In a biological tissue, light scattering is based on the size and type of scatterers seen as refractive index variations that describe the optical properties shown. In this paper, we have implemented the variable incidence angle technique of multiple angle of illumination experiment on tendon and cartilage samples whose dominant constituents are genetically different types of collagen fibers, type I and type II, respectively. It is found that tendon displays a much greater angular anisotropy in its optical backscattering coefficient than the healthy cartilage. We propose that this is due to a more uniform distribution of fine fibrils than is found in tendon. Rayleigh-Gans approximation is used to give qualitative support to this idea.

Brachytherapy is a form of radiation therapy commonly used in the treatment of prostate cancer wherein sustained radiation doses can be precisely targeted to the tumor area by the implantation of small radioactive seeds around the treatment area. Ultrasound is a popular imaging mode for seed implantation, but the seeds are difficult to distinguish from the tissue structure. In this work, we demonstrate the feasibility of photoacoustic imaging for identifying brachytherapy seeds in a tissue phantom, comparing the received intensity to endogenous contrast. We have found that photoacoustic imaging at 1064 nm can identify brachytherapy seeds uniquely at laser penetration depths of 5 cm in biological tissue at the ANSI limit for human exposure with a contrast-to-noise ratio of 26.5 dB. Our realtime combined photoacoustic-ultrasound imaging approach may be suitable for brachytherapy seed placement and post-placement verification, potentially allowing for realtime dosimetry assessment during implantation.

Photoacoustic tomography is a hybrid modality based on optical absorption excitation and ultrasonic detection. It is sensitive to melanin, one of the primary absorbers in skin. For cells that do not naturally contain melanin, melanin production can be induced by introducing the gene for tyrosinase, the primary enzyme responsible for expression of melanin in melanogenic cells. Optical resolution photoacoustic microscopy was used in the ex vivo study reported here, where the signal from transfected cells increased by more than 10 times over wild-type cells. A subsequent in vivo experiment was conducted to demonstrate the capability of photoacoustic microscopy to spectrally differentiate between tyrosinase-catalyzed melanin and various other absorbers in tissue.

We combined photoacoustic ophthalmoscopy (PAOM) with autofluorescence imaging for simultaneous in vivo imaging of dual molecular contrasts in the retina using a single light source. The dual molecular contrasts come from melanin and lipofuscin in the retinal pigment epithelium (RPE). Melanin and lipofuscin are two types of pigments and are believed to play opposite roles (protective versus exacerbate) in the RPE in the aging process. We have successfully imaged the retina of pigmented and albino rats at different ages. The experimental results showed that multimodal PAOM system can be a potentially powerful tool in the study of age-related degenerative retinal diseases.

Time-reversed ultrasonically encoded (TRUE) optical focusing was recently proposed to deliver light dynamically to a tight region inside a scattering medium. In this letter, we report the first development of a reflection-mode TRUE optical focusing system. A high numerical aperture light guide is used to transmit the diffusely reflected light from a turbid medium to a phase-conjugate mirror (PCM), which is sensitive only to the ultrasound-tagged light. From the PCM, a phase conjugated wavefront of the tagged light is generated and conveyed by the same light guide back to the turbid medium, subsequently converging to the ultrasonic focal zone. We present experimental results from this system, which has the ability to focus light in a highly scattering medium with a round-trip optical penetration thickness (extinction coefficient multiplied by round-trip depth) as large as 160.

Spatiotemporal activity patterns in local neural networks are fundamental to understanding how information is processed and stored in brain microcircuits. Currently, imaging techniques are able to map the directional activation of macronetworks across brain areas; however, these strategies still fail to resolve the activation direction for fine microcircuits with cellular spatial resolution. Here, we show the capability to identify the activation direction of a multicell network with a cellular resolution and millisecond precision by using fast two-photon microscopy and cross correlation procedures. As an example, we characterized a directional neuronal network in an epilepsy brain slice to provide different initiation delay among multiple neurons defined at a millisecond scale.

Here we introduce the existing fabrication techniques, detection methods, and related techniques for microfluidic sensing, with an emphasis on the detection techniques. A general survey and comparison of the fabrication techniques were given, including prototyping (hot embossing, inject molding, and soft lithography) and direct fabrication (laser micromachining, photolithography, lithography, and x-ray lithography) techniques. This is followed by an in-depth look at detection techniques: optical, electrochemical, mass spectrometry, as well as nuclear magnetic resonance spectroscopy-based sensing approaches and related techniques. In the end, we highlight several of the most important issues for future work on microfluidic sensing. This article aims at providing a tutorial review with both introductory materials and inspiring information on microfluidic fabrication and sensing for nonspecialists.

