We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

This is the first attempt devoted to the 1st–4th order statistic analysis of the complex degree of mutual polarization of biological tissues coherent images. The ensemble of diagnostic criteria (skewness and kurtosis of two-dimensional distributions of complex degree of mutual polarization) of pathological changes of muscular and connective (skin derma) tissues is defined.

We present a new quantitative method for investigating red blood cell morphology and dynamics. The instrument integrates quantitative phase microscopy with an inverted microscope, which makes it particularly suitable for the noninvasive assessment of live erythrocytes. In particular, we demonstrate the ability of this approach to quantify noninvasively cell volume and dynamic morphology. The subnanometer path-length sensitivity at the millisecond time scales is exemplified by measuring the hemoglobin flow out of the cell during hemolysis.

A continuous-wave (cw) near-infrared spectroscopy (NIRS) instrument has been developed to noninvasively quantify fetal cerebral blood oxygen saturation (StO₂). A linear Green's function formulism was used to analytically solve the photon diffusion equation and extract the time-varying fetal tissue oxy- and deoxy-hemoglobin concentrations from the NIR measurements. Here we explored the accuracy with which this instrument can be expected to perform over a range of fetal hypoxic states. We investigated the dependence of this accuracy on the accuracy of the reference optical properties chosen based on the literature. The fetal oxygenation of a pregnant ewe model was altered via maternal aortic occlusion. The NIR cw instrument was placed on the maternal abdomen directly above the fetal head, continuously acquiring diffuse optical measurements. Blood was sampled periodically from the fetus to obtain fetal arterial saturation (SaO₂) measurements from blood gas analysis. The NIR StO₂ values were compared with the fetal SaO₂ measurements. Variations in the NIR results due to uncertainty in the reference optical properties were relatively small within the fetal SaO₂ range of 30 to 80%. Under hypoxic conditions, however, the variability of the NIR StO₂ calculations with changes in the assumed reference properties became more significant.

Laser Doppler perfusion imaging (LDPI) enables superficial tissue perfusion assessment, but is sensitive to tissue motion not related to blood cells. The aim was to investigate if a polarization technique could reduce movement-induced artifacts. A linearly polarized laser and a cross-polarized filter, placed in front of the detectors, were used to block specular reflection. Measurements were performed with, and without, the polarization filter, at a single site during horizontal and vertical movement of skin tissue (index finger, twelve subjects, n=112) and of a flow model (n=432), with varying surface structures. Measurements were repeated during different flow conditions and at increased skin specular reflection. Statistical analysis was performed using ANOVA models. The perfusion signal was lower (p<0.001, skin and p<0.05, flow model) using the polarization filter, due to movement artifact reduction. No significant influence from surface structure was found when using the polarization filter. Movement artifacts were lower (p<0.05) in the vertical movement direction, however, depending on flow conditions for skin measurements. Increased skin specular reflection gave rise to large movement artifacts without the polarization filter. In conclusion, the polarized LDPI technique reduces movement artifacts and is particularly appropriate when assessing, e.g., ulcers and burns, where specular reflection is high.

A novel method for damage-free, artifact-free stimulation of neural tissue using pulsed, low-energy infrared laser light is presented. Optical stimulation elicits compound nerve and muscle potentials similar to responses obtained with conventional electrical neural stimulation in a rat sciatic nerve model. Stimulation and damage thresholds were determined as a function of wavelength using a tunable free electron laser source (λ=2 to 10 µm) and a solid state holmium:YAG laser (λ=2.12 µm). Threshold radiant exposure required for stimulation varies with wavelength from 0.312 J/cm² (λ=3 µm) to 1.22 J/cm² (λ=2.1 µm). Histological analysis indicates no discernable thermal damage with suprathreshold stimulation. The largest damage/stimulation threshold ratios (<6) were at wavelengths corresponding to valleys in the IR spectrum of soft tissue absorption (4 and 2.1 µm). Furthermore, optical stimulation can be used to generate a spatially selective response in small fascicles of the sciatic nerve that has significant advantages (e.g., noncontact, spatial resolution, lack of stimulation artifact) over conventional electrical methods in diagnostic and therapeutic procedures in neuroscience, neurology, and neurosurgery.

