transmittance, which lowers the imaging quality. In this case, an immersion method for optical clearing of biological tissues can provide a possible solution to this problem, because the application of biocompatible clearing agents can reduce light scattering. Optical clearing represents a promising approach to increasing the imaging depth for various techniques, for example, various spectroscopy and fluorescent methods, and optical coherence tomography (OCT). We investigate the improvement of light penetration depth in blood after application of polyethylene glycol, polypropylene glycol, propylene glycol, and hemoglobin solutions using an OCT system. Influence of clearing agents on light transport in tissues and blood was also investigated in the mouse tail vein.

The structure, composition and photocatalytic activity of TiO(2) nanoparticles annealed in various gas atmospheres (N(2), NH(3) and H(2)) were studied in this work. The effect of treatment on crystal structure, particle size, chemical composition and optical absorbance were assessed by means of x-ray diffraction, transmission electron microscopy, x-ray photoelectron spectroscopy and diffuse optical reflectance/transmittance measurements, respectively. Photocatalytic properties of the materials were evaluated by three different methods: degradation of methyl orange in water, killing of Staphylococcus aureus bacteria and photogeneration of radicals in the presence of 3-carboxy-2,2,5,5-tetramethyl pyrrolidine-1-oxyl (PCA) marker molecules. The results indicate that the correlation between pretreatment and the photocatalytic performance depends on the photocatalytic processes and cannot be generalized.

Photonic nanotechnologies have good perspectives to be widely used in biophotonics. In this study we have developed an approach for calculation of nanoparticle temperature field accounting for absorbed local intensity at pulse laser radiation of composite spherical nanoparticles (nanoshells). This approach allowed us to analyze spatial inhomogeneities of light field diffracted into a nanoshell and corresponding distribution of the absorption energy and to provide numerical solution of time-dependent heat conduction equation accounting for corresponding spatially inhomogeneous distribution of heating sources. We were able to predict the appearance of a novel thermal effect - hoop-shaped hot zone on the nanoshell surface. The observed effect has potential applications in cell biology and medicine for controlled cell optoporation and nanosurgery, as well as cancer cell killing., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The strong optical scattering of skin tissue makes it very difficult for optical coherence tomography (OCT) to achieve deep imaging in skin. Significant optical clearing of in vivo rat skin sites was achieved within 15 min by topical application of an optical clearing agent PEG-400, a chemical enhancer (thiazone or propanediol), and physical massage. Only when all three components were applied together could a 15 min treatment achieve a three fold increase in the OCT reflectance from a 300 μm depth and 31% enhancement in image depth Z(threshold).

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.

Heating of composite plasmon-resonant nanoparticles (spherical gold nanoshells) under pulse laser illumination is considered. The numerical solution of the time-dependent heat conduction equation accounting for spatial inhomogeneities of absorbed laser radiation is performed. Important features of temperature kinetics and thermal flux inside nanoparticles are analyzed. Possible applications of the observed effects in nanotechnology and medicine are discussed.

The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined.

Changes in the light transmission coefficient of human adipose cells as a result of photodynamic action have been detected and investigated. Based on the statistical processing of digital micrographs of thin tissue slices, the kinetics of processes occurring in the adipose tissue has been quantitatively estimated. It has been suggested that the effects observed are brought about through the lipolysis of adipose cells with the retention of their structure.

Flow cytometry (FCM) has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo FCM, which provides detection and imaging of circulating normal and abnormal cells directly in blood or lymph flow. The goal of this review is to provide a brief history, features, and challenges of this new generation of FCM methods and instruments. Spectrum of possibilities of in vivo FCM in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, and cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform., (Copyright © 2011 International Society for Advancement of Cytometry.)

Dehydration induced by optical clearing agents (OCAs) can improve tissue optical transmittance; however, current studies merely gave some qualitative descriptions. We develop a model to quantitatively evaluate water content with partial least-squares method based on the measurements of near-infrared reflectance spectroscopy and weight of porcine skin. Furthermore, a commercial spectrometer with an integrating sphere is used to measure the transmittance and reflectance of skin after treatment with different OCAs, and then the water content and optical properties of sample are calculated, respectively. The results show that both the reduced scattering coefficient and dehydration of skin decrease with prolongation of action of OCAs, but the relative change in former is larger than that in latter after a 60-min treatment. The absorption coefficient at 1450 nm decreases completely coincident with dehydration of skin. Further analysis illustrates that the correlation coefficient between the relative changes in the reduced scattering coefficient and dehydration is ∼1 during the 60-min treatment of agents, but there is an extremely significant difference between the two parameters for some OCAs with more hydroxyl groups, especially, glycerol or D-sorbitol, which means that the dehydration is a main mechanism of skin optical clearing, but not the only mechanism.

