A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.

High-throughput optical imaging is critical to obtain large-scale neural connectivity information of brain in neuroscience. Using a digital mirror device and a scientific complementary metal-oxide semiconductor camera, we report a significant speed improvement of structured illumination microscopy (SIM), which produces a maximum SIM net frame rate of 133 Hz. We perform three-dimensional (3-D) imaging of mouse brain slices at diffraction-limited resolution and demonstrate the fast 3-D imaging capability to a large sample with an imaging rate of 6.9×10 ⁷   pixel/s of our system, an order of magnitude faster than previously reported.

An autofluorescence lifetime wide-field imaging system that can generate contrast in underlying tissue structures of normal and malignant brain tissue samples with video rate acquisition and processing time is presented. Images of the investigated tissues were acquired with high resolution (∼35  μm ) using an algorithm to produce contrast based on differences in relative lifetimes. Sufficient contrast for delineation was produced without the computation of fluorescence decay times or Laguerre coefficients. The imaged tissues were sent for histological analysis that confirmed the detected imaged tissues morphological findings and correlations between relative lifetime maps and histology identified.

In recent years, the use of light emitting diodes (LEDs) has become commonplace in fluorescence microscopy. LEDs are economical and easy to couple to commercial microscopes, and they provide powerful and stable light that can be triggered by transistor-transistor logic pulses in the range of tens of microseconds or shorter. LEDs are usually installed on the epifluorescence port of the microscope to obtain whole-field illumination, which is ideal for fluorescence imaging. In contrast, photolysis or channelrhodopsin stimulation often requires localized illumination, typically achieved using lasers. Here we show that insertion of a long-pass (<411  nm ) filter with an appropriately sized pinhole in the epifluorescence pathway, combined with dual UV/visible illumination, can produce efficient whole-field visible illumination and spot UV illumination of 15 to 20 μm. We tested our system by performing calcium imaging experiments combined with L-glutamate or N -methyl-D-aspartic acid (NMDA) photorelease in hippocampal neurons from brain slices or dissociated cultures, demonstrating the ability to obtain local activation of NMDA receptors exclusively in the illuminated spot. The very inexpensive and simple system that we report here will allow many laboratories with limited budgets to run similar experiments in a variety of physiological applications.

Strain-dependent transmission data of nine iliotibial tract specimens are determined using a custom-built optical setup with a halogen light source and an industrial norm material testing machine. Polarized light microscopy and hematoxylin-eosin staining indicated that lateral contraction of collagen structures is responsible for total intensity variations during a 20-cycle preconditioning and a 5-cycle tensile test. Tensile force progress is opposite to total transmission progress. Due to dehydration, wavelength-specific radiation intensity shifting is determined during the test, primarily noticeable in a water absorption band between 1400 and 1500 nm. The results show the capability of integrating spectrophotometry technology into biomechanics for determining structural alterations of human collagen due to applied strain. Being more sensitive to drying, spectrophotometry may likely serve as a quality control in stress-strain testing of biological structures.

A time-domain multiwavelength (635 to 1060 nm) optical mammography was performed on 147 subjects with recent x-ray mammograms available, and average breast tissue composition (water, lipid, collagen, oxy- and deoxyhemoglobin) and scattering parameters (amplitude a and slope b ) were estimated. Correlation was observed between optically derived parameters and mammographic density [Breast Imaging and Reporting Data System (BI-RADS) categories], which is a strong risk factor for breast cancer. A regression logistic model was obtained to best identify high-risk (BI-RADS 4) subjects, based on collagen content and scattering parameters. The model presents a total misclassification error of 12.3%, sensitivity of 69%, specificity of 94%, and simple kappa of 0.84, which compares favorably even with intraradiologist assignments of BI-RADS categories.

Laser speckle imaging is widely used to monitor functional blood perfusion within tissue beds in vivo but traditionally has difficulty visualizing small blood vessels even when the exposure time of the detector is long. We report a simple method that uses the motion contrast of dynamic speckle patterns to noninvasively visualize the distribution of blood flow within tissue beds in vivo. We experimentally demonstrate that the motion contrast can significantly suppress the effect of static scattering, leading to enhanced visibility of the functional blood vessels, including capillaries when compared to the traditional laser speckle contrast imaging.

The skin is the biggest organ of the body, representing its barrier to the environment. It provides protection against water loss, keeps microorganisms from invading our body, and responds sensitively to external stimuli. The skin barrier is formed by the uppermost cell layer, i.e., the stratum corneum, consisting of dead horny cells. Underlying the stratum corneum are various layers of living cells. The capillary structures of the blood vessels appear from a depth of approximately 150 µm beneath the skin surface. The homogeneous structure of the skin surface is interrupted by hair follicles and sweat glands. As a sensory organ the skin is an essential means of interpersonal communication. This is why we devote particular attention to skin care and dermal treatment. In this context, diagnostics and therapy control play a decisive role. Easily accessible, the skin is an ideal object to be investigated by noninvasive optical and spectroscopic methods. An ample range of such methods is available for dermal analysis, including fluorescence, reflectance, Raman, and CARS measurements. Laser scanning microscopy has proven to be specifically suitable for investigating the skin barrier and the underlying living cell layers up to a depth of 200 µm depending on the wavelength applied, imaging both cellular and molecular structures. The primary purpose of these investigations is to analyze the integrity of the skin barrier, which is characterized by the organization of the cellular structures and by the composition of the lipid layers surrounding the cells. The application both of laser scanning microscopy and optical coherence tomography is focused mainly on the detection of dermal lesions, in particular of skin tumors, and their response to different therapies.

Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections and the effects of tissue processing. Both hand and thigh sections of human cadavers were analyzed in their unprocessed and formalin-fixed, paraffin-processed (FFPP), and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum (SC), intermediate underlying epithelium, and dermal layers for sections from both anatomical sites. The SC is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the SC, intermediate, and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks.

Maxillary defects resulting from cancer, trauma, and congenital malformation affect the chewing efficiency and retention of dentures in these patients. The use of implant-retained palatal obturator dentures has improved the self-esteem and quality of life of several subjects. We evaluate the stress distribution of implant-retained palatal obturator dentures with different attachment systems by using the photoelastic analysis images. Two photoelastic models of the maxilla with oral-sinus-nasal communication were fabricated. One model received three implants on the left side of the alveolar ridge (incisive, canine, and first molar regions) and the other did not receive implants. Afterwards, a conventional palatal obturator denture (control) and two implant-retained palatal obturator dentures with different attachment systems (O-ring; bar-clip) were constructed. Models were placed in a circular polariscope and a 100-N axial load was applied in three different regions (incisive, canine, and first molar regions) by using a universal testing machine. The results were photographed and analyzed qualitatively using a software (Adobe Photoshop). The bar-clip system exhibited the highest stress concentration followed by the O-ring system and conventional denture (control). Images generated by the photoelastic method help in the oral rehabilitator planning.