Fourier domain (FD) techniques have increasingly gained attention in optical coherence tomography (OCT). This is primarily due to their demonstrated sensitivity of two to three orders of magnitude over conventional time-domain techniques. FDOCT images are subject to two primary sources of artifacts. First, a complex conjugate ambiguity arises because the Fourier transform of the real-valued spectral interferometric signal is Hermitian symmetric. This ambiguity leads to artifactual superposition of reflectors at positive and negative pathlength differences between the sample and reference reflectors. Second, noninterferometric and sample autocorrelation terms appear at dc, obscuring reflectors at zero pathlength difference. We show that heterodyne detection in swept-source OCT (SSOCT) enables the resolution of complex conjugate ambiguity and the removal of noninterferometric and autocorrelation artifacts. We also show that complex conjugate ambiguity resolution via frequency shifting circumvents falloff induced by finite source linewidth in SSOCT when samples are shifted to large pathlength differences. We describe an efficient heterodyne SSOCT design that enables compensation of power losses from frequency-shifting elements. Last, we demonstrate this technique, coupled with wavenumber triggering and electronic demodulation, for in vivo imaging of the human anterior eye segment.

Nanoparticle phosphors made of lanthanide oxides are a promising new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long luminescence lifetime, and good photostability. We demonstrate the application of these nanoparticles to the visualization of protein micropatterns. Luminescent europium-doped gadolinium oxide (Eu:Gd₂O₃) nanoparticles are synthesized by spray pyrolysis. The size distribution is from 5 to 200 nm. The particles are characterized by means of laser-induced fluorescent spectroscopy and transmission electron microscopy (TEM). The main emission peak is at 612 nm. The nanoparticles are coated with avidin through physical adsorption. biotinylated bovine serum albumin (BSA-b) is patterned on a silicon wafer using a microcontact printing technique. The wafer is then incubated in a solution of avidin-coated nanoparticles. Fluorescent microscopic images reveal that the nanoparticles are organized onto designated area, as defined by the microcontact printing process. The luminescent nanoparticles do not suffer photobleaching during the observation, which demonstrates their suitability as luminescent labels for fluorescence microscopy studies. More detailed studies are preformed using atomic-force microscopy (AFM) at a single nanoparticle level. The specific and the nonspecific binding densities of the particles are qualitatively evaluated.

Fluorescence imaging of tissues has gained significant attention in recent years due to the emergence of appropriate reporter technologies that enable noninvasive sensing of molecular function in vivo. Two major approaches have been used so far for fluorescence molecular imaging, i.e., epi-illumination (reflectance) imaging and fluorescence molecular tomography. Transillumination is an alternative approach that has been employed for imaging tissues in the past and could be similarly beneficial for fluorescence molecular imaging. We investigate data normalization schemes in reflectance and transillumination mode and experimentally demonstrate that normalized transillumination offers significant advantages over planar reflectance imaging and over nonnormalized methods. Our observations, based on phantoms and on postmortem and in vivo mouse measurements display image quality improvement, superior depth sensitivity, and improved imaging accuracy over the nonnormalized methods examined. Normalized planar imaging retains implementation simplicity and could be used to improve on standard fluorescence reflectance imaging and as a simplified alternative to the more integrated and accurate tomographic methods.

