The reconstruction of images in photoacoustic tomography is reliant on specifying the speed of sound within the propagation medium. However, for in vivo imaging, this value is not normally accurately known. Here, an autofocus approach for automatically selecting the sound speed is proposed. This is based on maximizing the sharpness of the reconstructed image as quantified by a focus function. Several focus functions are investigated, and their performance is discussed. The method is demonstrated using phantom measurements made in a medium with a known sound speed and in vivo measurements of the vasculature in the flank of an adult mouse.

We designed a miniature laser speckle imager that weighs ∼20 g and is 3.1-cm high for full-field high-resolution imaging of cerebral blood flow (CBF) in freely moving animals. Coherent laser light illuminates the cortex through a multimode optical fiber bundle fixed onto the supporting frame of the imager. The reflected lights are then collected by a miniature macrolens system and imaged by a high-resolution CMOS camera at a high frame rate (50 fps). Using this miniature imager, we achieve high spatiotemporal resolution laser speckle contrast imaging of CBF in freely moving animals in real time.

Optical scattering coefficient from ex vivo unfixed normal and malignant ovarian tissue was quantitatively extracted by fitting optical coherence tomography (OCT) A-line signals to a single scattering model. 1097 average A-line measurements at a wavelength of 1310 nm were performed at 108 sites obtained from 18 ovaries. The average scattering coefficient obtained from the normal tissue group consisted of 833 measurements from 88 sites was 2.41 mm⁻¹ (±0.59), while the average coefficient obtained from the malignant tissue group consisted of 264 measurements from 20 sites was 1.55 ^mm−1 (±0.46). The malignant ovarian tissue showed significant lower scattering than the normal group (p < 0.001). The amount of collagen within OCT imaging depth was analyzed from the tissue histological section stained with Sirius Red. The average collagen area fraction (CAF) obtained from the normal tissue group was 48.4% (±12.3%), while the average CAF obtained from the malignant tissue group was 11.4% (±4.7%). A statistical significance of the collagen content was found between the two groups (p < 0.001). These results demonstrated that quantitative measurements of optical scattering coefficient from OCT images could be a potential powerful method for ovarian cancer detection.

A full ring ultrasonic array-based photoacoustic tomography system was recently developed for small animal brain imaging. The 512-element array is cylindrically focused in the elevational direction, and can acquire a two-dimensional (2D) image in 1.6 s. In this letter, we demonstrate the three-dimensional (3D) imaging capability of this system. A novel 3D reconstruction algorithm was developed based on the focal-line concept. Compared to 3D images acquired simply by stacking a series of 2D images, the 3D focal-line reconstruction method renders images with much less artifacts, and improves the elevational resolution by 30% and the signal-to-noise ratio by two times. The effectiveness of the proposed algorithm was first validated by numerical simulations and then demonstrated with a hair phantom experiment and an ex vivo mouse embryo experiment.

Photoacoustic microscopy (PAM) was used to detect small animal brain activation in response to drug abuse. Cocaine hydrochloride in saline solution was injected into the blood stream of Sprague Dawley rats through tail veins. The rat brain functional change in response to the injection of drug was then monitored by the PAM technique. Images in the coronal view of the rat brain at the locations of 1.2 and 3.4 mm posterior to bregma were obtained. The resulted photoacoustic (PA) images showed the regional changes in the blood volume. Additionally, the regional changes in blood oxygenation were also presented. The results demonstrated that PA imaging is capable of monitoring regional hemodynamic changes induced by drug abuse.

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca²⁺ dye, were monitored by imaging intracellular Ca²⁺. An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds.

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.

We propose and demonstrate a common-path low-coherence interferometry (CP-LCI) fiber-optic sensor guided precise microincision. The method tracks the target surface and compensates the tool-to-surface relative motion with better than ±5 μm resolution using a precision micromotor connected to the tool tip. A single-fiber distance probe integrated microdissector was used to perform an accurate 100 μm incision into the surface of an Intralipid phantom. The CP-LCI guided incision quality in terms of depth was evaluated afterwards using three-dimensional Fourier-domain optical coherence tomography imaging, which showed significant improvement of incision accuracy compared to free-hand-only operations.