Digital infrared thermal imaging is used to assess noninvasively the severity of burn wounds in 13 pediatric patients. A delta-T (ΔT ) parameter obtained by subtracting the temperature of a healthy contralateral region from the temperature of the burn wound is compared with the burn depth measured histopathologically. Thermal imaging results show that superficial dermal burns (IIa) show increased temperature compared with their contralateral healthy region, while deep dermal burns (IIb) show a lower temperature than their contralateral healthy region. This difference in temperature is statistically significant (p<0.0001 ) and provides a way of distinguishing deep dermal from superficial dermal burns. These results show that digital infrared thermal imaging could be used as a noninvasive procedure to assess burn wounds. An additional advantage of using thermal imaging, which can image a large skin surface area, is that it can be used to identify regions with different burn depths and estimate the size of the grafts needed for deep dermal burns.

Noncontact imaging photoplethysmography (PPG) can provide physiological assessment at various anatomical locations with no discomfort to the patient. However, most previous imaging PPG (iPPG) systems have been limited by a low sample frequency, which restricts their use clinically, for instance, in the assessment of pulse rate variability (PRV). In the present study, plethysmographic signals are remotely captured via an iPPG system at a rate of 200 fps. The physiological parameters (i.e., heart and respiration rate and PRV) derived from the iPPG datasets yield statistically comparable results to those acquired using a contact PPG sensor, the gold standard. More importantly, we present evidence that the negative influence of initial low sample frequency could be compensated via interpolation to improve the time domain resolution. We thereby provide further strong support for the low-cost webcam-based iPPG technique and, importantly, open up a new avenue for effective noncontact assessment of multiple physiological parameters, with potential applications in the evaluation of cardiac autonomic activity and remote sensing of vital physiological signs.

Raman spectra of the skin of subjects with nickel allergy are analyzed and compared to the spectra of healthy subjects to detect possible biochemical differences in the structure of the skin that could help diagnose metal allergies in a noninvasive manner. Results show differences between the two groups of Raman spectra. These spectral differences can be classified using principal component analysis. Based on these findings, a novel computational technique to make a fast evaluation and classification of the Raman spectra of the skin is presented and proposed as a noninvasive technique for the detection of nickel allergy.

Vibrational spectroscopy and imaging have been used to compare barrier properties in human skin, porcine skin, and two human skin equivalents, Epiderm 200X with an enhanced barrier and Epiderm 200 with a normal barrier. Three structural characterizations were performed. First, chain packing and conformational order were compared in isolated human stratum corneum (SC), isolated porcine SC, and in the Epiderm 200X surface layers. The infrared (IR) spectrum of isolated human SC revealed a large proportion of orthorhombically packed lipid chains at physiological temperatures along with a thermotropic phase transition to a state with hexagonally packed chains. In contrast, the lipid phase at physiological temperatures in both porcine SC and in Epiderm 200X, although dominated by conformationally ordered chains, lacked significant levels of orthorhombic subcell packing. Second, confocal Raman imaging of cholesterol bands showed extensive formation of cholesterol-enriched pockets within the human skin equivalents (HSEs). Finally, IR imaging tracked lipid barrier dimensions as well as the spatial disposition of ordered lipids in human SC and Epiderm 200X. These approaches provide a useful set of experiments for exploring structural differences between excised human skin and HSEs, which in turn may provide a rationale for the functional differences observed among these preparations.

Photodynamic therapy (PDT) is a noninvasive therapeutic method first introduced in the field of dermatology. It is mainly used for the treatment of precancerous and superficial malignant skin tumors. Today PDT finds new applications not only for nononcologic dermatoses but also in the field of other medical specialties such as otorhinolaryngology, ophthalmology, neurology, gastroenterology, and urology. We are witnessing a broadening of the spectrum of skin diseases that are treated by PDT. Since its introduction, PDT protocol has evolved significantly in terms of increasing method efficacy and patient safety. In this era of evidence-based medicine, it is expected that much effort will be put into creating a worldwide accepted consensus on PDT. A review on the current knowledge of PDT is given, and the historical basis of the method's evolution since its introduction in the 1900s is presented. At the end, future challenges of PDT are focused on discussing gaps that exist for research in the field.

Optical imaging techniques have shown tremendous potential for assessing cutaneous microcirculation, but the imaging depth and contrast is limited by the strong scattering of skin. Current skin windows have to be fulfilled by surgical operation and suffer from some side effects. In this study, a switchable skin window was developed by topical application of an optical clearing agent (OCA) and saline on rat skin in vivo. The validity of the skin window was evaluated by the laser speckle contrast imaging technique, and the safety of OCA to the body was tested through histologic examinations. The results indicated that administration of OCA or saline on rat skin in vivo can open or close the window of skin repeatedly for three days. With the repair effect of hyaluronic acid and Vaseline, it is able to repeatedly visualize the dermal blood vessels and flow distribution. Long-term observation shows that there is no abnormal reflection in micro-structure, body weight, organ coefficients, histopathologic lesions, or toxic reactions compared with a control group. This switchable window will provide an effective tool not only for cutaneous microcirculation with laser speckle contrast imaging, but also for diagnosis and treatment of peripheral vascular diseases, including tumor research with various optical imaging techniques.

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

Skin cancer is the most common cancer in the Western world. In order to accurately detect the disease, especially malignant melanoma-the most fatal form of skin cancer-at an early stage when the prognosis is excellent, there is an urgent need to develop noninvasive early detection methods. We believe that polarization speckle patterns, defined as a spatial distribution of depolarization ratio of traditional speckle patterns, can be an important tool for skin cancer detection. To demonstrate our technique, we conduct a large in vivo clinical study of 214 skin lesions, and show that statistical moments of the polarization speckle pattern could differentiate different types of skin lesions, including three common types of skin cancers, malignant melanoma, squamous cell carcinoma, basal cell carcinoma, and two benign lesions, melanocytic nevus and seborrheic keratoses. In particular, the fourth order moment achieves better or similar sensitivity and specificity than many well-known and accepted optical techniques used to differentiate melanoma and seborrheic keratosis.

Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

In scars arising from burns, objective assessment of vascularity is important in the early identification of pathological scarring, and in the assessment of progression and treatment response. We demonstrate the first clinical assessment and automated quantification of vascularity in cutaneous burn scars of human patients in vivo that uses optical coherence tomography (OCT). Scar microvasculature was delineated in three-dimensional OCT images using speckle decorrelation. The diameter and area density of blood vessels were automatically quantified. A substantial increase was observed in the measured density of vasculature in hypertrophic scar tissues (38%) when compared against normal, unscarred skin (22%). A proliferation of larger vessels (diameter≥100  μm) was revealed in hypertrophic scarring, which was absent from normal scars and normal skin over the investigated physical depth range of 600 μm. This study establishes the feasibility of this methodology as a means of clinical monitoring of scar progression.