We try a new approach with near-IR time-resolved spectroscopy, to separate optical signals originated in the upper layer from those in the lower layer and to selectively determine the absorption coefficient (µ_a) of each layer in a two-layered turbid medium. The difference curve in the temporal profiles of light attenuation between a target and a reference medium is divided into segments along the time axis, and a slope of each segment is calculated to determine the depth-dependent µ_a. The depth-dependent µ_a values are estimated under various conditions in which µ_a and the reduced scattering coefficient (µ′_s) of each layer are changed with a Monte Carlo simulation and in phantom experiments. Temporal variation of them represents the difference in µ_a between two layers when µ′_s of a reference is the same as that of the upper layer of the target. The discrepancies between calculated µ_a and the real µ_a depend on the ratio of the real µ_a of the upper layer to that of the lower layer, and our approach enables us to estimate the ratio of µ_a between the two layers. These results suggest the potential that µ_a of the lower layer can be determined by our procedure.

We developed a spectral technique that is independent of the light transport modality (diffusive or nondiffusive) to separate optical changes in scattering and absorption in the cat's brain due to the hemodynamic signal following visual stimulation. We observe changes in oxyhemoglobin and deoxyhemoglobin concentration signals during visual stimulation reminiscent of the functional magnetic resonance imaging (fMRI) blood oxygenation level dependence (BOLD) effect. Repeated measurements at different locations show that the observed changes are local rather than global. We also determine that there is an apparent large decrease in the water concentration and scattering coefficient during stimulation. We model the apparent change in water concentration on the separation of the optical signal from two tissue compartments. One opaque compartment is featureless (black), due to relatively large blood vessels. The other compartment is the rest of the tissue. When blood flow increases due to stimulation, the opaque compartment increases in volume, resulting in an overall decrease of tissue transmission. This increase in baseline absorption changes the apparent relative proportion of all tissue components. However, due to physiological effects, the deoxyhemoglobin is exchanged with oxyhemoglobin resulting in an overall increase in the oxyhemoglobin signal, which is the only component that shows an apparent increase during stimulation.

Diagnosis of brain edema is important to manage severe head injuries and diseases, but there is no method of noninvasive bedside monitoring of brain edema. We speculate that brain edema changes the optical properties of brain tissue. To verify this speculation, we perform transcranial measurement of diffuse light reflectance from cold-injured brains in rats. To induce edema, a liquid nitrogen-cooled copper probe of 3 mm diameter was applied to the right parietal bone for 60 s under anesthesia. The rat skull was irradiated with 633-nm HeNe laser light and 532-nm Nd:YVO₄ laser light, which were used to investigate changes in light scattering and blood flow, respectively. Reflectance intensity was measured as functions of time and position on the skull. We found that reflectance intensities were significantly decreased around the cold injury at both 633 and 532 nm, while reflectance was increased in the cold injury at 532 nm, indicating that cerebral blood volume was decreased in the cold injury. Under the condition of decreased cerebral blood volume, the decrease in reflectance intensity around the injury suggests that the scattering coefficient of brain tissue was reduced due to edema formation in this area.

We perform measurement of photoacoustic (PA) signals for burned skin in rats in the spectral range of 500 to 650 nm. The wavelength dependence of PA signal amplitude shows characteristics similar to those of the absorption spectrum of hemoglobin, suggesting that the PA signal originates from blood in the uninjured skin tissue under the injured tissue layer. High-contrast signals are obtained in the spectral range of 532 to 580 nm. At 550 nm, a PA detector is scanned on the wounds and PA tomograms are obtained. The tomograms clearly show the zones of stasis, demonstrating that a 2-D PA measurement is useful for burn depth assessment.

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkin's disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.

The surface stress on the real shape (biconcave disklike) of an erythrocyte under laser irradiation is theoretically studied according to the finite-difference time-domain (FDTD) method. The distribution of the surface stresses depends on the orientation of erythrocytes in the laser beam. Typically when the erythrocyte was irradiated from the side direction (the laser beam was perpendicular to the normal of the erythrocyte plane), the surface stresses were so asymmetrical and nonuniform that the magnitude of the surface stress on the back surface was three times higher than that on the front surface, and the highest-to-lowest ratio of the stress reached 16 times. For comparison, the surface stress was also calculated according to the ray optics (RO) method. The tendency of the stress distribution from the RO calculation was roughly similar to that of the FDTD method. However the RO calculation produced some unphysical results, such as the infinite stress on some surface region and the zero stress on the most parts of the erythrocyte surface, which is due to the neglecting of light diffraction. The results obtained from the FDTD calculation are believed quantitatively reliable, because the FDTD method automatically takes into account of the diffraction and interference effects of the light wave. Thus, the FDTD method is more suitable than the RO method for the stress study of erythrocytes.

Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE.

Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA₃) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images.

Despite advances in the measurement of lymphatic function, little is known about the actual velocities of flow in microlymphatic (~100 µm diam) vessels. In this work, video microscopy and particle tracking methods are adapted and integrated with an ultra-high-speed imaging camera to obtain measurements of lymph velocities throughout the entire lymphatic contraction cycle in the ratmesentery, something that previous systems were incapable of measuring. To determine the system's accuracy, calibration experiments are conducted across the hypothesized physiologically significant range of velocities for microlymphatic flow (up to 15 mm/sec). The system shows high accuracy, less than 2% error, when comparing actual with measured velocities. Microspheres flowing through 140-µm-diam tubing are imaged to demonstrate the system's ability to determine flow rates in these small vessels by measuring particle velocities. To demonstrate biological applicability, mesenteric microlymphatics in loops of the small intestine of three male Sprague-Dawley rats are exteriorized and imaged with the high-speed system at a rate of 500 frames/sec for several contraction sequences. Lymph velocity fluctuates cyclically with the vessel wall contractions, ranging from –1 to 7 mm/sec. These rates are higher than would be possible with standard video microscopy (3.75 mm/sec maximum).

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.

A 2-D finite element model of the cornea is developed to simulate corneal reshaping and the resulting deformation induced by refractive surgery. In the numerical simulations, linear and nonlinear elastic models are applied when stiffness inhomogeneities varying with depth are considered. Multiple simulations are created that employ different geometric configurations for the removal of the corneal tissue. Side-by-side comparisons of the different constitutive laws are also performed. To facilitate the comparison, the material property constants are identified from the same experimental data, which are obtained from mechanical tests on corneal strips and membrane inflation experiments. We then validate the resulting models by comparing computed refractive power changes with clinical results. Tissue deformations created by simulated corneal tissue removal using finite elements are consistent with clinically observed postsurgical results. The model developed provides a much more predictable refractive outcome when the stiffness inhomogeneities of the cornea and nonlinearities of the deformations are included in the simulations. Finite element analysis is a useful tool for modeling surgical effects on the cornea and developing a better understanding of the biomechanics of the cornea. The creation of patient-specific simulations would allow surgical outcomes to be predicted based on individualized finite element models.

The complex refractive index of highly concentrated hemoglobin solutions as they appear in red blood cells are determined in the wavelength range of 250 to 1100 nm using transmittance and Fresnel reflectance measurements. The determined real parts of the refractive indices are on average 0.02 units higher than the values found in the literature. The wavelength dependence of the measured data in the UV region differs from the calculated data using the Kramers-Kronig relation.

We describe a study of the discrimination of early melanoma from common and dysplastic nevus using fiber optic diffuse reflectance spectroscopy. Diffuse reflectance spectra in the wavelength range 550 to 1000 nm are obtained using 400-µm core multimode fibers arranged in a six-illumination-around-one-collection geometry with a single fiber-fiber spacing of 470 µm. Spectra are collected at specific locations on 120 pigmented lesions selected by clinicians as possible melanoma, including 64 histopathologically diagnosed as melanoma. These locations are carried through to the histopathological diagnosis, permitting a spatially localized comparison with the corresponding spectrum. The variations in spectra between groups of lesions with different diagnoses are examined and reduced to features suitable for discriminant analysis. A classifier distinguishing between benign and malignant lesions performs with sensitivity/specificity of between 64/69% and 72/78%. Classifiers between pairs of the group common nevus, dysplastic nevus, in situ melanoma, and invasive melanoma show better or similar performance than the benign/malignant classifier, and analysis provides evidence that different spectral features are needed for each pair of groups. This indicates that multiple discriminant systems are likely to be required to distinguish between melanoma and similar lesions.

