This PDF file contains the editorial “Optics in Neuroscience” for JBO Vol. 10 Issue 01

Developments in imaging technology now enable visualization of the functioning of individual ion channels in living cells: something previously possible only by the electrophysiological patch-clamp technique. We review techniques that track channel gating via changes in intracellular [Ca²⁺] resulting from openings of Ca²⁺-permeable channels. Spatial and temporal resolution are optimized by monitoring Ca²⁺ close to the channel mouth, and we describe the use of two imaging modalities: confocal laser scan microscopy (linescan CLSM) and total internal reflection fluorescence microscopy (TIRFM). Both currently achieve a kinetic resolution of <10 ms, provide a simultaneous and independent readout from many channels, and enable their locations to be mapped with submicrometer resolution. TIRFM provides 2-D images from a very thin (~100 nm) optical section, but it is restricted to channels in the plasma membrane of cells adhering close to a cover glass. In contrast, CLSM can image channels in intracellular membranes but, to achieve good temporal resolution, has been utilized only in a linescan mode with limited spatial information. We anticipate that imaging techniques will develop as a useful adjunct to patch-clamping for single-channel studies, with capabilities including simultaneous readout from multiple channels, high-resolution mapping of channel location, and mobility that is inaccessible by electrophysiological means. Optical single-channel recording is applicable to diverse voltage- and ligand-gated Ca²⁺-permeable channels and has potential for high-throughput functional analysis.

Optical stimulation techniques prove useful to map functional inputs in the in vitro brain slice preparation: Glutamate released by a focused beam of UV light induces action potentials, which can be detected in postsynaptic neurons. The direct activation effect is influenced by factors such as compound concentration, focus depth, light absorption in the tissue, and sensitivity of different neuronal domains. We analyze information derived from direct stimulation experiments in slices from rat barrel cortex and construct a computational model of a layer V pyramidal neuron that reproduces the experimental findings. The model predictions concerning the influence of focus depth on input maps and action potential generation are investigated further in subsequent experiments where the focus depth of a high-numerical-aperture lens is systematically varied. With our setup flashes from a xenon light source can activate neuronal compartments to a depth of 200 µm below the surface of the slice. The response amplitude is influenced both by tissue depth and focus plane. Specific somatodendritic structures can be targeted as the probability of action potential induction falls off exponentially with distance. Somata and primary apical dendrites are most sensitive to uncaged glutamate with locally increased sensitivity on proximal apical dendrites. We conclude that optical stimulation can be targeted with high precision

Cortical spreading depression (CSD) is a pronounced depolarization of neurons and glia that spreads slowly across the cortex followed by a period of depressed electrophysiological activity. The vascular changes associated with CSD are a large transient increase in blood flow followed by a prolonged decrease lasting greater than 1 h. Currently, the profile of functional vascular activity during this hypovolemic period has not been well characterized. Perfusion-based imaging techniques such as functional magnetic resonance imaging (fMRI) assume a tight coupling between changes in neuronal and vascular activity. Under normal conditions, these variables are well correlated. Characterizing the effect of CSD on this relationship is an important step to understand the impact acute pathophysiological events may have on neurovascular coupling. We examine the effect of CSD on functional changes in cerebral blood volume (CBV) evoked by cortical electrophysiological activity for 1 h following CSD induction. CBV signal amplitude, duration, and time to peak show little recovery at 60 min post-induction. Analysis of spontaneous vasomotor activity suggests a decrease in vascular reactivity may play a significant role in the disruption of normal functional CBV responses. Electrophysiological activity is also attenuated but to a lesser degree. CBV and evoked potentials are not well correlated following CSD, suggesting a breakdown of the neurovascular coupling relationship.