The purpose of this study was to evaluate the color stability of a maxillofacial elastomer with the addition of a nanoparticle pigment and/or an opacifier submitted to chemical disinfection and artificial aging. Specimens were divided into four groups (n = 30): group I: silicone without pigment or opacifier, group II: ceramic powder pigment, group III: Barium sulfate (BaSO₄) opacifier, and group IV: ceramic powder and BaSO₄ opacifier. Specimens of each group (n = 10) were disinfected with effervescent tablets, neutral soap, or 4% chlorhexidine gluconate. Disinfection was done three times a week during two months. Afterward, specimens were submitted to different periods of artificial aging. Color evaluation was initially done, after 60 days (disinfection period) and after 252, 504, and 1008 h of artificial aging with aid of a reflection spectrophotometer. Data were analyzed by three-way ANOVA and Tukey test (α = 0.05). The isolated factor disinfection did not statistically influence the values of color stability among groups. The association between pigment and BaSO₄ opacifier (GIV) was more stable in relationship to color change (△E). All values of △E obtained, independent of the disinfectant and the period of artificial aging, were considered acceptable in agreement with the norms presented in literature.

The ability of cells to sustain mechanical stress is largely modulated by the cytoskeleton. We present a new application of optical tweezers to study cell's mechanical properties. We trap a fibronectin-coated bead attached to an adherent H4II-EC3 rat hepatoma cell in order to apply the force to the cell surface membrane. The bead position corresponding to the cell's local mechanical response at focal adhesions is measured with a quadrant detector. We assessed the cell response by tracking the evolution of the equilibrium force for 40 cells selected at random and selected a temporal window to assess the cell initial force expression at focal adhesions. The mean value of the force within this time window over 40 randomly selected bead/cell bounds was 52.3 pN. Then, we assessed the responses of the cells with modulation of the cytoskeletons, namely the ubiquitous actin-microfilaments and microtubules, plus the differentiation-dependent keratin intermediate filaments. Notably, a destabilization of the first two networks led to around 50 and 30% reductions in the mean equilibrium forces, respectively, relative to untreated cells, whereas a loss of the third one yielded a 25% increase. The differences in the forces from untreated and treated cells are resolved by the optical tweezers experiment.

We developed a new theory-based azimuthal phase retardation microscope to visualize distributions of actin filaments in biological cells without having them with exogenous dyes, fluorescence labels, or stains. The azimuthal phase retardation microscope visualizes distributions of actin filaments by measuring the intensity variations of each pixel of a charge coupled device camera while rotating a single linear polarizer. Azimuthal phase retardation δ between two fixed principal axes was obtained by calculating the rotation angles of the polarizer at the intensity minima from the acquired intensity data. We have acquired azimuthal phase retardation distributions of human breast cancer cell, MDA MB 231 by our microscope and compared the azimuthal phase retardation distributions with the fluorescence image of actin filaments by the commercial fluorescence microscope. Also, we have observed movement of human umbilical cord blood derived mesenchymal stem cells by measuring azimuthal phase retardation distributions.

This study investigates the optical properties of human blood during the coagulation process under statics using optical coherence tomography (OCT). OCT signal slope (OCTSS) and 1/e light penetration depth (d_1/e) were obtained from the profiles of reflectance versus depth. Results showed that both OCTSS and d_1/e were able to sensitively differentiate various stages of blood properties during coagulating. After 1 h clotting, OCTSS decreased by 47.0%, 15.0%, 13.7%, and 8.5% and d_1/e increased by 34.7%, 29.4%, 24.3%, and 22.9% for the blood samples at HCT of 25%, 35%, 45%, and 55%, respectively. The slope of d_1/e versus time (Sr, ×10−4 mm/s), associated with clot formation rate decreased from 6.0±0.3, 3.7±0.5 to 2.3±0.4 with the increasing of HCT from 35%, 45%, to 55%. The clotting time (t_c) from the d_1/e evolution curves was estimated to be 1969±92 s, 375±12 s, 455±11 s, and 865±47 s for the blood of 25%, 35%, 45%, and 55%. This study demonstrates that the parameters (t_c and S_r) from the variations in d_1/e had better sensitivity and smaller standard deviation. Furthermore, blood hematocrit affecting backscattering properties of blood during coagulation was capable of being discerned by OCT parameters. It is concluded that OCT is a potential technique to quantify and follow the liquid-gel transition of blood during clotting.