Studies of basal cell carcinoma using terahertz pulsed imaging have revealed a significant difference between regions of tumor and healthy tissue. These differences are manifested in the reflected pulse due to what is thought to be changes in refractive index and absorption. We present measurements of the refractive index and absorption coefficient of excised normal tissue and basal cell carcinoma using terahertz (THz) transmission spectroscopy. We extract Debye parameters from these data and enter them into a finite difference time domain simulation to predict the shape of the waveforms reflected off the normal tissue and basal cell carcinoma and compare them with published in vivo data. Simulating the interaction of terahertz radiation with normal and cancerous tissue is a key step toward understanding the origin of contrast in terahertz images of skin cancer.

The selective retina treatment (SRT) targets retinal diseases associated with disorders in the retinal pigment epithelium (RPE). Due to the ophthalmoscopic invisibility of the laser-induced RPE effects, we investigate a noninvasive optoacoustic real-time dosimetry system. In vitro porcine RPE is irradiated with a Nd:YLF laser (527 nm, 1.7-µs pulse duration, 5 to 40 µJ, 30 pulses, 100-Hz repetition rate). Generated acoustic transients are measured with a piezoelectric transducer. During 27 patient treatments, the acoustic transients are measured with a transducer embedded in an ophthalmic contact lens. After treatment, RPE damage is visualized by fluorescein angiographic leakage. Below the RPE damage threshold, the optoacoustic transients show no pulse-to-pulse fluctuations within a laser pulse train. Above threshold, fluctuations of the individual transients among each other are observed. If optoacoustic pulse-to-pulse fluctuations are present, RPE leakage is observed in fluorescein angiography. In 96% of the irradiated areas, RPE leakage correlated with the optoacoustic defined threshold value. A noninvasive optoacoustic real-time dosimetry for SRT is developed and proved in vitro and during patient treatment. It detects the ophthalmoscopically invisible laser-induced damage of RPE cells and overcomes practical limitations of SRT for use in private practice.

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

A numerical algorithm based on a small-angle approximation of the radiative transfer equation (RTE) is developed to reconstruct scattering characteristics of biological tissues from optical coherence tomography (OCT) images. According to the algorithm, biological tissue is considered to be a layered random medium with a set of scattering parameters in each layer: total scattering coefficient, variance of a small-angle scattering phase function, and probability of backscattering, which fully describe the OCT signal behavior versus probing depth. The reconstruction of the scattering parameters is performed by their variation to fit the experimental OCT signal by the theoretical one using a time-saving genetic algorithm. The proposed reconstruction procedure is tested on model media with known scattering parameters. The possibility to estimate scattering parameters from OCT images is studied for various regimes of OCT signal decay. The developed algorithm is applied to reconstruct optical characteristics of epithelium and stroma for normal cervical tissue and its pathologies, and the potential to distinguish between the types of pathological changes in epithelial tissue by its OCT images is demonstrated.

We investigate the statistical polarization parameters of biological tissue histological section images with different morphological structure. First we outline the results of polarization coordinate mapping and analysis of the statistics of the first to fourth orders of biological tissue image polarization azimuth and ellipticities. Second, we study the statistics of the first to fourth orders of coordinate distributions of the complex degree of mutual polarization (CDMP) of biological tissue images. Finally, we consider the diagnostic possibilities of investigating 2-D distributions of CDMP of images that correspond to physiologically normal and degeneratively and/or dystrophycally changed biological tissues that are being analyzed.

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors.

Evaluation of molar dental restorations on enamel is performed using optical coherence tomography (OCT) with 10 µm resolution. Images of ~50 µm failure gaps in the restorations are demonstrated and the OCT images are compared with x-ray and optical microscopy pictures. The results demonstrate the potential of the technique for clinical evaluation of dental restorations.