Changes in optical properties of human adipose tissue cells after photodynamic exposure in vitro were found and investigated. Analysis of the kinetics of the process was realized by means of photomicrography of the object investigated. The statistical computer processing of digital photos obtained gave us an opportunity to estimate quantitatively the kinetics of photodynamic effect upon the tissue. Optical interpretation of the photos obtained indicates that the observed phenomenon corresponds to the partial lysis of adipose tissue cells without their complete destruction.

In this article, we discuss the optical immersion method based on refractive index matching of scatterers (e.g., collagen, elastin fibers, cells and cell compartments) and the ground material (interstitial fluid and/or cytoplasm) of tissue and blood under the action of exogenous optical clearing agents. We analyze the optical clearing of fibrous and cell-structured tissues and blood from the point of view of receiving more valuable, normally hidden, information from spectroscopic and polarization measurements, confocal microscopy, optical coherence and optical projection tomography, as well as from nonlinear spectroscopies, such as two-photon fluorescence and second-harmonic generation techniques. Some important applications of the immersion technique to glucose sensing, drug delivery monitoring, improvements of image contrast and imaging depth, nondistortive delivery of laser radiation and precision tissue laser photodisruption, among others, are also described.

It is known that the fibrous structure of muscle causes light scattering. This phenomenon occurs due to the refractive index discontinuities located between muscle fibers and interstitial fluid. To study the possibility of reducing light scattering inside muscle, we consider its spectral transmittance evolution during an immersion treatment with an optical clearing solution containing ethanol, glycerol, and distilled water. Our methodology consists of registering spectral transmittance of muscle samples while immersed in that solution. With the spectral data collected, we represent the transmittance evolution for some wavelengths during the treatment applied. Additionally, we study the variations that the treatment has caused on the samples regarding tissue refractive index and mass. By analyzing microscopic photographs of tissue cross section, we can also verify changes in the internal arrangement of muscle fibers caused by the immersion treatment. Due to a mathematical model that we develop, we can explain the variations observed in the studied parameters and estimate the amount of optical clearing agent that has diffused into the tissue samples during the immersion treatment. At the end of the study, we observe and explain the improvement in tissue spectral transmittance, which is approximately 65% after 20 min.

Background and Objectives: Tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs) can reduce the scattering and enhance the penetration of light in tissue. However, the barrier function of epidermis limits the penetration of OCAs, and hence is responsible for the poor optical clearing efficacy of skin by topical action. In this study, a variety of light irradiation was applied to increase permeability of agents in skin and improve the optical clearing efficacy., Study Design/materials and Methods: Different light sources with different dose, i.e, CO(2) laser, Nd:YAG laser (532 and 1,064 nm) with different pulse modes and Intense Pulsed Light (IPL) (400-700 and 560-950 nm) were used to irradiate rat skin in vivo, and then glycerol was applied onto the irradiated zone. VIS-NIR spectrometer was utilized to monitor the changes of reflectance. In vitro skin samples were also irradiated by Q-switched Nd:YAG laser (1,064 nm) and then treated by glycerol for 10-60 minutes. Based on the measurement of the reflectance and transmittance of the samples, the optical properties of skin and penetration depth of light were calculated., Results: Results show that photo-irradiation with appropriate dose combining with the following glycerol treatment is able to reduce in vivo skin reflectance. Compared with the control group, the maximal changes in reflectance are ninefold at 575 nm and eightfold at 615 nm, respectively, which were caused by Q-switched 1,064-nm Nd:YAG laser irradiation and following glycerol treatment. The results for in vitro skin demonstrate that the joint action can significantly increase the optical penetration depth in samples., Conclusions: The combination of Q-switched Nd:YAG (1,064 nm) laser and glycerol could enhance optical skin clearing efficacy significantly. This study provides a non-invasive way to improve the optical clearing of skin, which will benefit the skin optical therapy.

