The development of miniaturized nonlinear optical microscopy or endoscopy is essential to complement the current imaging modalities for diagnosis and monitoring of cancers. We report on a nonlinear optical endoscope based on a double-clad photonic crystal fiber and a two-dimensional (2-D) microelectromechanical system mirror, enabling the three-dimensional (3-D) nonlinear optical imaging through in vitro gastrointestinal tract tissue and human breast cancer tissue with a penetration depth of approximately 100 μm and axial resolution of 10 μm. The 3-D high-resolution and high-sensitive imaging ability of the nonlinear optical endoscope facilitates the visualization of 3-D morphologic and cell nuclei arrangement within tissue, and therefore will be important for histopathologic interpretation without the need of tissue excision.

This PDF file contains the editorial “Optical Coherence Tomography in Ophthalmology” for JBO Vol. 12 Issue 04

A feasibility study of ultrahigh-resolution full-field optical coherence tomography (FF-OCT) for a subcellular-level imaging of human donor corneas is presented. The FF-OCT system employed in this experiment is based on a white light interference microscope, where the sample is illuminated by a thermal light source and a horizontal cross-sectional (en face) image is detected using a charge coupled device (CCD) camera. A conventional four-frame phase-shift detection technique is employed to extract the interferometric image from the CCD output. A 95-nm-broadband full-field illumination yields an axial resolution of 2.0 μm, and the system covers an area of 850 μm×850 μm with a transverse resolution of 2.4 μm using a 0.3-NA microscope objective and a CCD camera with 512×512 pixels. Starting a measurement from the epithelial to the endothelial side, a series of en face images was obtained. From detected en face images, the epithelial cells, Bowman's layer, stromal keratocyte, nerve fiber, Descemet's membrane, and endothelial cell were clearly observed. Keratocyte cytoplasm, its nuclei, and its processes were also separately detected. Two-dimensional interconnectivity of the keratocytes is visualized, and the keratocytes existing between collagen lamellaes are separately extracted by exploiting a high axial resolution ability of FF-OCT.

The use of high-resolution optical coherence tomography (OCT) to visualize penetration kinetics during the initial phase of chemical eye burns is evaluated. The changes in scattering properties and thickness of rabbit cornea ex vivo were monitored after topical application of different corrosives by time-resolved OCT imaging. Eye burn causes changes in the corneal microstructure due to chemical interaction or change in the hydration state as a result of osmotic imbalance. These changes compromise the corneal transparency. The associated increase in light scattering within the cornea is observed with high spatial and temporal resolution. Parameters affecting the severity of pathophysiological damage associated with chemical eye burns like diffusion velocity and depth of penetration are obtained. We demonstrate the potential of high-resolution OCT for the visualization and direct noninvasive measurement of specific interaction of chemicals with the eye. This work opens new horizons in clinical evaluation of chemical eye burns, eye irritation testing, and product testing for chemical and pharmacological products.

Objective imaging of the optic nerve structure has become central to the management of patients with glaucoma. There is an urgent need in diagnosis and staging for reliable objective precursors and markers. Three-dimensional ultrahigh-resolution frequency domain optical coherence tomography (3D UHR OCT) holds particular promise in this respect since it enables volumetric assessment of intraretinal layers including tomographic data for the retinal nerve fiber layer (RNFL) and optic nerve head. The integrated analysis of this information and the resolution advantage has enabled the development of more informative indices of axonal damage in glaucoma compared with measurements of RNFL thickness and cup-to-disc ratio provided by commercial OCT devices. The potential for UHR OCT in enabling the combined analysis of tomographic and volumetric data on retinal structure is explored. A novel parameter was developed; the three-dimensional minimal distance as the optical correlate of true retinal nerve fiber layer thickness around the optic nerve head region. For the purposes of this pilot study, we present data from a normal subject and from two patients with characteristic optic nerve and retinal nerve fiber layer changes secondary to glaucoma.