Variations in hair shaft morphology reflect ethnical diversity, but may also indicate internal diseases, nutritional deficiency, or hair and scalp disorders. The measurement and the follow-up of the hair shaft thickness over a defined period of time would be a valuable diagnostic tool in clinical practice. Standard light microscopy (LM) measurements require the epilation of hair shafts and frequently yield inaccurate values caused by the elliptic geometry of human hair shafts. Optical coherence tomography (OCT) is a noninvasive investigation method based on the principles of Michelson interferometry with a detection depth of approximately 1 mm in human skin. Two-dimensional images of the cross sections of tissue samples at a resolution of approximately 10 μm are produced, which allows convenient calculation of hair shaft thickness. To evaluate this new methodology for hair shaft thickness measurements, hair shafts taken from 28 healthy volunteers were analyzed by in vivo OCT and compared to standard in vitro LM measurements of hair shaft thickness. OCT yielded highly reproducible measurements of hair shaft thickness with a distinctly reduced variation compared to standard LM. This technique offers a unique opportunity for in vivo measurement and a follow-up of the kinetics of hair shaft thickness in humans during medical therapy.

We report the development and application of a knowledge-based coherent anti-Stokes Raman scattering (CARS) microscopy system for label-free imaging, pattern recognition, and classification of cells and tissue structures for differentiating lung cancer from non-neoplastic lung tissues and identifying lung cancer subtypes. A total of 1014 CARS images were acquired from 92 fresh frozen lung tissue samples. The established pathological workup and diagnostic cellular were used as prior knowledge for establishment of a knowledge-based CARS system using a machine learning approach. This system functions to separate normal, non-neoplastic, and subtypes of lung cancer tissues based on extracted quantitative features describing fibrils and cell morphology. The knowledge-based CARS system showed the ability to distinguish lung cancer from normal and non-neoplastic lung tissue with 91% sensitivity and 92% specificity. Small cell carcinomas were distinguished from nonsmall cell carcinomas with 100% sensitivity and specificity. As an adjunct to submitting tissue samples to routine pathology, our novel system recognizes the patterns of fibril and cell morphology, enabling medical practitioners to perform differential diagnosis of lung lesions in mere minutes. The demonstration of the strategy is also a necessary step toward in vivo point-of-care diagnosis of precancerous and cancerous lung lesions with a fiber-based CARS microendoscope.

All living organisms emit spontaneous ultraweak photon emission as a result of cellular metabolic processes. In this study, the involvement of reactive oxygen species (ROS) formed as the byproduct of oxidative metabolic processes in spontaneous ultraweak photon emission was studied in human hand skin. The effect of molecular oxygen and ROS scavengers on spontaneous ultraweak photon emission from human skin was monitored using a highly sensitive photomultiplier tube and charged coupled device camera. When spontaneous ultraweak photon emission was measured under anaerobic conditions, the photon emission was decreased, whereas under hyperaerobic condition the enhancement in photon emission was observed. Spontaneous ultraweak photon emission measured after topical application of glutathione, α-tocopherol, ascorbate, and coenzyme Q10 was observed to be decreased. These results reveal that ROS formed during the cellular metabolic processes in the epidermal cells play a significant role in the spontaneous ultraweak photon emission. It is proposed that spontaneous ultraweak photon emission can be used as a noninvasive tool for the temporal and spatial monitoring of the oxidative metabolic processes and intrinsic antioxidant system in human skin.