Topical trans-dermal delivery of drugs has proven to be a promising route for treatment of many dermatological diseases. The aim of this study is to monitor and quantify the permeability rate of glucose solutions in rhesus monkey skin noninvasively in vivo as a primate model for drug diffusion. A time-domain Optical Coherence Tomography (OCT) system was used to image the diffusion of glucose in the skin of anesthetized monkeys for which the permeability rate was calculated. From 5 experiments on 4 different monkeys, the permeability for glucose-20% was found to be (4.41 +/- 0.28) 10(-6) cm/sec. The results suggest that OCT might be utilized for the noninvasive study of molecular diffusion in the multilayered biological tissues in vivo., (2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The penetration of light in biological tissues can be enhanced by using optical-clearing techniques. However, researches on the mechanism are limited to in vitro experiments. In this study, rat dorsal skin was used to discuss the mechanism for in vivo optical clearing. Glycerol solutions with different concentrations were applied by dermal injection; then the skin reflectance spectrum, SHG imaging and microstructural changes were monitored. The results showed that with the skin becoming transparent, the corresponding reflectance decreased, and the thickness of dermis and diameter of collagen fibril decreased, but no collagen fiber was dissolved or fractured. Hence, it can be concluded that the thickness decrease of dermis and corresponding more regular packing of tissue fibers plays an important role in the mechanism for glycerol-induced optical clearing of skin in vivo., (2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the major challenges in imaging biological tissues using optical techniques, such as optical coherence tomography (OCT), is the lack of light penetration due to highly turbid structures within the tissue. Optical clearing techniques enable the biological samples to be more optically homogeneous, allowing for deeper penetration of light into the tissue. This study investigates the effect of optical clearing utilizing various concentrations of glucose solution (10%, 30%, and 50%) on porcine skin. A gold-plated mirror was imaged beneath the tissue and percentage clearing was determined by monitoring the change in reflected light intensity from the mirror over time. The ratio of percentage clearing per tissue thickness for 10%, 30% and 50% glucose was determined to be 4.7 ± 1.6% mm(-1) (n = 6), 10.6 ± 2.0% mm(-1) (n = 7) and 21.8 ± 2.2% mm(-1) (n = 5), respectively. It was concluded that while higher glucose concentration has the highest optical clearing effect, a suitable concentration should be chosen for the purpose of clearing, considering the osmotic stress on the tissue sample.

Optical clearing agents (OCAs) with high refractive indices and hyperosmolarity can enhance the penetration of light in tissues by reducing scattering in tissues. However, the mechanism of tissue optical clearing is not much clear for the complex interaction between tissues and OCAs. In this work, Intralipid was mixed with different concentrations of OCAs, i.e. dimethyl sulfoxide (DMSO), glycerol, 1,4-butanediol, 1,2-propanediol, poly-ethylene glycol 200 (PEG200) and poly-ethylene glycol 400 (PEG400). Except for PEG200 and PEG400 that make aggregation of particles, the others kept the mixture uniform. The reduced scattering coefficients of uniform mixtures were predicted with Mie theory and measured by a commercially available spectrophotometer with an integrating sphere. The results show that all of the OCAs used enhance the optical clearing effect of Intralipid. If OCAs do not change the structure of Intralipid, Mie theory prediction matches well with the measurements. And the higher the refractive index of OCA, the smaller the reduced scattering coefficient. A simple formula deduced can quantitatively predict the optical clearing effect caused by OCAs. This work is helpful for clarifying the mechanism of tissue optical clearing, which will make the effect of optical clearing of tissue predictable and controllable.

This report introduces a novel diagnostic and therapeutic platform for in vivo non-invasive detection and treatment of metastases in sentinel lymph nodes (SLNs) at single cell level using an integrated system of multicolor photoacoustic (PA) lymph flow cytometry, PA lymphography, absorption image cytometry, and photothermal (PT) therapy. A melanoma-bearing mouse model was used to demonstrate the capability of this platform for real-time lymphatic mapping, counting of disseminated tumor cells (DTCs) in prenodal lymphatics, and detecting metastasis in SLNs and its purging. The detection and ablation of non-pigmented breast cancer cells in SLNs was achieved by labeling them with nanoparticles. The association between DTC count and SLN metastasis progression supports lymphatic DTCs as a novel prognostic marker of metastasis. The fiber-based portable PA device may replace the conventional SLN(s) excision and histology-based staging. The earliest detection of DTCs in the lymphatic vessels before the establishment of nodal metastasis may prevent metastasis by well-timed ablation of DTCs., (2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The formulation of the finite-difference time-domain (FDTD) approach is presented in the framework of its potential applications to in-vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive-index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in-vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open up a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets., (2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Kinetics, biodistribution, and histological studies were performed to evaluate the particle-size effects on the distribution of 15 nm and 50 nm PEG-coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance-scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15-30 min after injection. Rats were injected in the tail vein with PEG-coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment.