Accurate wavelength assignment of each spectral element for spectral-domain optical coherence tomography (SD-OCT) and optical frequency domain imaging (OFDI) is required for proper construction of biological tissue cross-sectional images. This becomes more critical for functional extensions of these techniques, especially in polarization-sensitive optical coherence tomography (PS-OCT), where incorrect wavelength assignment between the two orthogonal polarization channels leads to polarization artifacts. We present an autocalibration method for wavelength assignment that does not require separate calibration measurements and that can be applied directly on actual data. Removal of the birefringence artifact is demonstrated in a PS-OCT system with picometer accuracy in the relative wavelength assignment, resulting in a residual phase error of 0.25 deg/100 μm. We also demonstrate, for the first time, a quantitative birefringence map of an in vivo human retinal nerve fiber layer.

Recent developments in Fourier domain—optical coherence tomography (Fd-OCT) have increased the acquisition speed of current ophthalmic Fd-OCT instruments sufficiently to allow the acquisition of volumetric data sets of human retinas in a clinical setting. The large size and three-dimensional (3D) nature of these data sets require that intelligent data processing, visualization, and analysis tools are used to take full advantage of the available information. Therefore, we have combined methods from volume visualization, and data analysis in support of better visualization and diagnosis of Fd-OCT retinal volumes. Custom-designed 3D visualization and analysis software is used to view retinal volumes reconstructed from registered B-scans. We use a support vector machine (SVM) to perform semiautomatic segmentation of retinal layers and structures for subsequent analysis including a comparison of measured layer thicknesses. We have modified the SVM to gracefully handle OCT speckle noise by treating it as a characteristic of the volumetric data. Our software has been tested successfully in clinical settings for its efficacy in assessing 3D retinal structures in healthy as well as diseased cases. Our tool facilitates diagnosis and treatment monitoring of retinal diseases.

We present a computationally efficient, semiautomated method for analysis of posterior retinal layers in three-dimensional (3-D) images obtained by spectral optical coherence tomography (SOCT). The method consists of two steps: segmentation of posterior retinal layers and analysis of their thickness and distance from an outer retinal contour (ORC), which is introduced to approximate the normal position of external interface of the healthy retinal pigment epithelium (RPE). The algorithm is shown to effectively segment posterior retina by classifying every pixel in the SOCT tomogram using the similarity of its surroundings to a reference set of model pixels from user-selected area(s). Operator intervention is required to assess the quality of segmentation. Thickness and distance maps from the segmented layers and their analysis are presented for healthy and pathological retinas.

Optical coherence tomography (OCT) has already proven an important clinical tool for imaging and diagnosing retinal diseases. Concerning the standard commercial ophthalmic OCT systems, speckle noise is a limiting factor with respect to resolving relevant retinal features. We demonstrate successful suppression of speckle noise from mutually aligning a series of in vivo OCT recordings obtained from the same retinal target using the Stratus system from Humphrey-Zeiss. Our registration technique is able to account for the axial movements experienced during recording as well as small transverse movements of the scan line from one scan to the next. The algorithm is based on a regularized shortest path formulation for a directed graph on a map formed by interimage (B-scan) correlations. The resulting image enhancement typically increases the contrast-to-noise ratio (CNR) with a factor of three or more and facilitates segmentation and quantitative characterization of pathologies. The method is currently successfully being applied by medical doctors in a number of specific retinal case studies.

The development of improved segmentation algorithms for more consistently accurate detection of retinal boundaries is a potentially useful solution to the limitations of existing optical coherence tomography (OCT) software. We modeled artifacts related to operator errors that may normally occur during OCT imaging and evaluated their influence on segmentation results using a novel segmentation algorithm. These artifacts included: defocusing, depolarization, decentration, and a combination of defocusing and depolarization. Mean relative reflectance and average thickness of the automatically extracted intraretinal layers was then measured. Our results show that defocusing and depolarization errors together have the greatest altering effect on all measurements and on segmentation accuracy. A marked decrease in mean relative reflectance and average thickness was observed due to depolarization artifact in all intraretinal layers, while defocus resulted in a less-marked decrease. Decentration resulted in a marked but not significant change in average thickness. Our study demonstrates that care must be taken for good-quality imaging when measurements of intraretinal layers using the novel algorithm are planned in future studies. An awareness of these pitfalls and their possible solutions is crucial for obtaining a better quantitative analysis of clinically relevant features of retinal pathology.