Increasing evidence points to the beneficial effects of carotenoid antioxidants in the human body. Several studies, for example, support the protective role of lutein and zeaxanthin in the prevention of age-related eye diseases. If present in high concentrations in the macular region of the retina, lutein and zeaxanthin provide pigmentation in this most light sensitive retinal spot, and as a result of light filtering and/or antioxidant action, delay the onset of macular degeneration with increasing age. Other carotenoids, such as lycopene and beta-carotene, play an important role as well in the protection of skin from UV and short-wavelength visible radiation. Lutein and lycopene may also have protective function for cardiovascular health, and lycopene may play a role in the prevention of prostate cancer. Motivated by the growing importance of carotenoids in health and disease, and recognizing the lack of any accepted noninvasive technology for the detection of carotenoids in living human tissue, we explore resonance Raman spectroscopy as a novel approach for noninvasive, laser optical carotenoid detection. We review the main results achieved recently with the Raman detection approach. Initially we applied the method to the detection of macular carotenoid pigments, and more recently to the detection of carotenoids in human skin and mucosal tissues. Using skin carotenoid Raman instruments, we measure the carotenoid response from the stratum corneum layer of the palm of the hand for a population of 1375 subjects and develope a portable skin Raman scanner for field studies. These experiments reveal that carotenoids are a good indicator of antioxidant status.

Damage by pulsed lasers to the retina or other tissues containing strongly absorbing particles may occur through biophysical mechanisms other than simple heating. Shockwaves and bubbles have been observed experimentally, and depending on pulse duration, may be the cause of retinal damage at threshold fluence levels. We perform detailed calculations on the shockwave and bubble generation expected from pulsed lasers. For a variety of different laser pulse durations and fluences, we tabulate the expected strength of the shockwave and size of the bubble that will be generated. We also explain how these results will change for absorbing particles with different physical properties such as absorption coefficient, bulk modulus, or thermal expansion coefficient. This enables the assessment of biological danger, and possible medical benefits, for lasers of a wide range of pulse durations and energies, incident on tissues with absorbing particles with a variety of thermomechanical characteristics.

Noninvasive imaging techniques might be of particular diagnostic value for studying and monitoring cutaneous inflammatory conditions such as contact dermatitis. We evaluate acute allergic contact dermatitis (AACD) by means of optical coherence tomography (OCT) and correlate the clinical grading of patch test reactions with the findings obtained from OCT. Twenty positive patch test reactions (+,n=6;++,n=7;+++,n=7) are investigated using a conventional OCT scanner. In comparison to the control sites, OCT of AACD showed pronounced skin folds, thickened and/or disrupted entrance signals, and a significant increase in epidermal thickness. Moreover, clearly demarcated signal-free cavities within the epidermis and considerable reduction of dermal reflectivity are demonstrated by OCT. Notably, the latter findings strongly correlate with the clinical patch test grading. OCT may be a useful tool for visualization of micromorphological features of AACD. However, before OCT can be employed as an objective parameter in grading severity of patch test reactions, larger studies are required that correlate clinical patch test readings and OCT findings with histopathology.

A novel sensor fabrication concept using indicators trapped in nanoengineered ultrathin films deposited on fluorescent nanoparticles is demonstrated for oxygen sensing. The nanoscale systems are based on the quenching of tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) immobilized within polyelectrolyte multilayers deposited on the surface of nanoparticle templates. The ionically bound nanofilms create a porous scaffold into which controlled precipitation of the chromophore is achieved using a combination of electrostatic attraction and dye insolubility in water. The fluorescent nanoparticles act as physical scaffolds and also provide a complementary spectral signature for use as an internal intensity reference. Oxygen sensors created on 100-nm yellow-green fluorescent particles exhibit a linear Stern-Volmer behavior with a quenching constant of 1.06/mM and sensitivity of 60%, which demonstrates that the dye is still partially accessible to oxygen following immobilization. To demonstrate the feasibility of intracellular metabolic monitoring with such nanoprobes, the oxygen sensors were chemically delivered into human dermal fibroblasts with no apparent loss in cell viability. The results prove that the approach to sensor production is facile and leads to sensitive systems that can be further optimized for improved response, and these findings support the further development of similar self-referenced probes toward quantitative intracellular analysis.