Confocal laser scanning microscopy (CLSM) is a well-used microscopic tool that provides valuable morphological and functional information within cells and tissues. The application of CLSM to skin and the topical penetration of nanoparticles (NP) will be addressed. First, we describe the advantages of confocal microscopy compared to other techniques and its use relative to skin research. Second, we discuss the ability of CLSM to detect single NP. Regarding their interaction with skin, the appropriate method to retain nanoparticle localization in the tissue with minimal fixation is critically important. Also, the interaction of several different types of NP (quantum dots, fullerene and dendrimers) and their interaction with skin detected by CLSM under various conditions (flexed, tape stripped and abraded skin) is reviewed. Finally, human epidermal keratinocytes and dendritic cells that serve as appropriate in vitro models for skin cell interactions and cellular uptake of NP are also discussed. In conclusion, the unique functions of CLSM such as the ability to detect fluorescence, optical sectioning, three dimensional remodeling, as well as its use in the reflection mode in tandem with other methods, provides great promise with broad applications regarding the interactions of nanomaterials with skin.

Widespread applications of nanotechnology materials have raised safety concerns due to their possible penetration through skin and concomitant uptake in the organism. This calls for systematic study of nanoparticle transport kinetics in skin, where high-resolution optical imaging approaches are often preferred. We report on application of emerging luminescence nanomaterial, called upconversion nanoparticles (UCNPs), to optical imaging in skin that results in complete suppression of background due to the excitation light back-scattering and biological tissue autofluorescence. Freshly excised intact and microneedle-treated human skin samples were topically coated with oil formulation of UCNPs and optically imaged. In the first case, 8- and 32-nm UCNPs stayed at the topmost layer of the intact skin, stratum corneum. In the second case, 8-nm nanoparticles were found localized at indentations made by the microneedle spreading in dermis very slowly (estimated diffusion coefficient, Dnp=3-7×10−12  cm2·s−1). The maximum possible UCNP-imaging contrast was attained by suppressing the background level to that of the electronic noise, which was estimated to be superior in comparison with the existing optical labels.

Depth information is resolved from thick specimens using a modification of structured illumination. By projecting a random projection pattern with varied spatial frequencies that is rotated while capturing images, sectioning can be performed using an incoherent light source in reflectance only. This provides a low-cost solution to obtaining information similar to that produced in confocal microscopy and other methods of structured illumination, without the requirement of complex or elaborate equipment, coherent light sources, or fluorescence. The broad line width of the light emitting diode minimizes artifacts associated with speckle from the laser while also increasing the safety of the instrument. Single diffusers and cascaded diffusers are compared to provide the most efficient method for sectioning at depth. By using reflectance only, in vivo images are produced on a human subject, generating high-contrast images and providing depth information about subsurface objects.

Ultraviolet radiation from solar exposure is a key extrinsic factor responsible for premature skin aging (i.e., photo-aging). Recent advances using in vivo multiphoton tomography (MPT) demonstrate the efficacy of this approach to assess intrinsic and extrinsic skin aging as an alternative to existing invasive techniques. In this study, we measured changes in epidermal autofluorescence, dermal collagen second harmonic generation (SHG), and the redox state of solar-exposed and solar-protected human skin by MPT with fluorescence lifetime imaging (MPT-FLIM). Twenty-four volunteers across four age categories (20 to 29, 30 to 39, 40 to 49, and 50 to 59 years old; six volunteers each) were recruited for MPT-FLIM imaging of the dorsal (solar-exposed; photo-damaged) and volar (solar-protected) forearm. We demonstrate a higher intensity of dermal collagen SHG within the volar forearm compared to dorsal solar-exposed skin. Redox imaging of each epidermal skin stratum by FLIM demonstrates an increase in fluorescence lifetime in the solar-exposed dorsal forearm that is more apparent in aged skin. The results of this study suggest the redox state of the viable epidermis is a key marker in assessing intrinsic and photo-damage skin aging, in combination with changes in autofluorescence and SHG.

Owing to the limited source of human skin (HS) and the ethical restrictions of using animals in experiments, in vitro skin equivalents are a possible alternative for conducting particle penetration experiments. The conditions for conducting penetration experiments with model particles, 15-nm gold nanoparticles (AuNP), through nonsealed skin equivalents are described for the first time. These conditions include experimental setup, sterility conditions, effective applied dose determination, skin sectioning, and skin integrity check. Penetration at different exposure times (two and 24 h) and after tissue fixation (fixed versus unfixed skin) are examined to establish a benchmark in comparison to HS in an attempt to get similar results to HS experiments presented earlier. Multiphoton microscopy is used to detect gold luminescence in skin sections. λ_ex = 800 nm is used for excitation of AuNP and skin samples, allowing us to determine a relative index for particle penetration. Despite the observed overpredictability of penetration into skin equivalents, they could serve as a first fast screen for testing the behavior of nanoparticles and extrapolate their penetration behavior into HS. Further investigations are required to test a wide range of particles of different physicochemical properties to validate the skin equivalent-human skin particle penetration relationship.

Herd health programs for the maintenance of welfare and productivity in cattle need efficient tools for monitoring the health of individual animals. Recent reports demonstrate that the oxidative status is related to various stress conditions in dairy cows. Biomarkers, among other carotenoids, could serve as indicators of stress originating from the environment (e.g., heat stress or sun radiation) or from the animal itself (e.g., disease). To date, only invasive in vitro tests are available to assess the oxidative status in cattle. The present study compares the results of optical noninvasive in vivo measurements of dermal carotenoids in cattle udder skin using an LED-based miniaturized spectroscopic system (MSS) with those obtained by photometric analysis of beta carotene in whole blood samples using a portable device. Correlations between the concentrations of dermal and blood carotenoids were calculated under consideration of the nutritional status of the animals. Significant correlation (R = 0.86) was found for cattle with a moderate to obese body condition. Thus, the blood and skin concentrations of the marker substance beta carotene are comparable under stable stress conditions of the cattle. This demonstrates that the MSS is suitable for noninvasive assessment of dermal carotenoid concentrations in cattle.

We propose a method to visualize the arterial inflow, the vascular resistance, and the venous capacitance in the skin tissue from red, green, blue (RGB) digital color images. The arterial inflow and the venous capacitance in the skin tissue are visualized based on an increase in the rate of change in the total blood concentration and the change of the total blood concentration during upper limb occlusion at a pressure of 50 mmHg. The resultant arterial inflow with the measured mean arterial pressure also provides an image of the vascular resistance in human skin. The arterial inflow, the vascular resistance, and the venous capacitance acquired by the method are well correlated with those obtained from the conventional strain-gauge plethysmograph. The correlation coefficients R between the estimated values by the method and the measurements by the SPG are calculated to be 0.83 (P<0.001) for the arterial inflow, 0.77 (P<0.01) for the vascular resistance, and 0.77 (P<0.01) for the venous capacitance. The arterial inflow and the venous capacitance in the skin tissue are significantly higher in active subjects compared with the sedentary subjects, whereas the vascular resistance was significantly lower in the active subjects compared with the sedentary subjects. The results of the present study indicate the possibility of using the proposed method for evaluating the peripheral vascular functions in human skin.