We describe an application of plasmonic silica/gold nanoshells to produce a controllable laser hyperthermia in tissues with the aim of the enhancement of cancer photothermal therapy. Laser irradiation parameters are optimized on the basis of preliminary experimental studies using a test-tube phantom and laboratory rats. Temperature distributions on the animal skin surface at hypodermic and intramuscular injection of gold nanoparticle suspensions and affectations by the laser radiation are measured in vivo with a thermal imaging system. The results of temperature measurements are compared with tissue histology.

Compared with blood tests, cell assessment in lymphatics is not well-established. The goal of this work was to develop in vivo lymph tests using the principles of flow cytometry. Cells in living animals were counted by laser (420-2,300 nm) generation of photoacoustic (PA) signals in individual cells hydrodynamically focused by lymph valves into a single file flow, and using endogenous absorption as intrinsic cell-specific markers, or gold nanorods, nanoshells, and carbon nanotubes as multicolor probes. PA data were verified by high-speed transmission, photothermal, and fluorescent imaging. Counting of melanoma and immune-related cells in normal, apoptotic, and necrotic states in lymphatics in vivo was demonstrated to have the unprecedented sensitivity as one metastatic cell among millions of white blood cells. The time-resolved PA spectral identification of flowing cells was achieved using multicolor labels and laser pulses of different wavelengths and time delays. Multiparameter, noninvasive, portable flow cytometer can be used for preclinical studies on animals with the potential of translation to humans for in vivo PA mapping of colorless lymph vessels and sentinel nodes with simultaneous single cell detection and metastasis assessment without labeling or use of contrast dyes and/or novel low-toxic multicolor probes with different absorption spectra., (Copyright 2008 International Society for Advancement of Cytometry.)

Imaging of biological tissues with optical coherence tomography (OCT) poses a great interest for its capability to noninvasively outline subsurface microstructures within tissues. However, a major limitation for many optical imaging techniques is inadequate depth penetration of light in turbid media, which is bounded to just a few millimeters. There have been several attempts to improve light penetration depth in biological tissues, including application of different tissue optical clearing methods. In this study, an aqueous solution of glucose (40%) is added to rabbit sclera in vitro, where depth-resolved permeability coefficients and optical clearing are calculated with OCT. The permeability rate in regions in the upper 80- to 100-microm region is found to be different from that of regions in the deeper 100-microm region: (6.01+/-0.37)x10(-6) cmsec and (2.84+/-0.68)x10(-5) cmsec, respectively. A difference in percent clearing is also noted. Optical clearing of the upper region is about 10% and increased to 17 to 22% in the one beneath. These results demonstrate the capability of OCT-based methods to not only measure the diffusion rate and optical clearing of a tissue, but also its ability of functional differentiation between layers of epithelial tissues.

Accelerated diffusion of water and hyperosmotic optical clearing agents is studied as a result of enhanced epidermal permeability. A lattice of microzones (islets) of damage in stratum corneum is induced using a flash-lamp applique system. An optical clearing agent composed of 88% glycerol in aqueous solution is used for all experiments. Research of skin dehydration and glycerol delivery through epidermis at both intact and perforated stratum corneum is presented. The dehydration process induced by both stimuli of evaporation and osmotic agent action is studied by weight measurements. Dynamics of refractive index alteration of both glycerol solution and water during their interaction with skin samples is monitored. The amounts of water escaping from skin through the stratum corneum, due to hyperosmotic-agent action, and glycerol penetrating through the skin sample, are estimated. The results show that the proposed method allows for effective transepidermal water loss and delivery of optical clearing agents.

The coefficient of diffusion of methylene blue in pathologically changed human maxillary sinus mucosa in vitro has been estimated for the first time. The mean value of the diffusion coefficient is (4.8 +/- 2.9) x 10(-7) cm2/s. The method is based on the registration of the dynamics of reflectance of tissue samples under the action of the dye. The diffusion coefficient has been estimated by approximation of experimental data in the framework of the model presented.