Laser radiation provides a means to control the fields of temperature and thermo mechanical stress, mass transfer, and modification of fine structure of the cartilage matrix. The aim of this outlook paper is to review physical and biological aspects of laser-induced regeneration of cartilage and to discuss the possibilities and prospects of its clinical applications. The problems and the pathways of tissue regeneration, the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches for cartilage repair; consider possible mechanisms of laser-induced regeneration. Finally, we present the results in laser regeneration of joints and spine disks cartilages and discuss some future applications of lasers in regenerative medicine.

Experimental measurements of the reflected light intensity from two-layer phantoms are presented. We report, for the first time, an experimental observation of a typical reflected light intensity behavior for the two-layer structure characterized by two different slopes in the reflected light profile of the irradiated tissue. The point in which the first slope changes to the second slope, named as the crossover point, depends on the upper layer thickness as well as on the ratio between the absorption coefficients of the two layers. Since similar experiments from one-layer phantoms present a monotonic decay behavior, the existence and the location of the crossover point can be used as a diagnostic fingerprint for two-layer tissue structures. This pertains to two layers with greater absorptivity in the upper layer, which is the typical biological case in tissues like skin.

Metal nanoparticles can be functionalized with biomolecules to selectively localize in precancerous tissues and can act as optical contrast enhancers for reflectance-based diagnosis of epithelial precancer. We carry out Monte Carlo (MC) simulations to analyze photon propagation through nanoparticle-labeled tissues and to reveal the importance of using a proper form of phase function for modeling purposes. We first employ modified phase functions generated with a weighting scheme that accounts for the relative scattering strengths of unlabeled tissue and nanoparticles. To present a comparative analysis, we repeat our MC simulations with simplified functions that only approximate the angular scattering properties of labeled tissues. The results obtained for common optical sensor geometries and biologically relevant labeling schemes indicate that the exact form of the phase function used as model input plays an important role in determining the reflectance response and approximating functions often prove inadequate in predicting the extent of contrast enhancement due to labeling. Detected reflectance intensities computed with different phase functions can differ up to ∼60% and such a significant deviation may even alter the perceived contrast profile. These results need to be taken into account when developing photon propagation models to assess the diagnostic potential of nanoparticle-enhanced optical measurements.

We present a new Monte Carlo model of cylindrical diffusing fibers that is implemented with a graphics processing unit. Unlike previously published models that approximate the diffuser as a linear array of point sources, this model is based on the construction of these fibers. This allows for accurate determination of fluence distributions and modeling of fluorescence generation and collection. We demonstrate that our model generates fluence profiles similar to a linear array of point sources, but reveals axially heterogeneous fluorescence detection. With axially homogeneous excitation fluence, approximately 90% of detected fluorescence is collected by the proximal third of the diffuser for μs'/μa = 8 in the tissue and 70 to 88% is collected in this region for μs'/μa = 80. Increased fluorescence detection by the distal end of the diffuser relative to the center section is also demonstrated. Validation of these results was performed by creating phantoms consisting of layered fluorescent regions. Diffusers were inserted into these layered phantoms and fluorescence spectra were collected. Fits to these spectra show quantitative agreement between simulated fluorescence collection sensitivities and experimental results. These results will be applicable to the use of diffusers as detectors for dosimetry in interstitial photodynamic therapy.

The current paper describes the design and population testing of a flicker sensitivity assessment technique corresponding to the psychophysical approach for straylight measurement. The purpose is twofold: to check the subjects' capability to perform the straylight test and as a test for retinal integrity for other purposes. The test was implemented in the Oculus C-Quant straylight meter, using homemade software (MATLAB). The geometry of the visual field lay-out was identical, as was the subjects' 2AFC task. A comparable reliability criterion ("unc") was developed. Outcome measure was logTCS (temporal contrast sensitivity). The population test was performed in science fair settings on about 400 subjects. Moreover, 2 subjects underwent extensive tests to check whether optical defects, mimicked with trial lenses and scatter filters, affected the TCS outcome. Repeated measures standard deviation was 0.11 log units for the reference population. Normal values for logTCS were around 2 (threshold 1%) with some dependence on age (range 6 to 85 years). The test outcome did not change upon a tenfold (optical) deterioration in visual acuity or straylight. The test has adequate precision for checking a subject's capability to perform straylight assessment. The unc reliability criterion ensures sufficient precision, also for assessment of retinal sensitivity loss.