In previous publications we have reported on polarization-sensitive optical coherence tomography (PS-OCT) systems that measure and image retardation and axis orientation of birefringent samples with only a single input polarization state. This method requires that the sample is illuminated by circularly polarized light. In the case of retinal imaging, the retina is measured through the birefringent cornea, which causes a deviation of the sampling beam from the circular polarization state. To obtain undistorted birefringence patterns of the retina by PS-OCT, the corneal birefringence has to be compensated. We report on a software-based corneal birefringence compensation that uses the polarization state of the light backscattered at the retinal surface to measure the corneal birefringence. This information is used to numerically compensate the corneal birefringence. Contrary to hardware-based solutions, our method accounts for local variations of the corneal birefringence. We implemented the method in a state of the art spectral domain PS-OCT system and demonstrate it in a test sample and human retina in vivo.

Frequency domain optical coherence tomography (FD-OCT), based on an all-reflective high-speed InGaAs spectrometer, operating in the 1050 nm wavelength region for retinal diagnostics, enables high-speed, volumetric imaging of retinal pathologies with greater penetration into choroidal tissue is compared to conventional 800 nm three-dimensional (3-D) ophthalmic FD-OCT systems. Furthermore, the lower scattering at this wavelength significantly improves imaging performance in cataract patients, thereby widening the clinical applicability of ophthalmic OCT. The clinical performance of two spectrometer-based ophthalmic 3-D OCT systems compared in respect to their clinical performance, one operating at 800 nm with 150 nm bandwidth (~3 μm effective axial resolution) and the other at 1050 nm with 70 nm bandwidth (~7 μm effective axial resolution). Results achieved with 3-D OCT at 1050 nm reveal, for the first time, decisive improvements in image quality for patients with retinal pathologies and clinically significant cataract.

We use Fourier domain optical coherence tomography (OCT) data to assess retinal blood oxygen saturation. Three-dimensional disk-centered retinal tissue volumes were assessed in 17 normal healthy subjects. After removing DC and low-frequency a-scan components, an OCT fundus image was created by integrating total reflectance into a single reflectance value. Thirty fringe patterns were sampled; 10 each from the edge of an artery, adjacent tissue, and the edge of a vein, respectively. A-scans were recalculated, zeroing the DC term in the power spectrum, and used for analysis. Optical density ratios (ODRs) were calculated as ODR_Art=ln(Tissue₈₅₅/Art₈₅₅)/ln(Tissue₈₀₅/Art₈₀₅) and ODRVein=ln(Tissue₈₅₅/Vein₈₅₅)/ln(Tissue₈₀₅/Vein₈₀₅) with Tissue, Art, and Vein representing total a-scan reflectance at the 805- or 855-nm centered bandwidth. Arterial and venous ODRs were compared by the Wilcoxon signed rank test. Arterial ODRs were significantly greater than venous ODRs (1.007±2.611 and -1.434±4.310, respectively; p=0.0217) (mean±standard deviation). A difference between arterial and venous blood saturation was detected. This suggests that retinal oximetry may possibly be added as a metabolic measurement in structural imaging devices.

Resonant Doppler Fourier domain optical coherence tomography (FDOCT) is a functional imaging tool for extracting tissue flow. The method is based on the effect of interference fringe blurring in spectrometer-based FDOCT, where the path difference between structure and reference changes during camera integration. If the reference path length is changed in resonance with the Doppler frequency of the sample flow, the signals of resting structures will be suppressed, whereas the signals of blood flow are enhanced. This allows for an easy extraction of vascularization structure. Conventional flow velocity analysis extracts only the axial flow component, which strongly depends on the orientation of the vessel with respect to the incident light. We introduce an algorithm to extract the vessel geometry within the 3-D data volume. The algorithm calculates the angular correction according to the local gradients of the vessel orientations. We apply the algorithm on a measured 3-D resonant Doppler dataset. For validation of the reproducibility, we compare two independently obtained 3-D flow maps of the same volunteer and region.

Investigation of the autoregulatory mechanism of human retinal perfusion is conducted with a real-time spectral domain Doppler optical coherence tomography (SDOCT) system. Volumetric, time-sequential, and Doppler flow imaging are performed in the inferior arcade region on normal healthy subjects breathing normal room air and 100% oxygen. The real-time Doppler SDOCT system displays fully processed, high-resolution [512 (axial)×1000 (lateral) pixels] B scans at 17 frames/sec in volumetric and time-sequential imaging modes, and also displays fully processed overlaid color Doppler flow images comprising 512 (axial)×500 (lateral) pixels at 6 frames/sec. Data acquired following 5 min of 100% oxygen inhalation is compared with that acquired 5 min postinhalation for four healthy subjects. The average vessel constriction across the population is -16±26% after oxygen inhalation with a dilation of 36±54% after a return to room air. The flow decreases by -6±20% in response to oxygen and in turn increases by 21±28% as flow returns to normal in response to room air. These trends are in agreement with those previously reported using laser Doppler velocimetry to study retinal vessel autoregulation. Doppler flow repeatability data are presented to address the high standard deviations in the measurements.