The importance of dermatological noninvasive imaging techniques has increased over the last decades, aiming at diagnosing nonmelanoma skin cancer (NMSC). Technological progress has led to the development of various analytical tools, enabling the in vivo/in vitro examination of lesional human skin with the aim to increase diagnostic accuracy and decrease morbidity and mortality. The structure of the skin layers, their chemical composition, and the distribution of their compounds permits the noninvasive photodiagnosis of skin diseases, such as skin cancers, especially for early stages of malignant tumors. An important role in the dermatological diagnosis and disease monitoring has been shown for promising spectroscopic and imaging techniques, such as fluorescence, diffuse reflectance, Raman and near-infrared spectroscopy, optical coherence tomography, and confocal laser-scanning microscopy. We review the use of these spectroscopic techniques as noninvasive tools for the photodiagnosis of NMSC.

Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds.

Spectrophotometric intracutaneous analysis (SIAscopy) is an imaging technique developed for diagnostics of pigmented skin lesions. By image analysis, the displayed images indicate the potential distribution and position of melanin, blood, and collagen within the lesion. A topographic comparison was performed between SIAscopic findings and histopathology. In total, 60 patients with suspicious pigmented skin lesions were included. The lesions were SIAscopically imaged and documented before excision and histopathological preparation. Topographical comparisons between SIAscopy findings and histopathology were made. A sensitivity and specificity of 24% and 84%, respectively, were obtained for invasive melanomas. The positive and negative predicted values were 58% and 54%, respectively. The features indicating dermal melanin, blood displacement and collagen holes did only show “no” to “slight” agreement with histopathology, i.e., κ≤0.21 . It was concluded that (i) SIAscopy-based diagnosis has low diagnostic accuracy for melanoma, (ii) single SIAscopic features do not provide reliable diagnostic information relating to the lesions internal structure on histopathology examination and (iii) SIAscopy cannot be used as a guide for localizing the maximum tumor thickness when performing the histopathological examination. The importance of validating new optical tools for tumor diagnostics with histopathological findings was demonstrated.

Optical coherence tomography (OCT) is a noninvasive diagnostic method that offers a view into the superficial layers of the skin in vivo in real-time. An infrared broadband light source allows the investigation of skin architecture and changes up to a depth of 1 to 2 mm with a resolution between 15 and 3 μm, depending on the system used. Thus OCT enables evaluation of skin lesions, especially nonmelanoma skin cancers and inflammatory diseases, quantification of skin changes, visualization of parasitic infestations, and examination of other indications such as the investigation of nails. OCT provides a quick and useful diagnostic imaging technique for a number of clinical questions and is a valuable addition or complement to other noninvasive imaging tools such as dermoscopy, high-frequency ultrasound, and confocal laser scan microscopy.

The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

The ability to quantitatively and noninvasively detect nanoparticles nearby the skin surface has important implications on their development as an in vivo cancer diagnostic tool. The diffusion reflection (DR) method is a simple, noninvasive imaging technique which has been proven useful for the investigation of the optical parameters of the tissue. A new method is presented for the measurements of gold nanorod (GNR) concentration in tissue-like phantoms, based on DR measurement and intense light absorption of GNR. Monte Carlo simulations and tissue-like phantom measurements of the reflected light intensity are presented. The ability to extract optical properties of phantoms and their GNR concentrations from DR measurements is demonstrated, followed by a discussion about the best mathematical model for light propagation in tissues, based on the diffusion theory.

Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12  mm ² of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called “strip mosaicing,” which was demonstrated on a 10-×-10  mm² of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10  mm² of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5  cm ² piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Lasers have shown great advantages in enhancing transdermal drug delivery. However, the physical or physiological mechanisms are not clear, which limits the application in clinical medicine. Here, 1064 nm-Nd:YAG lasers with long-pulsed (LP, 15  J/cm² ) and Q-switched (QS, 0.5  J/cm² ) output modes inducing short- and long-term effects on the stratum corneum (SC) of skin are investigated. Infrared thermography is applied to monitor the dynamical temperature distribution of the skin surface, while histopathological analysis and two-photon fluorescence microscopy are employed to examine changes in the microstructure of skin and molecular constitution of SC, respectively. Results have shown that the LP laser irradiation increases skin temperature evidently and loosens keratin, making corneocytes fragile or exfoliative, whereas the QS laser irradiation disrupts the keratin or corneocytes completely, perforating some micropores on the SC. It can be concluded that the mechanisms of enhancing transdermal delivery caused by lasers depends on the output modes. The LP laser irradiation produces thermal effects on skin, which loosens the SC, while the QS laser induces mechanical effects on skin, which punches micropores on the SC. Moreover, the laser-induced enhancing effects on transdermal glycerol delivery can last for one week to wait for the recovery of SC.

The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with time-consuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.

Carotenoids are important substances for human skin due to their powerful antioxidant properties in reaction of neutralization of free radicals and especially reactive oxygen species, including singlet oxygen. Concentration of carotenoids in the skin could mirror the current redox status of the skin and should be investigated in vivo. Optical methods are ideally suited for determination of carotenoids in mammalian skin in vivo as they are both noninvasive and quick. Four different optical methods could be used for in vivo measurement of carotenoids in the human or animal skin: (1) resonance Raman spectroscopy; (2) Raman microscopy; (3) reflection spectroscopy; (4) skin color measurements. The advantages, shortcomings, and limitations of the above-mentioned optical methods are discussed.

Optical assessment of skin burns is possible with second-harmonic-generation (SHG) microscopy due to its high sensitivity to thermal denaturation of collagen molecules. In contrast to previous studies that were performed using excised tissue specimens ex vivo, in vivo observation of dermal collagen fibers in living rat burn models with SHG microscopy is demonstrated. Changes in signal vanishing patterns in the SHG images are confirmed to be dependent on the burn degree. Comparison of the SHG images with Masson’s trichrome-stained images indicated that the observed patterns were caused by the coexistence of molten and fibrous structures of dermal collagen fibers. Furthermore, a quantitative parameter for burn assessment based on the depth profile of the mean SHG intensity across the entire SHG image is proposed. These results and discussions imply a potential of SHG microscopy as a minimally invasive, highly quantitative tool for skin burn assessment.

The reflection and scattering properties of light incident on human skin covered with powder particles have been investigated. A three-layer skin structure with a pigmented area is modeled, and the propagation of light in the skin’s layers and in a layer of particles near the skin’s surface is simulated using the Monte Carlo method. Assuming that only single scattering of light occurs in the powder layer, the simulation results show that the reflection spectra of light from the skin change with the size of powder particles. The color difference between normal and discolored skin is found to decrease considerably when powder particles with a diameter of approximately 0.25 μm are present near the skin’s surface. The effects of the medium surrounding the particles, and the influence of the distribution of particle size (polydispersity), are also examined. It is shown that a surrounding medium with a refractive index close to that of the skin substantially suppresses the extreme spectral changes caused by the powder particles covering the skin surface.

The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. We show how bottom-up nanoantennas enhance the fluorescence intensity in a reduced hotspot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle (NP) dimers are positioned with nanometer precision. A maximum of almost 100-fold intensity enhancement is obtained using 100-nm gold NPs within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nanoantenna, we are able to perform single-molecule measurements at concentrations as high as 500 nM, which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic NPs with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single-molecule measurements at high concentrations where processes like DNA sequencing occur.