Conventional fluorescence lifetime imaging requires complicated algorithms to extract lifetimes of fluorophores and acquisition of multiple data points at progressively longer delay times to characterize tissues. To address diminishing signal-to-noise ratios at these progressively longer time delays, we report a time-resolved fluorescence imaging method, normalized fluorescence yield imaging that does not require the extraction of lifetimes. The concept is to extract the "contrast" instead of the lifetime value of the fluorophores by using simple mathematical algorithms. This process converts differences in decay times directly to different intensities. The technique was verified experimentally using a gated iCCD camera and an ultraviolet light-emitting diode light source. It was shown that this method can distinguish between chemical dyes (Fluorescein and Rhodamine-B) and biomedical samples, such as powders of elastin and collagen. Good contrast was obtained between fluorophores that varied by less than 6% in lifetime. Additionally, it was shown that long gate times up to 16 ns achieve good contrast depending upon the samples to be studied. These results support the feasibility of time-resolved fluorescence imaging without lifetime extraction, which has a potential clinical role in noninvasive real-time imaging.

We investigate the use of a frequency-domain reconstruction algorithm based on the nonuniform fast Fourier transform (NUFFT) for photoacoustic imaging (PAI). Standard algorithms based on the fast Fourier transform (FFT) are computationally efficient, but compromise the image quality by artifacts. In our previous work we have developed an algorithm for PAI based on the NUFFT which is computationally efficient and can reconstruct images with the quality known from temporal backprojection algorithms. In this paper we review imaging qualities, such as resolution, signal-to-noise ratio, and the effects of artifacts in real-world situations. Reconstruction examples show that artifacts are reduced significantly. In particular, image details with a larger distance from the detectors can be resolved more accurately than with standard FFT algorithms.

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

The primary pathophysiology of peripheral arterial disease is associated with impaired perfusion to the muscle tissue in the lower extremities. The lack of effective pharmacologic treatments that stimulate vessel collateralization emphasizes the need for an imaging method that can be used to dynamically visualize depth-resolved microcirculation within muscle tissues. Optical microangiography (OMAG) is a recently developed label-free imaging method capable of producing three-dimensional images of dynamic blood perfusion within microcirculatory tissue beds at an imaging depth of up to ∼2 mm, with an unprecedented imaging sensitivity of blood flow at ∼4 μm/s. In this paper, we demonstrate the utility of OMAG in imaging the detailed blood flow distributions, at a capillary-level resolution, within skeletal muscles of mice. By use of the mouse model of hind-limb ischemia, we show that OMAG can assess the time-dependent changes in muscle perfusion and perfusion restoration along tissue depth. These findings indicate that OMAG can represent a sensitive, consistent technique to effectively study pharmacologic therapies aimed at promoting the growth and development of collateral vessels.

We report the existence of polarization memory effect (PME) in optical coherence tomography and investigate its potential applications in dental imaging. We performed the study in three steps. First, microsphere scattering phantoms of different sizes were imaged in order to validate experimental results with PME theory. Both linearly and circularly polarized light were used to probe the samples. Second, healthy tooth samples were scanned and polarization memory effect was identified in dentin. In this step, specific verification and signal processing were performed to rule out possible image interpretation by birefringence effect. Third, we evaluated dentin demineralization with PME. Results show polarization memory can be useful to characterize this dynamic mineralization process for early caries detection and rehabilitation.

Transverse "chemical" interfaces are revealed with a conventional two beam narrowband coherent anti-Stokes Raman scattering microscopy setup in a collinear configuration. The exciting "pump" and "Stokes" beams are focused on the sample in two opposite directions. The subtraction of the two generated anti-Stokes signals gives rise to a signal that is directly proportional to the pure Raman spectrum of the resonant medium. This property is used to highlight an interface between glass and N,N-dimethylformamide (DMF) and recover the pure Raman spectrum of DMF around its 1408 cm⁻¹ vibrational band.

We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon "budget." These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

Skin cancer diagnosis depends not only on histopathological examination but also on visual inspection before and after the excision of suspected lesion. Neoplasm is accompanied with changes in birefringence of collagen, pleomorphicity, and hyperchromatic state of epithelial nuclei. These phenomena can be measured by spectral and polarization changes of light backscattered by the examined tissue. A new differential spectropolarimetric system is proposed using liquid crystal devices, one as a tunable filter and the other as a polarization rotator, both operating at wide spectral ranges from the visible to the near-infrared. Since collagen's fibrils texture orientation depends on its location in the skin and since it is not well organized, our system scans the bipolarization states by continuously rotating the linearly polarized light incident on a skin lesion, and collecting differential contrasts between sequenced images when simultaneously averaging the statistical readout of a video camera. This noninvasive method emphasizes areas on skin where the neoplasm, or tumor, minimizes the statistical polarization change of the scattered light from the lesion. The module can be considered as an assistant tool for epiluminescence microscopy. Images of skin tumors were captured in vivo before the patients having their surgery and compared to histopathological results.