The spatial variation in reflectance such as the blood-vessel pattern can be observed in the image of cerebral cortex. This spatial variation is mainly caused by the difference in concentrations of oxy- and deoxyhemoglobin in the tissue. We analyze the reflectance spectra obtained from multispectral images of pig cortex by principal component analysis to extract information that relates to physiological parameters such as the concentrations of oxy- and deoxyhemoglobin and physical parameters such as mean optical path length. The light propagation in a model of exposed pig cortex is predicted by Monte Carlo simulation to estimate the interpretation of physiological and physical meanings of the principal components. The spatial variance of reflectance spectra of the pig cortex can be approximately described by the first principal component. The first principal component reflects the spectrum of hemoglobin in the cortical tissue multiplied by the mean optical path length. These results imply that the wavelength dependence of mean optical path length can be experimentally estimated from the first principal component of the reflectance spectra obtained from multispectral image of cortical tissue.

The ability of ultra-high-resolution optical coherence tomography (UHR OCT) to discriminate between healthy and pathological human brain tissue is examined by imaging ex vivo tissue morphology of various brain biopsies. Micrometer-scale OCT resolution (0.9×2 µm, axial×lateral) is achieved in biological tissue by interfacing a state-of-the-art Ti:Al₂O₃ laser (λ_c=800 nm, Δλ=260 nm, and P_out=120 mW exfiber) to a free-space OCT system utilizing dynamic focusing. UHR OCT images are acquired from both healthy brain tissue and various types of brain tumors including fibrous, athypical, and transitional meningioma and ganglioglioma. A comparison of the tomograms with standard hematoxylin and eosin (H&E) stained histological sections of the imaged biopsies demonstrates the ability of UHR OCT to visualize and identify morphological features such as microcalcifications (<20 µm), enlarged nuclei of tumor cells (~8 to 15 µm), small cysts, and blood vessels, which are characteristic of neuropathologies and normally absent in healthy brain tissue.

Alzheimer's disease (AD) is characterized by the presence of aggregates of the amyloid-β (Aβ) peptide in the brain. These aggregates manifest themselves as senile plaques and cerebrovascular amyloid angiopathy (CAA). While traditional histochemical approaches can easily identify these deposits in postmortem tissue, only recently have specific ligands been developed to target Aβ in living patients using positron emission tomography (PET). Successful detection of Aβ pathology in patients will enable definitive preclinical diagnosis of AD, and enable quantitative evaluation of the efficacy of anti-Aβ therapeutics developed to treat the disease. PET scanning, however, has several disadvantages including high cost, low availability, and the requirement for radioactive tracers. We describe recent progress in the development of techniques for imaging Aβ deposits noninvasively using optical approaches. Successful development of an optical detection platform would enable inexpensive, accessible, nonradioactive detection of the Aβ deposits found in AD.

We review our most recent results on near-IR studies of human brain activity, which have been evolving in two directions: detection of neuronal signals and measurements of functional hemodynamics. We discuss results obtained so far, describing in detail the techniques we developed for detecting neuronal activity, and presenting results of a study that, as we believe, confirms the feasibility of neuronal signal detection. We review our results on near-IR measurements of cerebral hemodynamics, which are performed simultaneously with functional magnetic resonance imaging (MRI) These results confirm the cerebral origin of hemodynamic signals measured by optical techniques on the surface of the head. We also show how near-IR methods can be used to study the underlying physiology of functional MRI signals.

A recent workshop brought together a mix of researchers with expertise in optical physics, cerebral hemodynamics, cognitive neuroscience, and developmental psychology to review the potential utility of near-IR spectroscopy (NIRS) for studies of brain activity underlying cognitive processing in human infants. We summarize the key findings that emerged from this workshop and outline the pros and cons of NIRS for studying the brain correlates of perceptual, cognitive, and language development in human infants.