The anterior corneal asphericity (Q ) with the tangential radius is calculated, and a three-dimensional (3-D) anterior corneal model is constructed. Tangential power maps from Orbscan II are acquired for 66 young adult subjects. The Q -value of each semimeridian in the near-horizontal region is calculated with the tangential radius. Polynomial fitting is used to model the 360-semimeridional variation of Q -values, and to fit the Q -values in the near-vertical region. Furthermore, a customized 3-D anterior corneal model is constructed. The 360-semimeridional variation of Q -values is well fitted with a seventh-degree polynomial function for all subjects. The goodness of fit of the polynomial function was <0.9 , and the median value was 0.94. The Q -value distribution of the anterior corneal surface showed bimodal variation. Additionally, the Q -values gradually become less negative from the horizontal to the vertical semimeridians in the four quadrants. The 3-D surface plot of the anterior corneal surface approximated a prolate ellipsoid. Using a method to calculate the Q -value with the tangential radius combined with polynomial fitting, we are able to obtain the Q -value of any semimeridian. Compared with general models, this method generates a complete shape of the anterior corneal surface using asphericity.

Mirror therapy is a therapy to treat patients with pain syndromes or hemiparesis after stroke. However, the underlying neurophysiologic mechanisms are not clearly understood. In order to determine the effect of a mirror-like illusion (MIR) on brain activity using functional near-infrared spectroscopy, 20 healthy right-handed subjects were examined. A MIR was induced by a digital horizontal inversion of the subjects’ filmed hand. Optodes were placed on the primary motor cortex (M1) and the occipito-parietal cortex (precuneus, PC). Regions of interest (ROI) were defined a priori based on previous results of similar studies and confirmed by the analysis of effect sizes. Analysis of variance of the ROI signal revealed a dissociated pattern: at the PC, the MIR caused a significant inversion of a hemispheric lateralization opposite to the perceived hand, independent of the moving hand. In contrast, activity in M1 showed lateralization opposite to the moving hand, but revealed no mirror effect. These findings extend our understanding on interhemispheric rivalry and indicate that a MIR is integrated into visuomotor coordination similar to normal view, irrespective of the hand that is actually performing the task.

Calcifications are one of the most important indicators for early breast cancer detection. We explore the feasibility of deep-penetration photoacoustic (PA) imaging of calcifications based on a medical ultrasound array imaging platform. Intralipid and chicken breast phantoms embedded with different-sized hydroxyapatite (HA) particles, which are the major components of calcifications, were imaged to verify the equipment’s capability and penetration depth for the visualization of calcifications. An optimal near-infrared excitation wavelength was selected to maximize PA signals of HAs, resulting in a better HA signal-to-blood ratio. We demonstrated that PA imaging is capable of visualizing 0.5-mm HA particles at a depth of 3 cm in chicken breast phantoms. The noise-equivalent penetration depth of the system for visualizing 0.5-mm HA particles in the human breast was estimated to be about 2.9 to 3.5 cm, which is clinically relevant as calcifications are usually found at a depth of 0.6 to 3.0 cm. Moreover, the feasibility of differentiating HA from blood by the PA spectroscopic technique was presented and the mechanism of the HA signal generation was discussed. The results show that PA imaging is a promising technique for real-time visualization of breast calcifications.

Retinal hemoglobin oxygen saturation (sO ₂ ) level is believed to be associated with the pathophysiology of several leading blinding diseases. Methods to properly measure retinal sO ₂ have been investigated for decades; however, the accuracy of retinal oximetry is still considered to be limited. The Monte Carlo simulation of photon transport in retina to examine how the accuracy of retinal oximetry is affected by local parameters is discussed. Fundus photography was simulated in a multilayer retinal model, in which a single vessel segment with 0.7  sO ₂ was embedded, at six optical wavelengths. Then, 200 million photons were traced in each simulation to ensure statistically stable results. The optical reflectance and energy deposit were recorded to measure sO ₂ using both the reflection method (existing retinal oximetry) and a new absorption method, photoacoustic ophthalmoscopy (PAOM). By varying the vessel diameter and melanin concentration in the retinal pigment epithelium, the relative error of sO ₂ measurement in the reflection method increased with increasing vessel diameter and melanin concentration; in comparison, the sO ₂ measurement was insensitive to these two parameters in PAOM. The results suggest that PAOM potentially can be a more accurate tool in quantifying retinal sO ₂ .

In the last decade, coherent anti-Stokes Raman scattering (CARS) microscopy has emerged as a powerful multiphoton imaging technique offering label-free chemical sensitivity and high three-dimensional resolution. However, its widespread application in the life sciences has been hampered by the use of costly pulsed lasers, the existence of a nonresonant background requiring involved technical solutions for its efficient suppression, and the limited acquisition speed of multiplex techniques addressing several vibrational resonances, if improved chemical specificity is needed. We have recently reported a differential CARS technique (D-CARS), which simultaneously measures two vibrational frequencies, enhancing the chemical selectivity and sensitivity without introducing costly hardware, while maintaining fast acquisition. In this study, we demonstrate a compact, fully automated, cost-effective module, which integrates on hardware and software level with a commercial multiphoton microscope based on a single 100 fs Ti:Sapphire oscillator and enables D-CARS microscopy in a user-friendly format for applications in the life sciences.

A technique for generating en face parametric images of tissue birefringence from scans acquired using a fiber-based polarization-sensitive optical coherence tomography (PS-OCT) system utilizing only a single-incident polarization state is presented. The value of birefringence is calculated for each A-scan in the PS-OCT volume using a quadrature demodulation and phase unwrapping algorithm. The algorithm additionally uses weighted spatial averaging and weighted least squares regression to account for the variation in phase accuracies due to varying OCT signal-to-noise-ratio. The utility of this technique is demonstrated using a model of thermally induced damage in porcine tendon and validated against histology. The resulting en face images of tissue birefringence are more useful than conventional PS-OCT B-scans in assessing the severity of tissue damage and in localizing the spatial extent of damage.

Dual-axis confocal (DAC) microscopy has been found to exhibit superior rejection of out-of-focus and multiply scattered background light compared to conventional single-axis confocal microscopy. DAC microscopes rely on the use of separated illumination and collection beam paths that focus and intersect at a single focal volume (voxel) within tissue. While it is generally recognized that the resolution and contrast of a DAC microscope depends on both the crossing angle of the DAC beams, 2θ , and the focusing numerical aperture of the individual beams, α , a detailed study to investigate these dependencies has not been performed. Contrast and resolution are considered as two main criteria to assess the performance of a point-scanned DAC microscope (DAC-PS) and a line-scanned DAC microscope (DAC-LS) as a function of θ and α . The contrast and resolution of these designs are evaluated by Monte-Carlo scattering simulations and diffraction theory calculations, respectively. These results can be used for guiding the optimal designs of DAC-PS and DAC-LS microscopes.