In turbid media such as biological tissue, multiple scattering hinders direct light focusing at depths beyond one transport mean free path. As a solution to this problem, time-reversed ultrasonically encoded (TRUE) optical focusing is proposed based on ultrasonic encoding of diffused laser light and optical time reversal. In TRUE focusing, a laser beam of long coherence length illuminates a turbid medium, where the incident light undergoes multiple scattering and part of it gets ultrasonically encoded within the ultrasonic focal zone. A conjugated wavefront of the ultrasonically encoded light is then generated by a phase conjugate mirror outside the medium, which traces back the trajectories of the ultrasonically encoded diffused light and converges light to the ultrasonic focal zone. Here, we report the latest experimental improvement in TRUE optical focusing that increases its penetration in tissue-mimicking media from a thickness of 3.75 to 7.00 mm. We also demonstrate that the TRUE focus depends on the focal diameter of the ultrasonic transducer.

A method to locate an absorber embedded in a semi-infinite turbid medium by spatially-resolved continuous-wave (SRCW) diffuse reflectance measurements is introduced. The depth of the absorber is assessed by single wavelength SRCW diffuse reflectance measurements by two detectors in a radial row. The ratio of perturbations introduced by the defect at two detectors is used to be matched with the ratio-versus-depth curve, which are generated by approximate formulas of continuous wave diffuse reflectance. The error due to approximation and the error in depth assessment are studied for different cases revealing favorable source-detector placements with respect to planar position of the defect. The effect of lateral displacement of the source with respect to defect is studied. A strategy to overcome errors introduced by erroneous prediction of background medium optical properties is suggested. Theoretical results indicate that the depth of the absorber can be obtained with 0.1 mm precision independent of its absorption coefficient and its size for the values chosen in the study. The approach is tested experimentally and it is observed that theoretical results fit with experimental data.

Hypothermia can unintentionally occur in daily life, e.g., in cardiovascular surgery or applied as therapeutics in the neurosciences critical care unit. So far, the temperature-induced spatiotemporal responses of the neural function have not been fully understood. In this study, we investigated the functional change in cerebral blood flow (CBF), accompanied with neuronal activation, by laser speckle imaging (LSI) during hypothermia. Laser speckle images from Sprague-Dawley rats (n = 8, male) were acquired under normothermia (37°C) and moderate hypothermia (32°C). For each animal, 10 trials of electrical hindpaw stimulation were delivered under both temperatures. Using registered laser speckle contrast analysis and temporal clustering analysis (TCA), we found a delayed response peak and a prolonged response window under hypothermia. Hypothermia also decreased the activation area and the amplitude of the peak CBF. The combination of LSI and TCA is a high-resolution functional imaging method to investigate the spatiotemporal neurovascular coupling in both normal and pathological brain functions.

In order to visualize human skin hemodynamics, we investigated a method that is specifically developed for the visualization of concentrations of oxygenated blood, deoxygenated blood, and melanin in skin tissue from digital RGB color images. Images of total blood concentration and oxygen saturation can also be reconstructed from the results of oxygenated and deoxygenated blood. Experiments using tissue-like agar gel phantoms demonstrated the ability of the developed method to quantitatively visualize the transition from an oxygenated blood to a deoxygenated blood in dermis. In vivo imaging of the chromophore concentrations and tissue oxygen saturation in the skin of the human hand are performed for 14 subjects during upper limb occlusion at 50 and 250 mm Hg. The response of the total blood concentration in the skin acquired by this method and forearm volume changes obtained from the conventional strain-gauge plethysmograph were comparable during the upper arm occlusion at pressures of both 50 and 250 mm Hg. The results presented in the present paper indicate the possibility of visualizing the hemodynamics of subsurface skin tissue.

Multiphoton microscopy has been shown to be a useful tool in studying drug distribution in biological tissues. In addition, fluorescence lifetime imaging provides information about the structure and dynamics of fluorophores based on their fluorescence lifetimes. Fluorescein, a commonly used fluorescent probe, is metabolized within liver cells to fluorescein mono-glucuronide, which is also fluorescent. Fluorescein and its glucuronide have similar excitation and emission spectra, but different fluorescence lifetimes. In this study, we employed multiphoton fluorescence lifetime imaging to study the distribution and metabolism of fluorescein and its metabolite in vivo in rat liver. Fluorescence lifetime values in vitro were used to interpret in vivo data. Our results show that the mean fluorescence lifetimes of fluorescein and its metabolite decrease over time after injection of fluorescein in three different regions of the liver. In conclusion, we have demonstrated a novel method to study a fluorescent compound and metabolite in vivo using multiphoton fluorescence lifetime imaging.