Using both experimental and theoretical methods, we examine the contribution of different parts of the head to near-IR (NIR) signal. Time-resolved spectroscopy is employed to measure the mean optical path length (PL), and the absorption (µ_a) and reduced scattering (µ′_s) coefficients in multiple positions of the human head. Monte Carlo simulations are performed on four-layered head models based on an individual magnetic resonance imaging (MRI) scan to determine µ_a and µ′_s in each layer of the head by solving inverse problems, and to estimate the partial path length in the brain (p-PL) and the spatial sensitivity to regions in the brain at the source-detector separation of 30 mm. The PL is closely related to the thickness of the scalp, but not to that of other layers of the head. The p-PL is negatively related to the PL and its contribution ratio to the PL is 5 to 22% when the differential path length factor is 6. Most of the signal attributed to the brain comes from the upper 1 to 2 mm of the cortical surface. These results indicate that the NIR signal is very sensitive to hemodynamic changes associated with functional brain activation in the case that changes in the extracerebral tissue are ignorable.

Ultrahigh resolution optical coherence tomography (OCT) is an emerging imaging modality that enables noninvasive imaging of tissue with 1- to 3-µm resolutions. Initial OCT studies have typically been performed using harvested tissue specimens (ex vivo). No reports have investigated postexcision tissue degradation on OCT image quality. We investigate the effects of formalin fixation and commonly used cell culture media on tissue optical scattering characteristics in OCT images at different times postexcision compared to in vivo conditions. OCT imaging at 800-nm wavelength with 1.5-µm axial resolution is used to image the hamster cheek pouch in vivo, followed by excision and imaging during preservation in phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Media (DMEM), and 10% neutral-buffered formalin. Imaging is performed in vivo and at sequential time points postexcision from 15 min to 10 to 18 h. Formalin fixation results in increases in scattering intensity from the muscle layers, as well as shrinkage of the epithelium, muscle, and connective tissue of ~50%. PBS preservation shows loss of optical contrast within two hours, occurring predominantly in deep muscle and connective tissue. DMEM maintains tissue structure and optical scattering characteristics close to in vivo conditions up to 4 to 6 h after excision and best preserved tissue optical properties when compared to in vivo imaging.

Speckle in optical coherence tomography (OCT) images originates in the high spatial coherence of incident light that enables interference of light backscattered from spatially heterogenous tissue specimens. We report results of a numerical simulation and an experiment to test speckle reduction using a partially spatially coherent source. A Gaussian-Schell model for a partially spatially coherent source is used in the OCT simulation. For the experiment, such a source was generated by a spatially coherent boardband light source and a multimode fiber. The advantage of using a multimode fiber in combination with a broadband source is the large number of photons per coherence volume. To illustrate speckle reduction with a partially spatially coherent source, we record low-coherence interferograms of a scattering surface using single-mode and multimode source fibers. Interferograms recorded using a single-mode source fiber are indicative of those observed using conventional OCT. Speckle in OCT images recorded using a multimode source fiber is substantially reduced.

Many optical diagnostic approaches rely on changes in scattering and absorption properties to generate optical contrast between normal and diseased tissue. Recently, there has been increasing interest in using exogenous agents to enhance this intrinsic contrast with particular emphasis on the development for targeting specific molecular features of disease. Gold nanoshells are a class of core-shell nanoparticles with an extremely tunable peak optical resonance ranging from the near-UV to the mid-IR wavelengths. Using current chemistries, nanoshells of a wide variety of core and shell sizes can easily be fabricated to scatter and/or absorb light with optical cross sections often several times larger than the geometric cross section. Using gold nanoshells of different size and optical parameters, we employ Monte Carlo models to predict the effect of varying concentrations of nanoshells on tissue reflectance. The models demonstrate the importance of absorption from the nanoshells on remitted signals even when the optical extinction is dominated by scattering. Furthermore, because of the strong optical response of nanoshells, a considerable change in reflectance is observed with only a very small concentration of nanoshells. Characterizing the optical behavior of gold nanoshells in tissue will aid in developing nanoshells as contrast agents for optical diagnostics.