There is considerable interest in new methods for the assessment of retinal blood flow for the diagnosis of eye diseases. We present in vivo normal human volumetric retinal flow measurement using Fourier domain Doppler optical coherence tomography. We used a dual-plane scanning pattern to determine the angle between the blood flow and the scanning beam in order to measure total flow velocity. Volumetric flow in each blood vessel around the optic nerve head was integrated in one cardiac cycle in each measurement. Measurements were performed in the right eye of one human subject. The measured venous flow velocity ranged from 16.26 mm/s to 29.7 mm/s. The arterial flow velocity ranged from 38.35 mm/s to 51.13 mm/s. The total retinal venous and arterial flow both added up to approximately 54 µl/min. We believe this is the first demonstration of total retinal blood flow measurement using the OCT technique.

Nonlinear optical phenomena, such as two-photon fluorescence (2PF) and second harmonic generation (SHG), in combination with voltage sensitive dyes, can be used to acquire high-resolution spatio temporal maps of electrical activity in excitable cells and tissue. Developments in 1064-nm fiber laser technology have simplified the generation of high-intensity, long-wavelength, femtosecond light pulses, capable of penetrating deep into tissue.To merge these two advances requires the design and synthesis of new dyes that are optimized for longer wavelengths and that produce fast and sensitive responses to membrane potential changes. In this work, we have systematically screened a series of new dyes with varying chromophores and sidechains that anchor them in cell membranes. We discovered several dyes that could potentially be used for in vivo measurements of cellular electrical activity because of their rapid and sensitive responses to membrane potential. Some of these dyes show optimal activity for SHG; others for 2PF. This regulated approach to dye screening also allows significant insight into the molecular mechanisms behind both SHG and 2PF. In particular, the differing patterns of sensitivity and kinetics for these two nonlinear optical modalities indicate that their voltage sensitivity originates from differing mechanisms.

Characteristic changes in the organization of fibrillar collagen can potentially serve as an early diagnostic marker in various pathological processes. Tissue types containing collagen I can be probed by pulsed high-intensity laser radiation, thereby generating second harmonic light that provides information about the composition and structure at a microscopic level. A technique was developed to determine the essential second harmonic generation (SHG) parameters in a laser scanning microscope setup. A rat-tail tendon frozen section was rotated in the xy-plane with the pulsed laser light propagating along the z-axis. By analyzing the generated second harmonic light in the forward direction with parallel and crossed polarizer relative to the polarization of the excitation laser beam, the second-order nonlinear optical susceptibilities of the collagen fiber were determined. Systematic variations in SHG response between ordered and less ordered structures were recorded and evaluated. A 500μm-thick z-cut lithiumniobate (LiNbO3) was used as reference. The method was applied on frozen sections of malignant melanoma and normal skin tissue. Significant differences were found in the values of d₂₂, indicating that this parameter has a potential role in differentiating between normal and pathological processes.

We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations.

In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage.

Cardiovascular disease is the primary cause of death in the United States; the majority of these deaths are caused by the rupture of vulnerable plaques. An important feature of vulnerable plaques is the thickness of the fibrous cap that covers the necrotic core. A thickness of less than 65 μm has been proposed as a value that renders the plaque prone to rupture. This work shows that multiphoton microscopy (MPM) can image the plaque with µm resolution to a depth deeper than 65 μm. The fibrous cap emits primarily second harmonic generation due to collagen, in contrast to the necrotic core and healthy artery, which emits primarily two-photon excited fluorescence from elastin. This gives a good demarcation of the fibrous cap from underlying layers, facilitating the measurement of the fibrous cap thickness. Based on a measure of the collagen/elastin ratio, plaques were detected with a sensitivity of 65% and specificity of 81%. Furthermore, the technique gives detailed information on the structure of the collagen network in the fibrous cap. This network ultimately determines the mechanical strength of the plaque. A mechanical model based on this information could yield a measure of the propensity of the plaque to rupture.