Angular domain spectroscopic imaging (ADSI) is a novel technique for the detection and characterization of optical contrast in turbid media based on spectral characteristics. The imaging system employs a silicon micromachined angular filter array to reject scattered light traversing a specimen and an imaging spectrometer to capture and discriminate the largely remaining quasiballistic light based on spatial position and wavelength. The imaging modality results in hyperspectral shadowgrams containing two-dimensional (2D) spatial maps of spectral information. An ADSI system was constructed and its performance was evaluated in the near-infrared region on tissue-mimicking phantoms. Image-based spectral correlation analysis using transmission spectra and first order derivatives revealed that embedded optical targets could be resolved. The hyperspectral images obtained with ADSI were observed to depend on target concentration, target depth, and scattering level of the background medium. A similar analysis on a muscle and tumor sample dissected from a mouse resulted in spatially dependent optical transmission spectra that were distinct, which suggested that ADSI may find utility in classifying tissues in biomedical applications.

Oxygenation measurements are widely used in patient care. However, most clinically available instruments currently consist of contact probes that only provide global monitoring of the patient (e.g., pulse oximetry probes) or local monitoring of small areas (e.g., spectroscopy-based probes). Visualization of oxygenation over large areas of tissue, without a priori knowledge of the location of defects, has the potential to improve patient management in many surgical and critical care applications. In this study, we present a clinically compatible multispectral spatial frequency domain imaging (SFDI) system optimized for surgical oxygenation imaging. This system was used to image tissue oxygenation over a large area (16×12 cm) and was validated during preclinical studies by comparing results obtained with an FDA-approved clinical oxygenation probe. Skin flap, bowel, and liver vascular occlusion experiments were performed on Yorkshire pigs and demonstrated that over the course of the experiment, relative changes in oxygen saturation measured using SFDI had an accuracy within 10% of those made using the FDA-approved device. Finally, the new SFDI system was translated to the clinic in a first-in-human pilot study that imaged skin flap oxygenation during reconstructive breast surgery. Overall, this study lays the foundation for clinical translation of endogenous contrast imaging using SFDI.

Using a machine-learning classification algorithm applied to near-infrared spectroscopy (NIRS) signals, we classify a success (change detection) or a failure (change blindness) in detecting visual changes for a change-detection task. Five subjects perform a change-detection task, and their brain activities are continuously monitored. A support-vector-machine algorithm is applied to classify the change-detection and change-blindness trials, and correct classification probability of 70-90% is obtained for four subjects. Two types of temporal shapes in classification probabilities are found: one exhibiting a maximum value after the task is completed (postdictive type), and another exhibiting a maximum value during the task (predictive type). As for the postdictive type, the classification probability begins to increase immediately after the task completion and reaches its maximum in about the time scale of neuronal hemodynamic response, reflecting a subjective report of change detection. As for the predictive type, the classification probability shows an increase at the task initiation and is maximal while subjects are performing the task, predicting the task performance in detecting a change. We conclude that decoding change detection and change blindness from NIRS signal is possible and argue some future applications toward brain-machine interfaces.

In studies with near-infrared spectroscopy, the recorded signals contain information on the temporal interbeat intervals of the heart. If this cardiac information is needed exclusively and could directly be extracted, an additional electrocardiography device would be unnecessary. The aim was to estimate these intervals from signals measured with near-infrared spectroscopy with two novel approaches. In one approach, we model the heartbeat oscillations in these signals with a Fourier series where the coefficients and the fundamental frequency can continuously change over time. The time-dependent model parameters are estimated and used to calculate the interbeat intervals. The second approach uses empirical mode decomposition. The signal measured with near-infrared spectroscopy is empirically decomposed into a set of oscillatory components. The sum of a specific subset of them is an estimate of the pure heartbeat signal in which the diastolic peaks and consequential interbeat intervals are detected. We show in simultaneous electrocardiography and near-infrared spectroscopy measurements on 11 subjects (8 men and 3 woman with mean age 32.8 ± 8.1 yr), that the interbeat intervals (and the consequential pulse rate variability measures), estimated using the proposed approaches, are in high agreement with their correspondents from electrocardiography.