We demonstrate experimentally the possibility of reproducing the phase function, absorption, and scattering coefficients of a real biological tissue (adult brain white matter and liver) using a suspension of polystyrene microspheres with a fractal size distribution. The design of a light scattering goniometer with a cylindrical cell in air is discussed, and phase function measurements using the device are described. The scattering coefficient is measured using transmission spectrophotometry and the absorption and reduced scattering coefficients are measured using a time-resolved method. A good match between real tissue and phantom parameters is demonstrated.

Recently there has been a strong demand to protect human skin against negative effects of the UV solar light. This problem is interesting due to the increased frequency of human diseases caused by such radiation. We aim to evaluate how the optical properties of the horny layer of skin can be effectively changed by imbedding TiO₂ fine particles to achieve the maximal attenuation of the UV solar radiation. In-depth distribution of TiO₂ particles embedded into the skin by multiple administration of sunscreens is determined experimentally using the tape-stripping technique. A computer code implementing the Monte Carlo method is developed to simulate photon migration within the 20-µm-thick horny layer filled with nanosized TiO₂ spheres, 25 to 200 nm in diameter. Dependencies of the UV radiation of two wavelengths (310 and 400 nm) absorbed by and totally reflected from, as well as transmitted through the horny layer on the size of TiO₂ particles are obtained and analyzed. The most attenuating particles are found to be 62 and 122 nm in diameter for 310- and 400-nm light, respectively. The former could be suggested as the main fraction to be used in sunscreens to prevent erythema.

A scheme for fast 3-D laser scanning using acousto-optic deflectors is proposed and demonstrated. By employing counterpropagating acoustic waves that are both chirped and offset in their frequencies, we show that it is possible to simultaneously scan both axially and laterally with frame rates on the order of tens of kilohertz. This scheme was specifically designed for application with multiphoton imaging, particularly of neurons, where it will enable the concurrent monitoring of physiological signals at multiple locations within a microscopic 3-D volume (350×350×200 µm). When used for this purpose, we demonstrate how this scheme would also inherently compensate for spatial dispersion when ultrafast laser pulses are used in acousto-optic multiphoton microscopy.

Near-infrared measurements of glucose in human plasma are performed using a custom, rapid, high-throughput filter-based spectrometer covering a spectral range between 2080 and 2315 nm. Quality of the measured glucose signals is quantified through the use of two figures of merit: selectivity and limit of detection. Selectivity measures the uniqueness of the glucose spectrum from among the interfering spectra. Limit of detection measures the smallest glucose concentration change detectable. The proposed system, which includes the spectroscopic hardware and a spectral preprocessing algorithm, is shown to produce a selectivity value of 0.57, with zero being nonselective and one being fully selective, and a limit of detection value of 2.2 mM. Prediction of an independent dataset is also performed using net analyte signal-based and partial least-squares multivariate calibration techniques, which produce standard error of prediction values of 1.14 and 1.45 mM, respectively.

This PDF file contains the errata for “JBO Vol. 10 Issue 06 Paper 2147351” for JBO Vol. 10 Issue 06

This PDF file contains the errata for “JBO Vol. 10 Issue 06 Paper 2167067” for JBO Vol. 10 Issue 06

This PDF file contains the editorial “Book Review: Handbook of Coherent Domain Optical Methods: Biomedical Diagnostics, Environmental and Material Science, Volumes 1 and 2” for JBO Vol. 10 Issue 06