We present a multimodal optical microscope that incorporates six imaging modalities on one common platform. The imaging modalities include three staring modes, optical quadrature microscopy (OQM), differential interference contrast (DIC) microscopy, and epi-fluorescence microscopy, and three scanning modes, confocal reflectance microscopy (CRM), confocal fluorescence microscopy (CFM), and two-photon microscopy (2PM). OQM reconstructs the amplitude and phase of an optically transparent specimen within a modified Mach-Zehnder configuration. DIC microscopy images the phase gradient along a specified direction of an optically transparent specimen. CRM detects index of refraction changes that modulate backscatter. Epi-fluorescence microscopy, CFM, and 2PM detect endogenous and exogenous fluorophores within a specimen. The scanning modes are inherently capable of producing three-dimensional (3-D) images due to optical sectioning and localized probing. Illumination and imaging are performed coaxially with minimal changes of optical components between modes. Multimodal images of embryos are shown to demonstrate the microscope's imaging capabilities.

Optical coherence tomography (OCT) based on spectral interferometry has recently been examined, with authors often suggesting superior performance compared with time domain approaches. The technologies have similar resolutions and the spectral techniques may currently claim faster acquisition rates. Contrary to many current opinions, their detection parameters may be inferior. The dynamic range and signal-to-noise ratio (SNR) correlate with image penetration, the contrast as a function of depth. This work examines the theoretical sensitivity, dynamic range, and SNR of the techniques, within the practical limits of optoelectronics, taking into account often ignored or misunderstood classical factors that affect performance, such as low frequency noise, analog to digital (AD) conversion losses, and methods for potentially improving sensitivity, including fast laser sweeping. The technologies are compared relative to these parameters. While Fourier domain OCT has some advantages such as signal integration, it appears unlikely that its disadvantages can ultimately be overcome for nontransparent tissue. Ultimately, time-domain (TD)-OCT appears to have the superior performance with respect to SNR and dynamic range. This may not be the case for transparent tissue of the eye. Certain positive aspects of swept source OCT leave the possibility open that its performance may approach that of (TD)-OCT in nontransparent tissue.

We present spectral domain phase microscopy (SDPM) as a new tool for measurements at the cellular scale. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity in real time. Our goal was to use SDPM to investigate the mechanical properties of the cytoskeleton of MCF-7 cells. Magnetic tweezers were designed to apply a vertical force to ligand-coated magnetic beads attached to integrin receptors on the cell surfaces. SDPM was used to resolve cell surface motions induced by the applied stresses. The cytoskeletal response to an applied force is shown for both normal cells and those with compromised actin networks due to treatment with Cytochalasin D. The cell response data were fit to several models for cytoskeletal rheology, including one- and two-exponential mechanical models, as well as a power law. Finally, we correlated displacement measurements to physical characteristics of individual cells to better compare properties across many cells, reducing the coefficient of variation of extracted model parameters by up to 50%.

We describe the possibility of using a microresonance Raman spectrometer combined with a microfluidic system and optical tweezers to study Escherichia coli (E. coli) overexpressing wild type (wt) neuroglobin (NGB) and its E7Leu mutant, respectively. NGB is a recently discovered heme protein and its function still is a matter of debate. So far, the protein has been studied in its purified form, and in vivo measurements on the single cell level could give more information. To study the feasibility of the combined techniques, the possibilities of the setup are investigated by taking spectra from single cells and clusters of cells. We find that the microresonance Raman technique enables studies of the wt NGB protein in a living cell under fluctuating aerobic and anaerobic conditions. E. coli cells overexpressing wt NGB are stable, and the reversible oxygenation-deoxygenation can be studied over a long period of time. Further, the experiment indicates the presence of an enzymatic system in the bacteria reducing the ferric form NGB. The study of E. coli cells overexpressing E7Leu NGB, on the other hand, gives insight into limiting factors of the setup, such as cell lysis, photoinduced chemistry, and protein concentrations.