The functional morphology of biological fluids is a basically new branch of science, which is based on the study of a result of phase change processes in the substrate under study. The paper shows the specific features of plasma spatial organization in patients with chronic and acute coronary heart disease (CHD). The findings may be used as an additional criterion for the differential diagnosis of the above forms of CHD and open up a new line in the diagnosis and risk stratification in CHD patients.

The goal of this work is to develop in vivo photoacoustic (PA) flow cytometry (PAFC) for time-resolved detection of circulating absorbing objects, either without labeling or with nanoparticles as PA labels. This study represents the first attempt, to our knowledge, to demonstrate the capability of PAFC with tunable near-infrared (NIR) pulse lasers for real-time monitoring of gold nanorods, Staphylococcus aureus and Escherichia coli labeled with carbon nanotubes (CNTs), and contrast dye Lymphazurin in the microvessels of mouse and rat ears and mesenteries. PAFC shows the unprecedented threshold sensitivity in vivo as one gold nanoparticle in the irradiated volume and as one bacterium in the background of 10(8) of normal blood cells. The CNTs are demonstrated to serve as excellent new NIR high-PA contrast agents. Fast Lymphazurin diffusion in live tissue is observed with rapid blue coloring of a whole animal body. The enhancement of the thermal and acoustic effects is obtained with clustered, multilayer, and exploded nanoparticles. This novel combination of PA microscopy/spectroscopy and flow cytometry may be considered as a new powerful tool in biological research with the potential of quick translation to humans, providing ultrasensitive diagnostics of pathogens (e.g., bacteria, viruses, fungi, protozoa, parasites, helminthes), metastatic, infected, inflamed, stem, and dendritic cells, and pharmacokinetics of drug, liposomes, and nanoparticles in deep vessels (with focused transducers) among other potential applications.

The effect of temperature and lectin from bacteria of the genues Azospirillum with blocked activity on human adipose tissue cells has been studied. The temperature used was 43.5 +/- 0.5 degrees C. Comparative results are given for the effect of lectin with the blocked and active carbohydrate-binding centers on adipocytes during heating, and the time course of the structural changes of adipocytes is described. When lectin with the active carbohydrate-binding centers was used for treatment, the heat-treated cells of a healthy obesity-prone subject died on the average in 55 +/- 5 min, whereas cells treated with lectin with the L-fucose-blocked carbohydrate-binding centers died in 80 +/- 5 min. The heat-treated cells of a diabetic obesity-prone patient died in 150 +/- 10 min on average when exposed to both active and inactive lectin. Consequently, when the lectin center is blocked with L-fucose, the effectiveness of lectin action on adipose cells of healthy obesity-prone persons decreases.

Purpose: Noninvasive functional imaging, monitoring, and quantification of analytes transport in epithelial ocular tissues are extremely important for therapy and diagnostics of many eye diseases. In this study the authors investigated the capability of optical coherence tomography (OCT) for noninvasive monitoring and quantification of diffusion of different analytes in sclera and cornea of rabbit eyes., Methods: A portable time-domain OCT system with wavelength of 1310 +/- 15 nm, output power of 3.5 mW, and resolution of 25 mum was used in this study. Diffusion of different analytes was monitored and quantified in rabbit cornea and sclera of whole eyeballs. Diffusion of water, metronidazole (0.5%), dexamethasone (0.2%), ciprofloxacin (0.3%), mannitol (20%), and glucose solution (20%) were examined, and their permeability coefficients were calculated by using OCT signal slope and depth-resolved amplitude methods., Results: Permeability coefficients were calculated as a function of time and tissue depth. For instance, mannitol was found to have a permeability coefficient of (8.99 +/- 1.43) x 10(-6) cm/s in cornea and (6.18 +/- 1.08) x 10(-6) cm/s in sclera. The permeability coefficient of drugs with small concentrations (where water was the major solvent) was found to be in the range of that of water in the same tissue type, whereas permeability coefficients of higher concentrated solutions varied significantly., Conclusions: Results suggest that the OCT technique might be a powerful tool for noninvasive diffusion studies of different analytes in ocular tissues. However, additional methods of OCT signal acquisition and processing are required to study the diffusion of agents of small concentrations.