Using the multiresolution ability of wavelets and effectiveness of singular value decomposition (SVD) to identify statistically robust parameters, we find a number of local and global features, capturing spectral correlations in the co- and cross-polarized channels, at different scales (of human breast tissues). The copolarized component, being sensitive to intrinsic fluorescence, shows different behavior for normal, benign, and cancerous tissues, in the emission domain of known fluorophores, whereas the perpendicular component, being more prone to the diffusive effect of scattering, points out differences in the Kernel-Smoother density estimate employed to the principal components, between malignant, normal, and benign tissues. The eigenvectors, corresponding to the dominant eigenvalues of the correlation matrix in SVD, also exhibit significant differences between the three tissue types, which clearly reflects the differences in the spectral correlation behavior. Interestingly, the most significant distinguishing feature manifests in the perpendicular component, corresponding to porphyrin emission range in the cancerous tissue. The fact that perpendicular component is strongly influenced by depolarization, and porphyrin emissions in cancerous tissue has been found to be strongly depolarized, may be the possible cause of the above observation.

Tissue dynamics spectroscopy uses digital holography as a coherence gate to extract depth-resolved quasi-elastic dynamic light scattering from inside multicellular tumor spheroids. The temporal speckle contrast provides endogenous dynamical images of proliferating and hypoxic or necrotic tissues. Fluctuation spectroscopy similar to diffusing wave spectroscopy is performed on the dynamic speckle to generate tissue-response spectrograms that track time-resolved changes in intracellular motility in response to environmental perturbations. The spectrograms consist of several frequency bands that range from 0.005 to 5 Hz. The fluctuation spectral density and temporal autocorrelations show the signature of constrained anomalous diffusion, but with large fluctuation amplitudes caused by active processes far from equilibrium. Differences in the tissue-response spectrograms between the proliferating outer shell and the hypoxic inner core differentiate normal from starved conditions. The differential spectrograms provide an initial library of tissue-response signatures to environmental conditions of temperature, osmolarity, pH, and serum growth factors.

In medical near-infrared spectroscopy (NIRS), movements of the subject often cause large step changes in the baselines of the measured light attenuation signals. This prevents comparison of hemoglobin concentration levels before and after movement. We present an accelerometer-based motion artifact removal (ABAMAR) algorithm for correcting such baseline motion artifacts (BMAs). ABAMAR can be easily adapted to various long-term monitoring applications of NIRS. We applied ABAMAR to NIRS data collected in 23 all-night sleep measurements and containing BMAs from involuntary movements during sleep. For reference, three NIRS researchers independently identified BMAs from the data. To determine whether the use of an accelerometer improves BMA detection accuracy, we compared ABAMAR to motion detection based on peaks in the moving standard deviation (SD) of NIRS data. The number of BMAs identified by ABAMAR was similar to the number detected by the humans, and 79% of the artifacts identified by ABAMAR were confirmed by at least two humans. While the moving SD of NIRS data could also be used for motion detection, on average 2 out of the 10 largest SD peaks in NIRS data each night occurred without the presence of movement. Thus, using an accelerometer improves BMA detection accuracy in NIRS.

Oral habits like chewing and smoking are main causes of oral cancer, which has a higher mortality rate than many other cancer forms. Currently, the long term survival rate of oral cancer is less than 50%, as a majority of cases are detected very late. The clinician's main challenge is to differentiate among a multitude of red, white, or ulcerated lesions. Hence, new noninvasive, reliable, and fast techniques for the discrimination of oral cavity disorders are to be developed. This study includes autofluorescence spectroscopic screening of normal volunteers with and without lifestyle oral habits and patients with oral submucous fibrosis (OSF). The spectra from different sites of habitués, non-habitués, and OSF patients were analyzed using the intensity ratio, redox ratio, and linear discriminant analysis (LDA). The spectral disparities among these groups are well demonstrated in the emission regions of collagen and Flavin adenine dinucleotide. We observed that LDA gives better efficiency of classification than the intensity ratio technique. Even the differentiation of habitués and non-habitués could be well established with LDA. The study concludes that the clinical application of autofluorescence spectroscopy along with LDA, yields spontaneous screening among individuals, facilitating better patient management for clinicians and better quality of life for patients.