Fourier transform infrared (FTIR) spectroscopic imaging has been used to probe the biochemical composition of human renal tumor tissue and adjacent normal tissue. Freshly resected renal tumor tissue from surgery was prepared as a thin cryosection and examined by FTIR spectroscopic imaging. Tissue types could be discriminated by utilizing a combination of fuzzy k-means cluster analysis and a supervised classification algorithm based on a linear discriminant analysis. The spectral classification is compared and contrasted with the histological stained image. It is further shown that renal tumor cells have spread in adjacent normal tissue. This study demonstrates that FTIR spectroscopic imaging can potentially serve as a fast and objective approach for discrimination of renal tumor tissue from normal tissue and even in the detection of tumor infiltration in adjacent tissue.

We introduce a new method of rotational image acquisition for four-dimensional (4D) optical coherence tomography (OCT) of beating embryonic chick hearts. The rotational axis and the central A-scan of the OCT are identical. An out-of-phase image sequence covering multiple heartbeats is acquired at every angle of an incremental rotation of the deflection mirrors of the OCT system. Image acquisition is accomplished after a rotation of 180°. Comparison of a displayed live M-mode of the central A-scan with a reference M-mode allows instant detection of translational movements of the embryo. For calculation of 4D data sets, we apply an image-based retrospective gating algorithm using the phase information of the common central A-scan present in all acquired images. This leads to cylindrical three-dimensional data sets for every time step of the cardiac cycle that can be used for 4D visualization. We demonstrate this approach and provide a video of a beating Hamburger and Hamilton stage 16 embryonic chick heart generated from a 4D OCT data set using rotational image acquisition.

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p<1×10−⁸) and H&E+ (r = 0.74, p<0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity.

Early diagnosis of glaucoma, which is the second leading cause of blindness in the world, can halt or slow the progression of the disease. We propose an automated method for analyzing the optic disc and measuring the cup-to-disc ratio (CDR) on stereo retinal fundus images to improve ophthalmologists' diagnostic efficiency and potentially reduce the variation on the CDR measurement. The method was developed using 80 retinal fundus image pairs, including 25 glaucomatous, and 55 nonglaucomatous eyes, obtained at our institution. A disc region was segmented using the active contour method with the brightness and edge information. The segmentation of a cup region was performed using a depth map of the optic disc, which was reconstructed on the basis of the stereo disparity. The CDRs were measured and compared with those determined using the manual segmentation results by an expert ophthalmologist. The method was applied to a new database which consisted of 98 stereo image pairs including 60 and 30 pairs with and without signs of glaucoma, respectively. Using the CDRs, an area under the receiver operating characteristic curve of 0.90 was obtained for classification of the glaucomatous and nonglaucomatous eyes. The result indicates potential usefulness of the automated determination of CDRs for the diagnosis of glaucoma.

In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

The nonhuman primate model is suitable to study mechanisms of functional recovery following lesion of the cerebral cortex (motor cortex), on which therapeutic strategies can be tested. To interpret behavioral data (time course and extent of functional recovery), it is crucial to monitor the properties of the experimental cortical lesion, induced by infusion of the excitotoxin ibotenic acid. In two adult macaque monkeys, ibotenic acid infusions produced a restricted, permanent lesion of the motor cortex. In one monkey, the lesion was monitored over 3.5 weeks, combining laser speckle imaging (LSI) as metabolic readout (cerebral blood flow) and anatomical assessment with magnetic resonance imaging (T2-weighted MRI). The cerebral blood flow, measured online during subsequent injections of the ibotenic acid in the motor cortex, exhibited a dramatic increase, still present after one week, in parallel to a MRI hypersignal. After 3.5 weeks, the cerebral blood flow was strongly reduced (below reference level) and the hypersignal disappeared from the MRI scan, although the lesion was permanent as histologically assessed post-mortem. The MRI data were similar in the second monkey. Our experiments suggest that LSI and MRI, although they reflect different features, vary in parallel during a few weeks following an excitotoxic cortical lesion.