The capacity to represent the world in terms of numerically distinct objects (i.e., object individuation) is a milestone in early cognitive development and forms the foundation for more complex thought and behavior. Over the past 10 to 15 yr, infant researchers have expended a great deal of effort to identify the origins and development of this capacity. In contrast, relatively little is known about the neural mechanisms that underlie the ability to individuate objects, in large part because there are a limited number of noninvasive techniques available to measure brain functioning in human infants. Recent research suggests that near-IR spectroscopy (NIRS), an optical imaging technique that uses relative changes in total hemoglobin concentration and oxygenation as an indicator of neural activation, may be a viable procedure for assessing the relation between object processing and brain function in human infants. We examine the extent to which increased neural activation, as measured by NIRS, could be observed in two neural areas known to be involved in object processing, the primary visual cortex and the inferior temporal cortex, during an object processing task. Infants aged 6.5 months are presented with a visual event in which two featurally distinct objects emerge successively to opposite sides of an occluder and neuroimaging data are collected. As predicted, increased neural activation is observed in both the primary visual and inferior cortex during the visual event, suggesting that these neural areas support object processing in the young infant. The outcome has important implications for research in cognitive development, developmental neuroscience, and optical imaging.

The neonatal rabbit brain shows prolonged postnatal development both structurally and physiologically. We use noninvasive near-IR frequency-domain optical spectroscopy (NIRS) and magnetic resonance imaging (MRI) to follow early developmental changes in cerebral oxygenation and anatomy, respectively. Four groups of animals are measured: NIRS in normals, MRI in normals, and both NIRS and MRI with hypoxia-ischemia (HI) (diffusion MRI staging). NIRS and/or MRI are performed from P3 (postnatal day=P) up to P76. NIRS is performed on awake animals with a frequency-domain tissue photometer. Absolute values of oxyhemoglobin concentration ([HbO₂]), deoxyhemoglobin concentration ([HbR]), total hemoglobin concentration (HbT), and hemoglobin saturation (StO₂) are calculated. The brains of all animals appeared to be maturing as shown in the diffusion tensor MRI. Mean optical coefficients (reduced scattering) remained unchanged in all animals throughout. StO2 increased in all animals (40% at P9 to 65% at P43) and there are no differences between normal, HI controls, and HI brains. The measured increase in StO2 is in agreement with the reported increase in blood flow during the first 2 months of life in rabbits. HbT, which reflects blood volume, peaked at postnatal day P17, as expected since the capillary density increases up to P17 when the microvasculature matures.

The letter-fluency task-induced response over the prefrontal cortex is investigated bilaterally on eight subjects using a recently developed compact, eight-channel, time-resolved, near-IR system. The cross-subject mean values of prefrontal cortex oxygen saturation (SO2) were 68.8±3.2% (right) and 71.0±3.6% (left), and of total hemoglobin concentration (tHb) were 69.6±9.6 µM (right) and 69.5±9.9 µM (left). The typical cortical activation response to the cognitive task [characterized by an increase in oxyhemoglobin (O₂Hb) with a concurrent decrease in deoxyhemoglobin (HHb)] at each measurement point is observed in only four subjects. In this subset, the amplitude of the O₂Hb increase and HHb decrease is uniform over each prefrontal cortex area and comparable between the two hemispheres. These findings agree with previous studies using continuous wave functional near-IR spectroscopy and functional magnetic resonance imaging, therefore demonstrating the potential of a time-resolved spectroscopy approach. In addition, a significant increase in SO₂ levels was observed in the right (1.1±0.5%) compared to left side of the prefrontal cortex (0.9±0.5%) (P=0.005). A different pattern of cortical activation (characterized by the lack of HHb decrease or even increased HHb) was observed in the remaining subjects.

Time domain (TD) diffuse optical measurement systems are being applied to neuroimaging, where they can detect hemodynamics changes associated with cerebral activity. We show that TD systems can provide better depth sensitivity than the more traditional continuous wave (CW) systems by gating late photons, which carry information about deep layers of the brain, and rejecting early light, which is sensitive to the superficial physiological signal clutter. We use an analytical model to estimate the contrast due to an activated region of the brain, the instrumental noise of the systems, and the background signal resulting from superficial physiological signal clutter. We study the contrast-to-noise ratio and the contrast-to-background ratio as a function of the activation depth and of the source-detector separation. We then present experimental results obtained with a time-gated instrument on the motor cortex during finger-tapping exercises. Both the model and the experimental results show a similar contrast-to-noise ratio for CW and TD, but that estimation of the contrast is experimentally limited by background fluctuations and that a better contrast-to-background ratio is obtained in the TD case. Finally, we use the time-gated measurements to resolve in depth the brain activation during the motor stimulus.