Background: Photothermal (PT) cytometry has recently demonstrated great potential for the label-free detection of nonfluorescent cells under static conditions. The goal of our investigation was to expand this technique to the detection of flowing cells in vitro., Methods: Cells in flow were irradiated with short, tunable laser pulses (420-2,300 nm, 8 ns), and the absorbed energy was detected by monitoring of the temperature-dependent variations in the refractive index in the cells with a second, collinear probe beam in two modes: (a) PT imaging of single cells with a pulsed probe beam (639 nm, 13 ns) and (b) thermolens monitoring of the integral PT responses from individual cells as whole with a continuous-wave probe beam (633 nm, 2 mW)., Results: PT flow cytometry at the current speed of analysis of 10 cell/s, with the capability to image selected cells of interest flowing at velocities up to 2 m/s, demonstrated the capability for (a) label-free detection of flowing single cells (e.g., blood and cancer cells) on the basis of the differences in their endogenous absorption properties, (b) identification of cells labeled with gold nanoparticles, (c) rapid cell viability testing, (d) aggregation immunoassay, and (e) optimization of selective nanophotothermolysis., Conclusions: PT cytometry can be extended to the study of cells in flow. This new technique increases the speed of cell analysis approximately 10(2) times over that of conventional PT technique, with the potential to achieve a rate of 10(4)-10(5) cells/s in specific PT applications, which has previously been realized only with cells under static conditions., ((c) 2007 International Society for Analytical Cytology.)

The transport properties of dense random media such as rutile powder layers and polyball suspensions are analyzed in visible and near infrared on the basis of experimental data on coherent backscattering, diffuse transmittance, and low-coherence interferometry. The developed technique of retrieval of the transport parameters of examined scattering media allows the evaluation of the transport mean free path l* and the effective refractive index n(ef) of the medium without a priori knowledge of the optical properties of the scattering particles. It is found that with decreasing wavelength lambda(0) the value of localization parameter 2pin(ef)l*/lambda(0) of the studied rutile samples abruptly drops and approaches approximately 2.6 at 473 nm. This peculiarity is caused by the very large scattering efficiency of scatterers in the vicinity of the first Mie resonance.

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cellos functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Background: Rat mesentery has been widely used to study microvascular functions. The goal of this work is to extend this animal model to monitor blood and lymph microvessel function during lymphedema., Methods and Results: Lymphedema is created by microsurgical removal of regional lymph nodes (lymphadenectomy) or ligation of the collecting vein. Water volume in mesenteric tissue, microvessel diameters, phasic contraction, valve function, lymph flow velocity, and cell migration were analyzed during lymphedema development. Dynamic observation of water amount after lymphadenectomy revealed increasing edema from 30 min to 1 week; greatest degree of edema at one week, and gradual decrease in edema from 1 to 11 weeks. These effects were accompanied by acute constriction of lymph vessels and slowing of lymph flow velocity, switching to dilation and appearance of new blood capillaries at week 1, progressing to dilation and degenerative changes of the microlymphatic wall at week 4, and, finally, leading to lymphatic fibrosis and lymphangiogenesis at week 11. Acute venous insufficiency (30 min after vein ligation) led to significant edema, decreasing blood flow velocity to stasis, and output of erythrocytes from venules to interstitium, with further movement to microlymphatics and regional lymph nodes., Conclusions: Rat mesentery as an animal model in combination with an advanced optical imaging system is valuable in studying microlymphatic disturbances in mesentery during the development of experimental lymphedema from latent period to chronic stages, including monitoring of individual cell dislocation with high resolution optical imaging.

A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 10(7) normal blood cells.

Pilot results on the application of the finite-difference time-domain (FDTD) approach for studying the implementation of the optical immersion technique for the visualization of single and multiple gold nanoparticles in biological cells are presented. We focus on two different scenarios considering single biological cells containing (1) cytoplasm, nucleus, and membrane and (2) cytoplasm, nucleus, gold nanoparticles, and membrane. To the best of our knowledge, this is the first time the cell membrane thickness and gold nanoparticle effects on the forward scattered light from biological cells are discussed. The applicability and the potential of the FDTD approach for studying optical immersion technique enhanced bioimaging is demonstrated.