A high-speed and hardware-only algorithm using a center of mass method has been proposed for single-detector fluorescence lifetime sensing applications. This algorithm is now implemented on a field programmable gate array to provide fast lifetime estimates from a 32 × 32 low dark count 0.13 μm complementary metal-oxide-semiconductor single-photon avalanche diode (SPAD) plus time-to-digital converter array. A simple look-up table is included to enhance the lifetime resolvability range and photon economics, making it comparable to the commonly used least-square method and maximum-likelihood estimation based software. To demonstrate its performance, a widefield microscope was adapted to accommodate the SPAD array and image different test samples. Fluorescence lifetime imaging microscopy on fluorescent beads in Rhodamine 6G at a frame rate of 50 fps is also shown.

The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC₅₀ > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

Functional neuroimaging is becoming a valuable tool in cognitive research and clinical applications. The clinical context brings specific constraints that include the requirement of a high channel count to cover the whole head, high sensitivity for single event detection, and portability for long-term bedside monitoring. For epilepsy and stroke monitoring, the combination of electroencephalography (EEG) and functional near-infrared spectroscopy (NIRS) is expected to provide useful clinical information, and efforts have been deployed to create prototypes able to simultaneously acquire both measurement modalities. However, to the best of our knowledge, existing systems lack portability, NIRS sensitivity, or have low channel count. We present a battery-powered, portable system with potentially up to 32 EEG channels, 32 NIRS light sources, and 32 detectors. Avalanche photodiodes allow for high NIRS sensitivity and the autonomy of the system is over 24 h. A reduced channel count prototype with 8 EEG channels, 8 sources, and 8 detectors was tested on phantoms. Further validation was done on five healthy adults using a visual stimulation protocol to detect local hemodynamic changes and visually evoked potentials. Results show good concordance with literature regarding functional activations and suggest sufficient performance for clinical use, provided some minor adjustments were made.

We present a multicolor fluorescence imaging modality to visualize in real-time tissue structures emitting multispectral fluorescent light from different focal depths. Each designated spectrum of fluorescent emission from a specific depth within a volumetric tissue is probed by a depth-spectrum selective holographic grating. The grating for each fluorescent color are multiplexed within a volume hologram, which enables simultaneously obtaining multicolored fluorescent information at different depths within a biological tissue sample. We demonstrate the imaging modality's ability to obtain laser-induced multicolored fluorescence images of a biological sample from different depths without scanning. We also experimentally demonstrate that the imaging modality can be simultaneously operated at both fluorescent and bright field modes to provide complementary information of volumetric tissue structures at different depths in real-time.

The specificity of molecular and functional photoacoustic (PA) images depends on the accuracy of the photoacoustic absorption spectroscopy. The PA signal is proportional to the product of the optical absorption coefficient and local light fluence; quantitative PA measurements of the optical absorption coefficient therefore require an accurate estimation of optical fluence. Light-modeling aided by diffuse optical tomography (DOT) can be used to map the required fluence and to reduce errors in traditional PA spectroscopic analysis. As a proof-of-concept, we designed a tissue-mimicking phantom to demonstrate how fluence-related artifacts in PA images can lead to misrepresentations of tissue properties. To correct for these inaccuracies, the internal fluence in the tissue phantom was estimated by using DOT to reconstruct spatial distributions of the absorption and reduced scattering coefficients of multiple targets within the phantom. The derived fluence map, which only consisted of low spatial frequency components, was used to correct PA images of the phantom. Once calibrated to a known absorber, this method reduced errors in estimated absorption coefficients from 33% to 6%. These results experimentally demonstrate that combining DOT with PA imaging can significantly reduce fluence-related errors in PA images, while producing quantitatively accurate, high-resolution images of the optical absorption coefficient.

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG/THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium/stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.