In our previous studies, we have shown that the diffusing probe geometry can be used in conjunction with a two-layer diffusion model to accurately recover the absorption and scattering properties of skin in vivo. By modifying the original design to the diffusing probe with planar source (DPPS) geometry, we have also demonstrated that the efficiency of the accompanying multilayer diffusion model is comparable to that of a standard semi-infinite diffusion model; thus, precise quantification of superficial tissue optical properties in real time using a diffusion model becomes possible. In this study, the performance of the DPPS diffusion model is evaluated using Monte Carlo simulations and phantom measurements. It is found that the DPPS geometry is advantageous over the conventional planar source illumination geometry in interrogating superficial volumes of samples. In addition, our simulation results have shown that the DPPS geometry is capable of accurately recovering the optical properties of 50-μm thick epidermis and could be very useful in detecting cutaneous melanoma that has a radius as small as 250 μm.

Ambulatory near-infrared spectroscopy (aNIRS) enables recording of systemic or tissue-specific hemodynamics and oxygenation during a person's normal activities. It has particular potential for the diagnosis and management of health problems with unpredictable and transient hemodynamic symptoms, or medical conditions requiring continuous, long-duration monitoring. aNIRS is also needed in conditions where regular monitoring or imaging cannot be applied, including remote environments such as during spaceflight or at high altitude. One key to the successful application of aNIRS is reducing the impact of motion artifacts in aNIRS recordings. In this paper, we describe the development of a novel prototype aNIRS monitor, called NINscan, and our efforts to reduce motion artifacts in aNIRS monitoring. Powered by 2 AA size batteries and weighting 350 g, NINscan records NIRS, ECG, respiration, and acceleration for up to 14 h at a 250 Hz sampling rate. The system's performance and resistance to motion is demonstrated by long term quantitative phantom tests, Valsalva maneuver tests, and multiparameter monitoring during parabolic flight and high altitude hiking. To the best of our knowledge, this is the first report of multiparameter aNIRS monitoring and its application in parabolic flight.

While Raman spectroscopy provides a powerful tool for noninvasive and real time diagnostics of biological samples, its translation to the clinical setting has been impeded by the lack of robustness of spectroscopic calibration models and the size and cumbersome nature of conventional laboratory Raman systems. Linear multivariate calibration models employing full spectrum analysis are often misled by spurious correlations, such as system drift and covariations among constituents. In addition, such calibration schemes are prone to overfitting, especially in the presence of external interferences that may create nonlinearities in the spectra-concentration relationship. To address both of these issues we incorporate residue error plot-based wavelength selection and nonlinear support vector regression (SVR). Wavelength selection is used to eliminate uninformative regions of the spectrum, while SVR is used to model the curved effects such as those created by tissue turbidity and temperature fluctuations. Using glucose detection in tissue phantoms as a representative example, we show that even a substantial reduction in the number of wavelengths analyzed using SVR lead to calibration models of equivalent prediction accuracy as linear full spectrum analysis. Further, with clinical datasets obtained from human subject studies, we also demonstrate the prospective applicability of the selected wavelength subsets without sacrificing prediction accuracy, which has extensive implications for calibration maintenance and transfer. Additionally, such wavelength selection could substantially reduce the collection time of serial Raman acquisition systems. Given the reduced footprint of serial Raman systems in relation to conventional dispersive Raman spectrometers, we anticipate that the incorporation of wavelength selection in such hardware designs will enhance the possibility of miniaturized clinical systems for disease diagnosis in the near future.

We report on the use of diffuse optical spectroscopy analysis of breast spectra acquired in the wavelength range from 500 to 1600 nm with a fiber optic probe. A total of 102 ex vivo samples of five different breast tissue types, namely adipose, glandular, fibroadenoma, invasive carcinoma, and ductal carcinoma in situ from 52 patients were measured. A model deriving from the diffusion theory was applied to the measured spectra in order to extract clinically relevant parameters such as blood, water, lipid, and collagen volume fractions, β-carotene concentration, average vessels radius, reduced scattering amplitude, Mie slope, and Mie-to-total scattering fraction. Based on a classification and regression tree algorithm applied to the derived parameters, a sensitivity-specificity of 98%-99%, 84%-95%, 81%-98%, 91%-95%, and 83%-99% were obtained for discrimination of adipose, glandular, fibroadenoma, invasive carcinoma, and ductal carcinoma in situ, respectively; and a multiple classes overall diagnostic performance of 94%. Sensitivity-specificity values obtained for discriminating malignant from nonmalignant tissue were compared to existing reported studies by applying the different classification methods that were used in each of these studies. Furthermore, in these reported studies, either lipid or β-carotene was considered as adipose tissue precursors. We estimate both chromophore concentrations and demonstrate that lipid is a better discriminator for adipose tissue than β-carotene.

Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ∼0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.