Oblique back-illumination microscopy (OBM) is a new technique for simultaneous, independent measurements of phase gradients and absorption in thick scattering tissues based on widefield imaging. To date, OBM has been used with sequential camera exposures, which reduces temporal resolution, and can produce motion artifacts in dynamic samples. Here, a variation of OBM that allows single-exposure operation with wavelength multiplexing and image splitting with a Wollaston prism is introduced. Asymmetric anamorphic distortion induced by the prism is characterized and corrected in real time using a graphics-processing unit. To demonstrate the capacity of single-exposure OBM to perform artifact-free imaging of blood flow, video-rate movies of microcirculation in ovo in the chorioallantoic membrane of the developing chick are presented. Imaging is performed with a high-resolution rigid Hopkins lens suitable for endoscopy.

The model-based algorithm is an effective reconstruction method for photoacoustic imaging (PAI). Compared with the analytical reconstruction algorithms, the model-based algorithm is able to provide a more accurate and high-resolution reconstructed image. However, the relatively heavy computational complexity and huge memory storage requirement often impose restrictions on its applications. We incorporate the discrete cosine transform (DCT) in PAI reconstruction and establish a new photoacoustic model. With this new model, an efficient algorithm is proposed for PAI reconstruction. Relatively significant DCT coefficients of the measured signals are used to reconstruct the image. As a result, the calculation can be saved. The theoretical computation complexity of the proposed algorithm is figured out and it is proved that the proposed method is efficient in calculation. The proposed algorithm is also verified through the numerical simulations and in vitro experiments. Compared with former developed model-based methods, the proposed algorithm is able to provide an equivalent reconstruction with the cost of much less time. From the theoretical analysis and the experiment results, it would be concluded that the model-based PAI reconstruction can be accelerated by using the proposed algorithm, so that the practical applicability of PAI may be enhanced.

Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs, and fluorescence optical tomography has tremendous potential to provide clinically useful, functional information by identifying tumor hypoxia. The synthesis of a 2-nitroimidazole-indocyanine green conjugate using a piperazine linker (piperazine-2-nitroimidazole-ICG) capable of robust fluorescent imaging of tumor hypoxia is described. In vivo mouse tumor imaging studies were completed and demonstrate an improved imaging capability of the new dye relative to an earlier version of the dye that was synthesized with an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG). Mouse tumors located at imaging depths of 1.5 and 2.0 cm in a turbid medium were imaged at various time points after intravenous injection of the dyes. On average, the reconstructed maximum fluorescence concentration of the tumors injected with piperazine-2-nitroimidazole-ICG was twofold higher than that injected with ethanolamine-2-nitroimidazole-ICG within 3 h postinjection period and 1.6 to 1.7 times higher beyond 3 h postinjection. The untargeted bis-carboxylic acid ICG completely washed out after 3 h postinjection. Thus, the optimal window to assess tumor hypoxia is beyond 3 h postinjection. These findings were supported with fluorescence images of histological sections of tumor samples and an immunohistochemistry technique for identifying tumor hypoxia.

Frequent monitoring of early-stage burns is necessary for deciding optimal treatment and management. Both superficial and full thickness burns are relatively easy to diagnose based on clinical observation. In between these two extremes are superficial-partial thickness and deep-partial thickness burns. These burns, while visually similar, differ dramatically in terms of clinical treatment and are known to progress in severity over time. The objective of this study was to determine the potential of spatial frequency domain imaging (SFDI) for noninvasively mapping quantitative changes in chromophore and optical properties that may be an indicative of burn wound severity. A controlled protocol of graded burn severity was developed and applied to 17 rats. SFDI data was acquired at multiple near-infrared wavelengths over a course of 3 h. Burn severity was verified using hematoxylin and eosin histology. From this study, we found that changes in water concentration (edema), deoxygenated hemoglobin concentration, and optical scattering (tissue denaturation) to be statistically significant at differentiating superficial partial-thickness burns from deep-partial thickness burns.

Modern surgical procedures often have a fusion of video and other imaging modalities to provide the surgeon with information support. This requires interventional guidance equipment and surgical navigation systems to register different tools and devices together, such as stereoscopic endoscopes and ultrasound (US) transducers. In this work, the focus is specifically on the registration between these two devices. Electromagnetic and optical trackers are typically used to acquire this registration, but they have various drawbacks typically leading to target registration errors (TRE) of approximately 3 mm. We introduce photoacoustic markers for direct three-dimensional (3-D) US-to-video registration. The feasibility of this method was demonstrated on synthetic and ex vivo porcine liver, kidney, and fat phantoms with an air-coupled laser and a motorized 3-D US probe. The resulting TRE for each experiment ranged from 380 to 850 μm with standard deviations ranging from 150 to 450 μm. We also discuss a roadmap to bring this system into the surgical setting and possible challenges along the way.

The design of inhomogeneous phantoms for diffuse optical imaging purposes using totally absorbing objects embedded in a diffusive medium is proposed and validated. From time-resolved and continuous-wave Monte Carlo simulations, it is shown that a given or desired perturbation strength caused by a realistic absorbing inhomogeneity of a certain absorption and volume can be approximately mimicked by a small totally absorbing object of a so-called equivalent black volume (equivalence relation). This concept can be useful in two ways. First, it can be exploited to design realistic inhomogeneous phantoms with different perturbation strengths simply using a set of black objects with different volumes. Further, it permits one to grade physiological or pathological changes on a reproducible scale of perturbation strengths given as equivalent black volumes, thus facilitating the performance assessment of clinical instruments. A set of plots and interpolating functions to derive the equivalent black volume corresponding to a given absorption change is provided. The application of the equivalent black volume concept for grading different optical perturbations is demonstrated for some examples.

The adoption of axially oriented line illumination patterns for fluorescence excitation in small animals for fluorescence surface imaging (FSI) and fluorescence optical tomography (FOT) is being investigated. A trimodal single-photon-emission-computed-tomography/computed-tomography/optical-tomography (SPECT-CT-OT) small animal imaging system is being modified for employment of point- and line-laser excitation sources. These sources can be arbitrarily positioned around the imaged object. The line source is set to illuminate the object along its entire axial direction. Comparative evaluation of point and line illumination patterns for FSI and FOT is provided involving phantom as well as mouse data. Given the trimodal setup, CT data are used to guide the optical approaches by providing boundary information. Furthermore, FOT results are also being compared to SPECT. Results show that line-laser illumination yields a larger axial field of view (FOV) in FSI mode, hence faster data acquisition, and practically acceptable FOT reconstruction throughout the whole animal. Also, superimposed SPECT and FOT data provide additional information on similarities as well as differences in the distribution and uptake of both probe types. Fused CT data enhance further the anatomical localization of the tracer distribution in vivo. The feasibility of line-laser excitation for three-dimensional fluorescence imaging and tomography is demonstrated for initiating further research, however, not with the intention to replace one by the other.