This study investigates the ability of a flexible fiberoptic-based fluorescence lifetime imaging microscopy (FLIM) technique to resolve biochemical features in plaque fibrotic cap associated with plaque instability and based solely on fluorescence decay characteristics. Autofluorescence of atherosclerotic human aorta (11 autopsy samples) was measured at 48 locations through two filters, F377: 377/50 and F460: 460/60 nm (center wavelength/bandwidth). The fluorescence decay dynamic was described by average lifetime (τ) and four Laguerre coefficients (LECs) retrieved through a Laguerre deconvolution technique. FLIM-derived parameters discriminated between four groups [elastin-rich (ER), elastin and macrophage-rich (E+M), collagen-rich (CR), and lipid-rich (LR)]. For example, τF377 discriminated ER from CR (R = 0.84); τF460 discriminated E+M from CR and ER (R = 0.60 and 0.54, respectively); LEC-1F377 discriminated CR from LR and E+M (R = 0.69 and 0.77, respectively); P < 0.05 for all correlations. Linear discriminant analysis was used to classify this data set with specificity >87% (all cases) and sensitivity as high as 86%. Current results demonstrate for the first time that clinically relevant features (e.g., ratios of lipid versus collagen versus elastin) can be evaluated with a flexible-fiber based FLIM technique without the need for fluorescence intensity information or contrast agents.

In vivo imaging of microcirculation can improve our fundamental understanding of cerebral microhemodynamics under various physiological challenges, such as hypoxia and hyperoxia. However, existing techniques often involve the use of invasive procedures or exogenous contrast agents, which would inevitably perturb the intrinsic physiologic responses of microcirculation being investigated. We report ultrahigh sensitive optical microangiography (OMAG) for label-free monitoring of microcirculation responses challenged by oxygen inhalation. For the first time, we demonstrate that OMAG is capable of showing the impact of acute hypoxia and hyperoxia on microhemodynamic activities, including the passive and active modulation of microvascular density and flux regulation, within capillary and noncapillary vessels in rodents in vivo. The ability of OMAG to functionally image the intact microcirculation promises future applications for studying cerebral diseases.

Optical mapping of cardiac propagation has traditionally been hampered by motion artifact, chiefly due to changes in photodetector-to-tissue registration as the heart moves. We have developed an optical mapping technique to simultaneously record electrical waves and mechanical contraction in isolated hearts. This allows removal of motion artifact from transmembrane potential (V_m) recordings without the use of electromechanical uncoupling agents and allows the interplay of electrical and mechanical events to be studied at the whole organ level. Hearts are stained with the voltage-sensitive dye di-4-ANEPPS and ring-shaped markers are attached to the epicardium. Fluorescence, elicited on alternate frames by 450 and 505 nm light-emitting diodes, is recorded at 700 frames/ per second by a camera fitted with a 605±25 nm emission filter. Marker positions are tracked in software. A signal, consisting of the temporally interlaced 450 and 505 nm fluorescence, is collected from the pixels enclosed by each moving ring. After deinterlacing, the 505 nm signal consists of V_m with motion artifact, while the 450 nm signal is minimally voltage-sensitive and contains primarily artifacts. The ratio of the two signals estimates V_m. Deformation of the tissue enclosed by each set of 3 rings is quantified using homogeneous finite strain.

The pathological status of the sentinel lymph node is important for accurate melanoma staging, ascertaining prognosis and planning treatment. The standard procedure involves biopsy of the node and histopathological assessment of its status. Drawbacks of this examination include a finite sampling of the node with the likelihood of missing metastases, and a significant time-lag before histopathological results are available to the surgeon. We studied the applicability of photoacoustic computed tomographic imaging as an intraoperative modality for examining the status of resected human sentinel lymph nodes. We first applied the technique to image ex vivo pig lymph nodes carrying metastases-simulating melanoma cells using multiple wavelengths. The experience gained was applied to image a suspect human lymph node. We validated the photoacoustic imaging results by comparing a reconstructed slice with a histopathological section through the node. Our results suggest that photoacoustics has the potential to develop into an intraoperative imaging method to detect melanoma metastases in sentinel lymph nodes.