Attenuated total reflection Fourier transform infrared spectroscopic imaging was applied to study human stratum corneum (SC) tissue, the outermost layer of the skin. This imaging approach was combined with a controlled environment cell to demonstrate the possibility of obtaining chemical images of SC exposed to a wide range of relative humidities and diffusion of ethanol through the SC tissue with a specially designed liquid cell. The effect of water vapor sorbed into the SC on the distribution of other components in the SC was studied. Principal component analysis was applied in conjunction with univariate analysis to differentiate the distribution of different components in the SC. Swelling of the SC, a heterogeneous distribution of natural moisturizing factor and water, was detected upon the increase of relative humidity. The approach to image the penetration of liquid ethanol into the SC was also demonstrated and showed good potential and implications for studying transdermal drug delivery.

The present study focuses on enhancing the sensitivity and specificity of spectral diagnosis in a stratified architecture that models human cervical epithelia by experimentally demonstrating the efficacy of using angularly variable fiber geometry to achieve the desired layer selection and probing depths. The morphological and biochemical features of epithelial tissue vary in accordance with tissue depths; consequently, the accuracy of spectroscopic diagnosis of epithelial dysplasia may be enhanced by probing the optical properties of this tissue. In the case of cellular dysplasia, layer-specific changes in tissue optical properties may be optimally determined by reflectance spectroscopy when specifically coupled with angularly variable fiber geometry. This study addresses the utility of using such angularly variable fiber geometry for resolving spatially specific spectra of a two-layer epithelial tissue phantom. Spectral sensitivity to the scattering particles embedded in the epithelial phantom layer is shown to significantly improve as the obliquity of the collection fibers increases from 0 to 40 deg. Conversely, the orthogonal fibers are found to be more sensitive to changes in the stromal phantom layer.

A key component of accurate spectroscopic-based cancer diagnostics is the ability to differentiate spectral variations resulting from epithelial tissue dysplasia. Such measurement may be enhanced by discretely probing the optical properties of the epithelial tissue where the morphological and biochemical features vary according to tissue depths. More precisely, layer-specific changes in tissue optical properties correlated to cellular dysplasia can be determined by conventional reflectance spectroscopy when it is coupled with angularly variable fiber geometry. Thus, this study addresses how angularly variable fiber geometry can resolve spatially specific spectral signatures of tissue pathology by interpreting and analyzing the reflectance spectra of increasingly dysplastic epithelial tissue in reflectance-mode Monte Carlo simulation. Specifically, by increasing the obliquity of the collection fibers from 0 to 40 deg in the direction facing toward the illumination fiber, the spectral sensitivity to tissue abnormalities in the epithelial layer is thereby improved, whereas orthogonal fibers are more sensitive to the changes in the stromal layer.

The sun protection factor (SPF) describes the protective behavior of sunscreens insufficiently, because this factor takes into account only the UVB spectral range, and strains the volunteers during its determination by invasively invoking an erythema. A new noninvasive method is proposed that is based on the UV spectroscopic measurement of tape strips taken from a sunscreen-treated skin area. The resulting sum transmission spectra of the tape strips reflect the in-vivo distribution of the absorber on the skin and quantify the protective efficacy of the applied sunscreens over the complete UV spectral range. The spectroscopic data provide a basis for the calculation of a universal sun protection factor (USPF). The comparison of the concrete values of USPF and SPF results in the following statements. 1. An unique functional correlation is not to be expected because a different UVB / UVA dependence exists. 2. The size of the differences between both values is influenced clearly by the intensity relation of the average sum transmission in the UVB in comparison to the UVA range. 3. The USPF values objectively assess the efficacy of sunscreens considering a protection against all irradiation injuries.

The sensitivity of near-infrared spectroscopy (NIRS) to evoked brain activity is reduced by physiological interference in at least two locations: 1. the superficial scalp and skull layers, and 2. in brain tissue itself. These interferences are generally termed as "global interferences" or "systemic interferences," and arise from cardiac activity, respiration, and other homeostatic processes. We present a novel method for global interference reduction and real-time recovery of evoked brain activity, based on the combination of a multiseparation probe configuration and adaptive filtering. Monte Carlo simulations demonstrate that this method can be effective in reducing the global interference and recovering otherwise obscured evoked brain activity. We also demonstrate that the physiological interference in the superficial layers is the major component of global interference. Thus, a measurement of superficial layer hemodynamics (e.g., using a short source-detector separation) makes a good reference in adaptive interference cancellation. The adaptive-filtering-based algorithm is shown to be resistant to errors in source-detector position information as well as to errors in the differential pathlength factor (DPF). The technique can be performed in real time, an important feature required for applications such as brain activity localization, biofeedback, and potential neuroprosthetic devices.