Diffuse optical imaging is an effective technique for noninvasive functional brain imaging. However, the measurements respond to systemic hemodynamic fluctuations caused by the cardiac cycle, respiration, and blood pressure, which may obscure or overwhelm the desired stimulus-evoked response. Previous work on this problem employed temporal filtering, estimation of systemic effects from background pixels, or modeling of interference signals with predefined basis functions, with some success. However, weak signals are still lost in the interference, and other complementary methods are desirable. We use the spatial behavior of measured baseline signals to identify the interference subspaces. We then project signals components in this subspace out of the stimulation data. In doing so, we assume that systemic interference components will be more global spatially, with higher energy, than the stimulus-evoked signals of interest. Thus, the eigenvectors corresponding to the largest eigenvalues of an appropriate correlation matrix form the basis for an interference subspace. By projecting the data onto the orthogonal nullspace of these eigenvectors, we can obtain more localized response, as reflected in improved contrast-to-noise ratio and correlation coefficient maps.

In near-IR spectroscopy, the concentration change in oxy- and deoxyhemoglobin in tissue is calculated from the change in the detected intensity of light at two wavelengths by solving the simultaneous equation based on the modified Lambert-Beer law. The wavelength-independent constant or mean optical path length is usually assigned to the term of partial optical path length in the simultaneous equation. This insufficient optical path length in the calculation causes crosstalk between the concentration change in oxy- and deoxyhemoglobin. We investigate the crosstalk in the dual-wavelength measurement of oxy- and deoxyhemoglobin theoretically by Monte Carlo simulation to discuss the optimal wavelength pair to minimize the crosstalk. The longer wavelength of the dual-wavelength measurement is fixed at 830 nm and the shorter wavelength is varied from 650 to 780 nm. The optimal wavelength range for pairing with 830 nm for the dual-wavelength measurement of oxy- and deoxyhemoglobin is from 690 to 750 nm. The mean optical path length, which can be obtained by time- and phase-resolved measurement, is effective to reduce the crosstalk in the results of dual-wavelength measurement.

It is often necessary to replace pit and fissure sealants and composite restorations. This task is complicated by the necessity for complete removal of the remaining composite to enable suitable adhesion of new composite. Previous studies have shown that 355-nm laser pulses from a frequency-tripled Nd:YAG laser can selectively remove residual composite after orthodontic bracket removal on enamel surfaces. Our objective is to determine if such laser pulses are suitable for selective removal of composite pit and fissure sealants and restorations. Optical coherence tomography is used to acquire optical cross sections of the occlusal topography nondestructively before sealant application, after sealant application, and after sealant removal. Thermocouples are used to monitor the temperature in the pulp chamber during composite removal under clinically relevant ablation rates, i.e., 30 Hz and 30 mJ/pulse. At an irradiation intensity of 1.3 J/cm², pit and fissure sealants are completely removed without visible damage to the underlying enamel. At intensities above 1.5 J/cm², incident laser pulses remove the resin layer while at the same time preferentially etching the surface of the enamel. Temperature excursions in the pulp chamber of extracted teeth are limited to less than 5°C if air-cooling is used during the rapid removal (1 to 2 min) of sealants, water-cooling is not necessary. Selective removal of composite restorative materials is possible without damage to the underlying sound tooth structure.

We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology.

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four β cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.