Refractive index of biotissue is a useful optical parameter in the biomedical field. An extended differential total reflection method is introduced to determine the complex refractive index. The real part is directly determined by differential of the reflectance curve, and the imaginary part is obtained from nonlinear fitting. The method is verified by a series of tissue-mimicking phantoms, porcine muscle and porcine adipose.

Otitis media diagnosis can be assisted by measuring the shape of the tympanic membrane. We have developed an ear speculum for an otoscope, including spatially distributed source and detector optical fibers, to generate source-detector intensity matrices (SDIMs), representing the curvature of surfaces. The surfaces measured were a model ear with a latex membrane and harvested temporal bones including intact tympanic membranes. The position of the tympanic membrane was shifted from retracted to bulging by air pressure and that of the latex membrane by water displacement. The SDIM was normalized utilizing both external (a sheared flat plastic cylinder) and internal references (neutral position of the membrane). Data was fitted to a two-dimensional Gaussian surface representing the shape by its amplitude and offset. Retracted and bulging surfaces were discriminated for the model ear by the sign of the Gaussian amplitude for both internal and external reference normalization. Tympanic membranes were separated after a two-step normalization: first to an external reference, adjusted for the distance between speculum and the surfaces, and second by comparison with an average normally positioned SDIM from tympanic membranes. In conclusion, we have shown that the modified otoscope can discriminate between bulging and retracted tympanic membranes in a single measurement, given a two-step normalization.

Knowledge of the optical properties of neonatal skin is invaluable when developing new, or improving existing optical techniques for use at the neonatal intensive care. In this article, we present in vivo measurements of the absorption μa and reduced scattering coefficient μs′ of neonatal skin between 450 and 600 nm and assess the influence of age and skin pigmentation on the optical properties. The optical properties were measured using a spatially resolved, steady state diffuse reflectance spectroscopy setup, combined with a modified spatially resolved diffusion model. The method was validated on phantoms with known values for the absorption and reduced scattering coefficient. Values of μa and μs′ were obtained from the skin at four different body locations (forehead, sternum, hand, and foot) of 60 neonates with varying gestational age, postnatal age, and skin pigmentation. We found that μa ranged from 0.02 to 1.25 mm&^#8722;1 and μs′ was in the range of 1 to 2.8 mm⁻¹ (5th to 95th percentile of the patient population), independent of body location. In contrast to previous studies, no to very weak correlation was observed between the optical properties and gestational maturity, but a strong dependency of the absorption coefficient on postnatal age was found for dark skinned patients.

Early detection of cerebral hypoxemia is an important aim in neonatology. A relevant parameter to assess brain oxygenation may be the cerebral tissue oxygen saturation (StO₂) measured by near-infrared spectroscopy (NIRS). So far the reproducibility of StO2 measurements was too low for clinical application, probably due to inhomogeneities. The aim of this study was to test a novel sensor geometry which reduces the influence of inhomogeneities. Thirty clinically stable newborn infants, with a gestational age of median 33.9 (range 26.9 to 41.9) weeks, birth weight of 2220 (820 to 4230) g, postnatal age of 5 (1 to 71) days were studied. At least four StO₂ measurements of 1 min duration were carried out using NIRS on the lateral head. The sensor was repositioned between measurements. Reproducibility was calculated by a linear mixed effects model. The mean StO₂ was 79.99 ± 4.47% with a reproducibility of 2.76% and a between-infant variability of 4.20%. Thus, the error of measurement only accounts for 30.1% of the variability. The novel sensor geometry leads to considerably more precise measurements compared to previous studies with, e.g., ∼5% reproducibility for the NIRO 300. The novel StO₂ values hence have a higher clinical relevance.