Although glucocorticoids are frequently prescribed for the symptomatic management of inflammatory disorders such as rheumatoid arthritis, extended glucocorticoid exposure is the leading cause of physician-induced osteoporosis and leaves patients at a high risk of fracture. To study the biochemical effects of glucocorticoid exposure and how they might affect biomechanical properties of the bone, Raman spectra were acquired from ex vivo tibiae of glucocorticoid- and placebo-treated wild-type mice and a transgenic mouse model of rheumatoid arthritis. Statistically significant spectral differences were observed due to both treatment regimen and mouse genotype. These differences are attributed to changes in the overall bone mineral composition, as well as the degree of phosphate mineralization in tibial cortical bone. In addition, partial least squares regression was used to generate a Raman-based prediction of each tibia's biomechanical strength as quantified by a torsion test. The Raman-based predictions were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density. These results suggest that Raman spectroscopy could be a valuable tool for monitoring bone biochemistry in studies of bone diseases such as osteoporosis, including tests of drugs being developed to combat these diseases.

The aim is to study cerebral vascular functional connectivity during motor tasks and resting state using multichannel frequency-domain near-infrared spectrophotometry. Maps of 5.7 × 10.8 cm size displaying changes in cerebral oxyhemoglobin (O₂Hb), deoxyhemoglobin (HHb), and total hemoglobin (tHb) concentrations were measured in the motor cortex in 12 subjects (mean age of 28.8±12.7 yrs) during resting state and during two palm squeezing tasks with different timing. For each condition, phase plane plots, cross correlation functions, and connectivity indices were generated for O₂Hb, HHb, and tHb. The amplitude of the concentration changes in O₂Hb and HHb depends on the age of the subject. We found large regions of connectivity, which were similar for resting state and task conditions. This means the spatial relationships during resting state, when changes in O₂Hb, HHb, and tHb corresponded to spontaneous oscillations, were correlated to the spatial patterns during the activation tasks, when changes in O₂Hb, HHb, and tHb concentration were related to the alternation of stimulation and rest. Thus, the vascular functional connectivity was also present during resting state. The findings suggest that the vascular response to functional activation may be a nonlinear synchronization phenomenon and that resting state processes are more important than previously expected.

Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm² (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm² (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.

Gold nanoshells (GNs) are new materials that have an optical response dictated by the plasmon resonance. The wavelength at which the resonance occurs depends on the core and shell sizes. The purposes of this study were to use the combination of indocyanine green (ICG) and different concentration of gold nanoshells for skin tissue soldering and also to examine the effect of laser soldering parameters on the properties of repaired skin. Two mixtures of albumin solder and different combinations of ICG and gold nanoshells were prepared. A full thickness incision of 2 × 20 mm² was made on the surface and after addition of mixtures it was irradiated by an 810 nm diode laser at different power densities. The changes of tensile strength (σ_t) due to temperature rise, number of scan (Ns), and scan velocity (Vs) were investigated. The results showed at constant laser power density (I), σ_t of repaired incisions increases by increasing the concentration of gold nanoshells in solder, Ns, and decreasing Vs. It was demonstrated that laser soldering using combination of ICG + GNs could be practical provided the optothermal properties of the tissue are carefully optimized. Also, the tensile strength of soldered skin is higher than skins that soldered with only ICG or GNs. In our case, this corresponds to σ_t = 1800 g cm^-2 at I ∼ 47 Wcm−², T ∼ 85 ºC, Ns = 10, and Vs = 0.3 mms−¹.

The purpose of this study was to assess photodynamic antimicrobial chemotherapy (PACT) via irradiation, using a low power laser associated with a photosensitization dye, as an alternative to remove cariogenic microorganisms by drilling. Remaining dentinal samples in deep carious lesions on permanent molars (n = 26) were treated with 0.01% methylene blue dye and irradiated with a low power laser (InGaAIP - indium gallium aluminum phosphide; λ = 660 nm; 100 mW; 320 Jcm⁻²; 90 s; 9J). Samples of dentin from the pulpal wall region were collected with a micropunch before and immediately after PACT and kept in a transport medium for microbiological analysis. Samples were cultured in plates of Brucella blood agar, Mitis Salivarius Bacitracin agar and Rogosa SL agar to determine the total viable bacteria, mutans streptococci and Lactobacillus spp. counts, respectively. After incubation, colony-forming units were counted and microbial reduction was calculated for each group of bacteria. PACT led to statistically significant reductions in mutans streptococci (1.38 log), Lactobacillus spp. (0.93 log), and total viable bacteria (0.91 log). This therapy may be an appropriate approach for the treatment of deep carious lesions using minimally invasive procedures.