We evaluate Photofrin-mediated photodynamic therapy (PDT) in a phase 2 clinical trial as an adjuvant to surgery to treat peritoneal carcinomatosis. We extract tissue optical [reduced scattering (µ'_s), absorption (µ_a), and attenuation coefficients (µ_eff)] and physiological [blood oxygen saturation (%S_tO₂), total hemoglobin concentration (THC), and photosensitizer concentration (c_Photofrin)] properties in 12 patients using a diffuse reflectance instrument and algorithms based on the diffusion equation. Before PDT, in normal intraperitoneal tissues %S_tO₂ and THC ranged between 32 to 100% and 19 to 263 µM, respectively; corresponding data from tumor tissues ranged between 11 to 44% and 61 to 224 µM. Tumor %S_tO₂ is significantly lower than oxygenation of normal intraperitoneal tissues in the same patients. The mean (±standard error of mean) penetration depth (δ) in millimeters at 630 nm is 4.8(±0.6) for small bowel, 5.2 (±0.67) for large bowel, 3.39(±0.29) for peritoneum, 5.19(±1.4) for skin, 1.0(±0.1) for liver, and 3.02(±0.66) for tumor. c_Photofrin in micromolars is 4.9(±2.3) for small bowel, 4.8(±2.3) for large bowel, 3.0 (±1.0) for peritoneum, 2.5(±0.9) for skin, and 7.4(±2.8) for tumor. In all tissues examined, mean c_Photofrin tends to decrease after PDT, perhaps due to photobleaching. These results provide benchmark in-vivo tissue optical property data, and demonstrate the feasibility of in-situ measurements during clinical PDT treatments.

The oxygenation process of a human erythrocyte is monitored using a Raman microimaging technique. Raman images of the 1638 cm^–1 band are recorded in the oxygenated and deoxygenated state using only 120 s of laser exposure and ~1 mW of defocused laser power. The images show hemoglobin oxygenating and deoxygenating within the cell. Prolonged laser imaging exposure (<180 s) at low temperatures results in photoinduced and/or thermal degradation. The effect of thermal degradation is investigated by recording spectra of erythrocytes as a function of temperature between 4 and 52°C. Five bands at 1396, 1365, 1248, 972, and 662 cm^–1 are identified as markers for heme aggregation. Raman images recorded of cells after prolonged laser exposure appear to show heme aggregation commencing in the middle and moving toward the periphery of the cell. UV-visible spectra of erythrocytes show the Soret band to be broader and red shifted (~3 nm) at temperatures between 45 and 55° indicative of excitonic interactions. It is postulated that the enhancement of the aggregation marker bands observed at 632.8-nm excitation results primarily from excitonic interactions between the aggregated hemes in response to protein denaturation. The results have important medical implications in detecting and monitoring heme aggregation associated with hemopathies such as sickle cell disease.

Photothermal (PT) responses of individual intact cells are studied with a thermal lens dual-laser scheme. A multiparameter model for analysis of PT responses as a function of cell size, structure, and optical properties is suggested and verified experimentally for living cells, red blood cells, lymphocytes, tumor cells (K 562), hepatocytes, and miocytes, by applying pulsed laser radiation at 532 nm for 10-ns duration. PT responses for noninvasive and damaging modes of laser-cell interaction are investigated. It is shown theoretically and experimentally that specific optical and structural features of cells influence the polarity, shape, front, and tail lengths of their PT responses. Common for different cells, features of PT responses are evaluated. It is found that in cells with a highly heterogeneous light-absorbing structure, the PT response of a whole cell differs from that of the local absorbing area. The model suggested allows us to interpret PT responses from single cells and to compare cells in terms of their diameter, degree of spatial heterogeneity of light absorbance, and laser-induced damage thresholds.

A 3-D code for solving the set of Maxwell equations with the finite-difference time-domain method is developed for simulating the propagation and scattering of light in biological cells under realistic conditions. The numerical techniques employed in this code include the Yee algorithm, absorbing boundary conditions, the total field/scattered field formulation, the discrete Fourier transformation, and the near-to-far field transform using the equivalent electric and magnetic currents. The code is capable of simulating light scattering from any real cells with complex internal structure at all angles, including backward scattering. The features of the scattered light patterns in different situations are studied in detail with the objective of optimizing the performance of cell diagnostics employing cytometry. A strategy for determining the optimal angle for measuring side scattered light is suggested. It is shown that cells with slight differences in their intrastructure can be distinguished with two-parameter cytometry by measuring the side scattered light at optimal angles.