Background and Objectives: Strong light scattering in skin prevents precise targeting of optical energy in therapeutic and diagnostic applications. Optical immersion based on matching refractive index of scattering centers with that of surrounding matter through introduction of an exogenous index-matching agent can alleviate the problem. However, slow diffusion of the index-matching agent through skin barrier makes practical implementation of this approach difficult. We propose a method of accelerating penetration of the index-matching compounds by enhancing skin permeability through creating a lattice of micro-zones (islets) of limited thermal damage in the stratum corneum (SC)., Study Design/materials and Methods: A flash lamp (intense pulsed light) system and an island mask with a pattern of absorbing centers (center size approximately 75-120 microm, lattice pitch approximately 450-500 microm) were used to create the lattice of islets of damage (LID). Index-matching agents, such as glucose solution, propylene glycol solution, and glycerol solution, were applied., Results: Experimental results of optical clearing ex vivo rat and pig skin, and ex vivo and in vivo human skin are presented. Optical transmission spectra of the skin samples with LID were measured during some 2 hours after application of index-matching chemical agents. In order to assess and compare the clearing rate under different treatment and clearing agents we calculated the quantity that we call "relative transmittance": T(rel) = I(t)(lambda)/I(0)(lambda), were I(t)(lambda) is the intensity measured at elapsed time t. The dynamics of relative transmittance of skin samples at 470 and 650 nm shows that the implementation of limited thermal damage technique leads to a 3-10-fold increase of optical clearing (rise of transmittance) rate compared to the results obtained when the samples were treated with high-intensity light pulses but without the use of island damage mask (IDM). It was observed from the plotted spectra of relative transmittance that the maximum increase of transmitted light intensity has been obtained with glucose solution as a clearing agent. Noteworthy is the difference in the trend of spectral curves: relative transmittance spectrum for glycerol reveals, on the whole, a greater slope which may be indicative of higher extent of index matching between the scattering centers and base material for this index-matching agent. Under the transillumination of the skin sample by the wide flat beam the more effective clearing (the increase of transmitted intensity) is attained within the hemoglobin absorption bands; with the narrow quasi-collimated beam the higher relative transmittance was observed over the intervals of minimum absorption., Conclusions: The use of specially designed island mask combined with non-laser intensive pulse irradiation produces a lattice of islands of limited thermal damage in SC that substantially enhances the penetration rate of topically applied index-matching agents. The suggested technique gave comparable magnitudes of clearing dynamics enhancement for glucose solution, glycerol solution, and propylene glycol solution applied to mammalian skin.

In vivo, label-free, high-speed (up to 10,000 with the potential for 40,000 frames per second), high-resolution (up to 300 nm) real-time continuous imaging with successive framing of circulating individual erythrocytes, leukocytes, and platelets in fast blood flow is developed. This technique, used in an animal model, reveals the extremely high dynamic deformability of erythrocytes in natural flow. Potential applications of this technique are discussed with focus on time-resolved monitoring of the cell deformation dynamics in the native biological environment, which may have diagnostic value for the early diagnosis of diseases.

We demonstrate the capability of the optical coherence tomography (OCT) technique for depth-resolved monitoring and quantifying of glucose diffusion in fibrous tissues (sclera). The depth-resolved and average permeability coefficients of glucose were calculated. We found that the glucose diffusion rate is not uniform throughout the tissue and is increased from approximately 2.39+/-0.73 x 10(-6) cm/s at the epithelial side to 8.63+/-0.27 x 10(-6) cm/s close to the endothelial side of the sclera. Results demonstrated that the OCT technique is capable of depth-resolved monitoring and quantification of glucose diffusion in sclera with a resolution of approximately 40 mum.

Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments., ((c) 2006 Wiley-Liss, Inc.)

The high spatial resolution (approximately 350 nm) transmission digital microscopy (TDM) was developed for real time in vivo imaging of microlymphatics of rat mesentery at a single cell level without any contrast agent. The main mesenteric microstructures (lymph-vessel diameter, valve geometry, cells, etc.) and their dynamics (wall motion, valve function, cell velocity, etc.) were monitored with TDM. Depending on structure size, different magnifications were used to image relatively large whole lymphangion (x4 to x10) as well as to image single cells (x40 to x100) in lymph and blood flow including estimation of their shape, size, and aggregation state. Various potential applications of the TDM for in vivo studies are discussed, including visualization of circulating cells in lymph and blood flows, studying the kinetics of platelets, leukocyte rolling, as well as imaging absorbing nonfluorescent mesentery structures and leukocytes with a high optical resolution.