Probing tissue with near-infrared radiation (NIR) simultaneously produces remitted fluorescence and Raman scattering (IE) plus Rayleigh/Mie light scattering (EE) that noninvasively give chemical and physical information about the materials and objects within. We model tissue as a three-phase system: plasma and red blood cell (RBC) phases that are mobile and a static tissue phase. In vivo, any volume of tissue naturally experiences spatial and temporal fluctuations of blood plasma and RBC content. Plasma and RBC fractions may be discriminated from each other on the basis of their physical, chemical, and optical properties. Thus, IE and EE from NIR probing yield information about these fractions. Assuming there is no void volume in viable tissue, or that void volume is constant, changes in plasma and RBC volume fractions may be calculated from simultaneous measurements of the two observables, EE and IE. In a previously published analysis we showed the underlying phenomenology but did not provide an algorithm for calculating volume fractions from experimental data. Now, we present a simple analysis that allows monitoring of fluid fraction and hematocrit (Hct) changes by measuring IE and EE, and apply it to some experimental in vivo measurements.

Low-coherence enhanced backscattering (LEBS) is a depth-selective self-interference phenomenon that originates from light traveling time-reversed paths in a scattering medium. The depth selectivity of LEBS and its sensitivity to optical properties of the scattering medium has made it a promising technique for probing the structure of biological tissue with applications to disease diagnosis and, in particular, precancerous conditions. The ability to accurately predict the penetration depth of the LEBS signal is important in targeting an optimal tissue depth for detecting precancerous cells. This prediction is further complicated by the variation in optical properties of different tissue types. In this paper, the effects of the reduced scattering coefficient (μs'), the phase function and the instrument spatial coherence length (Lsc) on the LEBS penetration depth are quantified. It is determined that the LEBS penetration depth is primarily dependent on Lsc, μs', and the anisotropy factor (g), but has minimal dependence on higher moments of the phase function. An empirical expression, having a similar form as the double scattering approximation for LEBS, is found to accurately predict the average penetration depth in the multiple scattering regime. The expression is shown to be accurate for a broad range of experimentally relevant optical properties and spatial coherence lengths.

We describe an algorithm to calculate an index that characterizes spatial differences in broadband near-infrared [(NIR), 650-1000 nm] absorption spectra of tumor-containing breast tissue. Patient-specific tumor spatial heterogeneities are visualized through a heterogeneity spectrum function (HS). HS is a biomarker that can be attributed to different molecular distributions within the tumor. To classify lesion heterogeneities, we built a heterogeneity index (HI) derived from the HS by weighing the HS in specific NIR absorption bands. It is shown that neoadjuvant chemotherapy (NAC) response is potentially related to the tumor heterogeneity. Therefore, we correlate the heterogeneity index obtained prior to treatment with the final response to NAC. From a pilot study of 15 cancer patients treated with NAC, pathological complete responders (pCR) were separated from non-pCR according to their HI (-44 ± 12 and 43 ± 17, p = 3 × 10⁻⁸, respectively). We conclude that the HS function is a biomarker that can be used to visualize spatial heterogeneities in lesions, and the baseline HI prior to therapy correlates with chemotherapy pathological response.

Many laser wavelengths have been used in photodynamic therapy (PDT) for port wine stains (PWS). However, how these wavelengths result in different PDT outcomes has not been clearly illuminated. This study is designed to analyze which wavelengths would be the most advantageous for use in PDT for PWS. The singlet oxygen yield in PDT-treated PWS skin under different wavelengths at the same photosensitizer dosage was simulated and the following three situations were simulated and compared: 1. PDT efficiency of 488, 532, 510, 578, and 630 nm laser irradiation at clinical dosage (100 mW/cm², 40 min); 2. PDT efficiency of different wavelength for PWS with hyperpigmentation after previous PDT; 3. PDT efficiency of different wavelengths for PWS, in which only deeply located ectatic vessels remained. The results showed that singlet oxygen yield is the highest at 510 nm, it is similar at 532 nm and 488 nm, and very low at 578 nm and 630 nm. This result is identical to the state in clinic. According to this theoretical study, the optimal wavelength for PDT in the treatment of PWS should near the absorption peaks of photosensitizer and where absorption from native chromophores (haemoglobin and melanin) is diminished.

We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.