The influence of the synergy effects between organic and inorganic UV filter substances on the sun protection factor (SPF) of topically applied sunscreen formulations is investigated. The medium is considered to have reflection, absorption, and scattering properties. The distribution of photons in this medium is investigated by Monte Carlo calculation. Typical optical parameters of the skin and substances are used to characterize the synergy effect. The results of the model calculation are checked by in vitro and in vivo measurements investigating the influence of different types of scattering microparticles on the absorption efficacy of topically applied formulations. It is found that the inorganic filter substances act as scattering microparticles in the upper skin layers. They increase the optical pathway of the photons in the topically applied absorbing formulation also localized there. In this way, more photons are absorbed, increasing the SPF. The results obtained are important for the optimization of the SPF of sunscreen formulation containing organic and inorganic UV-filter components.

Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band.

We analyze and experimentally test the concept of laser polarization biotissue probing. The methods of increasing the SNR in coherent images of the optically anisotropic architectonics of the morphological biotissue structure are considered. The possibilities of polarization selection and contrasting of such images screened by other biotissues are examined. The influence of the depolarization degree of the scattered background on the SNR is investigated. The possibilities of polarization correction of the probing beam for contrasting biotissue images are analyzed.

Multispectral polarized light imaging (MSPLI) enables rapid inspection of a superficial tissue layer over large surfaces, but does not provide information on cellular microstructure. Confocal microscopy (CM) allows imaging within turbid media with resolution comparable to that of histology, but suffers from a small field of view. In practice, pathologists use microscopes at low and high power to view tumor margins and cell features, respectively. Therefore, we study the combination of CM and MSPLI for demarcation of nonmelanoma skin cancers. Freshly excised thick skin samples with nonmelanoma cancers are rapidly stained with either toluidine or methylene blue dyes, rinsed in acetic acid, and imaged using MSPLI and CM. MSPLI is performed at 630, 660, and 750 nm. The same specimens are imaged by reflectance CM at 630, 660, and 830 nm. Results indicate that CM and MSPLI images are in good correlation with histopathology. Cytological features are identified by CM, and tumor margins are delineated by MSPLI. A combination of MSPLI and CM appears to be complementary. This combined in situ technique has potential to guide cancer surgery more rapidly and at lower cost than conventional histopathology.

Anisotropy of mouse and human skin is investigated in vivo using polarized videoreflectometry. An incident beam (linearly polarized, wavelength 650 nm) is focused at the sample surface. Two types of tissuelike media are used as controls to verify the technique: isotropic delrin and highly anisotropic demineralized bone with a priori knowledge of preferential orientation of collagen fibers. Equi-intensity profiles of light, backscattered from the sample, are fitted with ellipses that appear to follow the orientation of the collagen fibers. The ratio of the ellipse semiaxes is well correlated with the ratio of reduced scattering coefficients obtained from radial intensity distributions. Variation of equi-intensity profiles with distance from the incident beam is analyzed for different initial polarization states of the light and the relative orientation of polarization filters for incident and backscattered light. For the anisotropic media (demineralized bone and human and mouse skin), a qualitative difference between intensity distributions for cross- and co-polarized orientations of the polarization analyzer is observed up to a distance of 1.5 to 2.5 mm from the entry point. The polarized videoreflectometry of the skin may be a useful tool to assess skin fibrosis resulting from radiation treatment.