We report the application of an optical microscope equipped with a high-resolution dark-field condenser for detecting dynamic responses of cellular nanostructures in real time. Our system provides an easy-to-use technique to visualize biological specimens without any staining. This system can visualize the dynamic behavior of nanospheres and nanofibers, such as F-actin, at the leading edges of adjacent neuronal cells. We confirmed that the nanofibers imaged with this high-resolution optical microscopic technique are F-actin by using fluorescence microscopy after immunostaining the F-actin of fixed cells. Furthermore, cellular dynamics are enhanced by applying noncontact electric field stimulation through a transparent graphene electric field stimulator. High-resolution label-free optical microscopy enables the visualization of nanofiber dynamics initiated by filopodial nanofiber contacts. In conclusion, our optical microscopy system allows the visualization of nanoscale cellular dynamics under various external stimuli in real time without specific staining.

Diagnosis of neuromuscular diseases is based on subjective visual assessment of biopsies from patients by the pathologist specialist. A system for objective analysis and classification of muscular dystrophies and neurogenic atrophies through muscle biopsy images of fluorescence microscopy is presented. The procedure starts with an accurate segmentation of the muscle fibers using mathematical morphology and a watershed transform. A feature extraction step is carried out in two parts: 24 features that pathologists take into account to diagnose the diseases and 58 structural features that the human eye cannot see, based on the assumption that the biopsy is considered as a graph, where the nodes are represented by each fiber, and two nodes are connected if two fibers are adjacent. A feature selection using sequential forward selection and sequential backward selection methods, a classification using a Fuzzy ARTMAP neural network, and a study of grading the severity are performed on these two sets of features. A database consisting of 91 images was used: 71 images for the training step and 20 as the test. A classification error of 0% was obtained. It is concluded that the addition of features undetectable by the human visual inspection improves the categorization of atrophic patterns.

When laser light illuminates a diffuse object, it produces a random interference effect known as a speckle pattern. If there is movement in the object, the speckles fluctuate in intensity. These fluctuations can provide information about the movement. A simple way of accessing this information is to image the speckle pattern with an exposure time longer than the shortest speckle fluctuation time scale—the fluctuations cause a blurring of the speckle, leading to a reduction in the local speckle contrast. Thus, velocity distributions are coded as speckle contrast variations. The same information can be obtained by using the Doppler effect, but producing a two-dimensional Doppler map requires either scanning of the laser beam or imaging with a high-speed camera: laser speckle contrast imaging (LSCI) avoids the need to scan and can be performed with a normal CCD- or CMOS-camera. LSCI is used primarily to map flow systems, especially blood flow. The development of LSCI is reviewed and its limitations and problems are investigated.

Optical nerve stimulation (ONS) may be useful as a diagnostic tool for intraoperative identification and preservation of the prostate cavernous nerves (CN), responsible for erectile function, during prostate cancer surgery. Successful ONS requires elevating the nerve temperature to within a narrow range (∼42 to 47°C) for nerve activation without thermal damage to the nerve. This preliminary study explores a prototype temperature-controlled optical nerve stimulation (TC-ONS) system for maintaining a constant (±1°C ) nerve temperature during short-term ONS of the rat prostate CNs. A 150-mW, 1455-nm diode laser was operated in continuous-wave mode, with and without temperature control, during stimulation of the rat CNs for 15 to 30 s through a fiber optic probe with a 1-mm-diameter spot. A microcontroller opened and closed an in-line mechanical shutter in response to an infrared sensor, with a predetermined temperature set point. With TC-ONS, higher laser power settings were used to rapidly and safely elevate the CNs to a temperature necessary for a fast intracavernous pressure response, while also preventing excessive temperatures that would otherwise cause thermal damage to the nerve. With further development, TC-ONS may provide a rapid, stable, and safe method for intraoperative identification and preservation of the prostate CNs.

Fluorescence and diffuse reflectance spectroscopy are powerful tools to differentiate normal and malignant tissue based on the emissions from endogenous fluorophores and diffuse reflection of absorbers such as hemoglobin. However, separate analytical methods are used for the identification of fluorophores and hemoglobin. The estimation of fluorophores and hemoglobin simultaneously using a single technique of autofluorescence spectroscopy is reported, and its diagnostic potential on clinical tissue samples is potentially exploited. Surgically removed brain tissues from patients that are later identified pathologically as astrocytoma, glioma, meningioma, and schwannoma are studied. The emissions from prominent fluorophores collagen, flavin adenine dinucleotide, phospholipids, and porphyrin are analyzed at 320 and 410 nm excitations. The hemoglobin concentration is also calculated from the ratio of fluorescence emissions at 500 and 570 nm. A better classification of normal and tumor tissues is yielded for 410 nm excitation compared to 320 nm when diagnostic algorithm based on linear discriminant analysis is used. The potential of fluorescence spectroscopy as a single entity to evaluate the prominent fluorophores as well as the hemoglobin concentration within normal and tumor brain tissues is emphasized.

Photoacoustic (PA) techniques have been exploited for monitoring thermal treatments. However, PA signals depend not only on tissue temperature but also on tissue optical properties which indicate tissue status (e.g., native or coagulated). The changes in temperature and tissue status often occur simultaneously during thermal treatments, so both effects cause changes to PA signals. A new dual-wavelength PA technique to monitor tissue status independent of temperature is performed. By dividing the PA signal intensities obtained at two wavelengths at the same temperature, a ratio, which only depends on tissue optical properties, is obtained. Experiments were performed with two experimental groups, one with untreated tissue samples and the other with high-intensity focused ultrasound treated tissue samples including thermal coagulated lesion, using ex vivo porcine myocardium specimens to test the technique. The ratio of PA signal intensities obtained at 700 and 800 nm was constant for both groups from 25 to 43°C, but with distinct values for the two groups. Tissue alteration during thermal treatment was then studied using water bath heating of tissue samples from 35 to 60°C. We found that the ratio stayed constant before it exhibited a marked increase at around 55°C, indicating tissue changes at this temperature.

The feasibility of early detection of gastric cancer using near-infrared (NIR) Raman spectroscopy (RS) by distinguishing premalignant lesions (adenomatous polyp, n=27 ) and cancer tissues (adenocarcinoma, n=33 ) from normal gastric tissues (n=45 ) is evaluated. Significant differences in Raman spectra are observed among the normal, adenomatous polyp, and adenocarcinoma gastric tissues at 936, 1003, 1032, 1174, 1208, 1323, 1335, 1450, and 1655  cm⁻¹ . Diverse statistical methods are employed to develop effective diagnostic algorithms for classifying the Raman spectra of different types of ex vivo gastric tissues, including principal component analysis (PCA), linear discriminant analysis (LDA), and naive Bayesian classifier (NBC) techniques. Compared with PCA-LDA algorithms, PCA-NBC techniques together with leave-one-out, cross-validation method provide better discriminative results of normal, adenomatous polyp, and adenocarcinoma gastric tissues, resulting in superior sensitivities of 96.3%, 96.9%, and 96.9%, and specificities of 93%, 100%, and 95.2%, respectively. Therefore, NIR RS associated with multivariate statistical algorithms has the potential for early diagnosis of gastric premalignant lesions and cancer tissues in molecular level.