Currently, measuring Raman spectra of tissues of living patients online and in real time, collecting the spectra in a very short measurement time, and allowing diagnosis immediately after the spectrum is recorded from any body region, are specific advantages that fiber optic near-infrared Raman spectroscopy (NIR RS) might represent for in vivo clinical applications in dermatology. We discuss various methodological aspects and state of the art of fiber optic NIR RS in clinical and experimental dermatology to outline its present advantages and disadvantages for measuring skin in vivo, particularly its water content. Fiber optic NIR Fourier transform (FT) RS has been introduced to dermatological diagnostics to obtain information regarding the molecular composition of the skin up to several hundred micrometers below the skin surface in a relatively fast nondestructive manner. This has been especially important for probing for in vivo assessment of cutaneous (intradermal) edema in patients patch test reactions. Fiber optic NIR FT Raman spectrometers still require further technological developments and optimization, extremely accurate water concentration determination and its intensity calculation in skin tissue, and for clinical applications, a reduction of measurement time and their size. Another promising option could be the possibility of applying mobile and compact fiber optic charge-coupled device (CCD)-based equipment in clinical dermatology.

Picosecond time-resolved Raman spectroscopy in equine cortical bone tissue is demonstrated. Using 400-nm pulsed laser excitation (1 ps at 1 kHz) it is shown that Kerr cell gating with a 4-ps window provides simultaneously time-resolved rejection of fluorescence and time-resolved Raman scatter enabling depth profiling through tissue. The Raman shifts are the same as those observed by conventional cw Raman spectroscopy using deep-red or near-infrared lasers. The time decay of Raman photons is shown to fit an inverse square root of time function, suggesting propagation by a diffusive mechanism. Using polystyrene behind a bone specimen, it is shown that the 400-nm laser light penetrates at least 0.31 mm below the surface of a fully mineralized bone tissue specimen and generates observable bone Raman scatter (approximately 415 to 430 nm) through most of this depth. These novel results demonstrate great promise for in vivo applications for studying diseased bone tissue, and ways to optimize the setup are discussed.

Collagenase treatment of cartilage serves as an in vitro model of the pathological collagen degradation that occurs in the disease osteoarthritis (OA). Fourier transform infrared imaging spectroscopic (FT-IRIS) analysis of collagenase-treated cartilage is performed to elucidate the molecular origin of the spectral changes previously found at the articular surface of human OA cartilage. Bovine cartilage explants are treated with 0.1% collagenase for 0, 15, or 30 min. In situ collagen cleavage is assessed using immunofluorescent staining with an antibody specific for broken type II collagen. The FT-IRIS analysis of the control and treated specimens mirrors the differences previously found between normal and OA cartilage using an infrared fiber optic probe (IFOP). With collagenase treatment, the amide II/1338 cm^–1 area ratio increases while the 1238 cm^–1/1227 cm^–1 peak ratio decreases. In addition, polarized FT-IRIS demonstrates a more random orientation of the collagen fibrils that correlate spatially with the immunofluorescent-determined regions of broken type II collagen. We can therefore conclude that the spectral changes observed in the collagenase-treated cartilage, and similarly in OA cartilage, arise from changes in collagen structure. These findings support the use of mid-infrared spectral analysis, in particular the minimally invasive IFOP, as potential techniques for the diagnosis and management of degenerative joint diseases such as osteoarthritis.

Analysis of time-of-flight (TOF) data is sometimes limited by the instrumental response function, and optical parameters are extracted from the observed response curve by several mathematical methods, such as deconvolution. In contrast to this, we demonstrate that a method using shifts of the peak time of the response curve with different source-detector separations can yield the average path length of the light traveling in a tissue-like sample without deconvolution. In addition, combining the intensity information allows us to separate the scattering and absorption coefficients. This simple method is more robust in signal-to-noise ratio than the moment analysis, which also does not require the deconvolution procedure, because the peak position is not significantly dependent on the baseline fluctuation and the contamination of the scattering. The analysis is demonstrated by TOF measurements of an Intralipid solution at 800 nm, and is applied to the measurements at 1.29 µm, where the temporal response of photomultiplier tubes is not sufficiently good.

This PDF file contains the editorial “Biophotonics” for JBO Vol. 10 Issue 01