Oblique incidence reflectometry (OIR) is an established technique for the estimation of tissue optical properties. However, a sensing footprint of a few transport mean-free paths is often needed when diffusion-regime-based algorithms are used. Smaller-footprint probes require improved light-propagation models and inversion schemes for diffuse reflectance close to the point of entry but might enable micro-endoscopic form factors for clinical assessments of cancers and precancers. The phase-function corrected diffusion theory presented by Vitkin et al. [Nat. Commun. 2, 587 (2011)] to the case of pencil beams obliquely incident on a semi-infinite turbid medium is extended. The model requires minimal computational resources and offers improved accuracy over more traditional diffusion-theory approximations models when validated against Monte Carlo simulations. The computationally efficient nature of the models lends itself to rapid fitting procedures for inverse problems. The analytical model is used in a nonlinear fitting algorithm to demonstrate the recovery of optical properties using a measurement footprint that is significantly smaller than needed in previous diffusion-regime OIR methods.

Transcranial magnetic stimulation (TMS) modulates processing in the human brain and is therefore of interest as a treatment modality for neurologic conditions. During TMS administration, an electric current passing through a coil on the scalp creates a rapidly varying magnetic field that induces currents in the cerebral cortex. The effects of low-frequency (1 Hz), repetitive TMS (rTMS ) on motor cortex cerebral blood flow (CBF) and tissue oxygenation in seven healthy adults, during/after 20 min stimulation, is reported. Noninvasive optical methods are employed: diffuse correlation spectroscopy (DCS) for blood flow and diffuse optical spectroscopy (DOS) for hemoglobin concentrations. A significant increase in median CBF (33%) on the side ipsilateral to stimulation was observed during rTMS and persisted after discontinuation. The measured hemodynamic parameter variations enabled computation of relative changes in cerebral metabolic rate of oxygen consumption during rTMS , which increased significantly (28%) in the stimulated hemisphere. By contrast, hemodynamic changes from baseline were not observed contralateral to rTMS administration (all parameters, p<0.29 ). In total, these findings provide new information about hemodynamic/metabolic responses to low-frequency rTMS and, importantly, demonstrate the feasibility of DCS/DOS for noninvasive monitoring of TMS-induced physiologic effects.

Raman spectroscopy is a unique optical technique that can probe the changes of vibrational modes of biomolecules associated with tissue premalignant transformation. This study evaluates the clinical utility of confocal Raman spectroscopy over near-infrared (NIR) autofluorescence (AF) spectroscopy and composite NIR AF/Raman spectroscopy for improving early diagnosis of cervical precancer in vivo at colposcopy. A rapid NIR Raman system coupled with a ball-lens fiber-optic confocal Raman probe was utilized for in vivo NIR AF/Raman spectral measurements of the cervix. A total of 1240 in vivo Raman spectra [normal (n=993 ), dysplasia (n=247 )] were acquired from 84 cervical patients. Principal components analysis (PCA) and linear discriminant analysis (LDA) together with a leave-one-patient-out, cross-validation method were used to extract the diagnostic information associated with distinctive spectroscopic modalities. The diagnostic ability of confocal Raman spectroscopy was evaluated using the PCA-LDA model developed from the significant principal components (PCs) [i.e., PC4, 0.0023%; PC5, 0.00095%; PC8, 0.00022%, (p<0.05 )], representing the primary tissue Raman features (e.g., 854, 937, 1095, 1253, 1311, 1445, and 1654  cm ⁻¹ ). Confocal Raman spectroscopy coupled with PCA-LDA modeling yielded the diagnostic accuracy of 84.1% (a sensitivity of 81.0% and a specificity of 87.1%) for in vivo discrimination of dysplastic cervix. The receiver operating characteristic curves further confirmed that the best classification was achieved using confocal Raman spectroscopy compared to the composite NIR AF/Raman spectroscopy or NIR AF spectroscopy alone. This study illustrates that confocal Raman spectroscopy has great potential to improve early diagnosis of cervical precancer in vivo during clinical colposcopy.

Early detection of pathological changes and progression in glaucoma and other neuroretinal diseases remains a great challenge and is critical to reduce permanent structural and functional retina and optic nerve damage. Raman spectroscopy is a sensitive technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, spectroscopic analysis was conducted on the retinal tissues of seven beagles with acute elevation of intraocular pressure (AEIOP), six beagles with compressive optic neuropathy (CON), and five healthy beagles. Spectroscopic markers were identified associated with the different neuropathic conditions. Furthermore, the Raman spectra were subjected to multivariate discriminate analysis to classify independent tissue samples into diseased/healthy categories. The multivariate discriminant model yielded an average optimal classification accuracy of 72.6% for AEIOP and 63.4% for CON with 20 principal components being used that accounted for 87% of the total variance in the data set. A strong correlation (R ² <0.92 ) was observed between pattern electroretinography characteristics of AEIOP dogs and Raman separation distance that measures the separation of spectra of diseased tissues from normal tissues; however, the underlining mechanism of this correlation remains to be understood. Since AEIOP mimics the pathological symptoms of acute/early-stage glaucoma, it was demonstrated that Raman spectroscopic screening has the potential to become a powerful tool for the detection and characterization of early-stage disease.

Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

We report a blood pressure evaluation methodology by recording the radial arterial pulse waveform in real time using a fiber Bragg grating pulse device (FBGPD). Here, the pressure responses of the arterial pulse in the form of beat-to-beat pulse amplitude and arterial diametrical variations are monitored. Particularly, the unique signatures of pulse pressure variations have been recorded in the arterial pulse waveform, which indicate the systolic and diastolic blood pressure while the patient is subjected to the sphygmomanometric blood pressure examination. The proposed method of blood pressure evaluation using FBGPD has been validated with the auscultatory method of detecting the acoustic pulses (Korotkoff sounds) by an electronic stethoscope.

The effects of laser etching on dentin are studied by microenergy-dispersive x-ray fluorescence spectrometry (μ-EDXRF) and scanning electron microscopy (SEM) to establish the correlation of data obtained. Fifteen human third molars are prepared, baseline μ-EDXRF mappings are performed, and ten specimens are selected. Each specimen received four treatments: acid etching (control-CG) or erbium:yttrium–aluminum–garnet (Er:YAG) laser irradiation (I—100 mJ, II—160 mJ, and III—220 mJ), and maps are done again. The Ca and P content are significantly reduced after acid etching (p<0.0001 ) and increased after laser irradiation with 220 mJ (Ca: p<0.0153 and P: p=0.0005 ). The Ca/P ratio increased and decreased after CG (p=0.0052 ) and GI (p=0.0003 ) treatments, respectively. CG treatment resulted in lower inorganic content (GI: p<0.05 , GII: p<0.01 , and GIII: p<;0.01 ) and higher Ca/P ratios than laser etching (GI: p<;0.001 , GII: p<0.01 , and GIII: p<0.01 ). The SEM photomicrographies revealed open (CG) and partially open dentin tubules (GI, GII, and GIII). μ-EDXRF mappings illustrated that acid etching created homogeneous distribution of inorganic content over dentin. Er:YAG laser etching (220 mJ) produced irregular elemental distribution and changed the stoichiometric proportions of hydroxyapatite, as showed by an increase of mineral content. Decreases and increases of mineral content in the μ-EDXRF images are correlated to holes and mounds, respectively, as found in SEM images.