A modified Monte Carlo method was used for numerical modeling of the propagation of near-infrared radiation (NIR) within the anatomical layers of the human head. The distribution of NIR transmission between particular anatomical layers in the measurement region (frontal tubers) of the head was obtained. The study demonstrates the effect of the cardiac pump function-dependent changes in the width of the subarachnoid space (SAS) on the intensity of the backscattered radiation. It was proved that the influence of this factor increases with increasing distance between the observation point and the location of the NIR source placed on the surface of the head. Moreover, with sufficiently small NIR detector-source distance, the contribution of the optic radiation propagated within the SAS to the total signal received is negligibly low, which gives a basis for estimation of the modulatory influence of blood circulation within the superficial skin layer on the total intensity of the backscattered radiation. The dimensions of anatomical layers used in the study are real values measured in a female patient, in whom-due to unique circumstances-it was possible to make measurements followed by recordings in clinical conditions, a situation essential for verification of the results of numerical modeling.

The study presents comparison of near-infrared light propagation and near-infrared backscattered radiation power, as simulated with numerical modeling and measured live in a patient in clinical conditions with the use of the near-infrared transillumination/backscattering sounding (NIR-T/BSS) technique. A unique chance for such precise comparative analysis was available to us in a clinical case of a female patient with scalp removed from one half of the head due to injury. The analysis performed indicates that the difference between the intensity of the signals in numerical modeling and live measurements is less than 4 dB. Analysis of the theoretical model also provides hints on the positioning of the two detectors relative to the source of radiation. Correctness of these predicted values is confirmed in practical application, when changes of signals received by the detectors are recorded, along with changes of the width of the subarachnoid space. What is more, the power distribution of the spectrum of near-infrared backscattered radiation returning to the detectors is confirmed in the real recording in the patient. An abridged description of the new method of NIR-T/BSS is presented.

Quantitative data on cell structure, shape, and size distribution are obtained by optical measurement of normal peripheral blood granulocytes and lymphocytes in a cell suspension. The cell nuclei are measured in situ. The distribution laws of the cell and nuclei sizes are estimated. The data gained are synthesized to construct morphometric models of a segmented neutrophilic granulocyte and a lymphocyte. Models of interrelation between the cell and nucleus metric characteristics for granulocyte and lymphocyte are obtained. The discovered interrelation decreases the amount of cell-nucleus size combinations that have to be considered under simulation of cell scattering patterns. It allows faster analysis of light scattering to discriminate cells in a real-time scale. Our morphometric data meet the requirements of scanning flow cytometry dealing with the high rate analysis of cells in suspension. Our findings can be used as input parameters for the solution of the direct and inverse light-scattering problems in scanning flow cytometry, dispensing with a costly and time-consuming immunophenotyping of the cells, as well as in turbidimetry and nephelometry. The cell models developed can ensure better interpretations of scattering patterns for an improvement of discriminating capabilities of immunophenotyping-free scanning flow cytometry.

Intraocular scattering can become an important source of optical degradation in the aging eye. To evaluate its relative contribution to the ocular modulation transfer function (MTF), a compact, dual experimental system comprising a laser ray tracing (LRT) wavefront sensor and a double-pass setup is used. An aberrometric MTF is estimated from aberration measurements, whereas a second MTF is derived from the double-pass point-spread function. While the former only accounts for the effect of aberrations (up to seventh order), the double-pass MTF includes the combined effect of both scattering and aberrations. A 532-nm laser light source is used to minimize choroidal scattering. Measurements are done on 19 normal, healthy eyes from three groups of subjects of different ages. The two MTFs are obtained for a 6-mm pupil diameter and partial refractive compensation. Intraocular scattering is modeled as a random wavefront aberration characterized by its variance and correlation length. These parameters are fitted from the differences between both MTFs. Our results show that double-pass and LRT techniques provide similar MTFs for most normal eyes, although small amounts of scattering, or high-order aberrations, could be measured in some eyes. A gradual increase in intraocular scattering with age is also observed.

Cardiac fluorescent optical imaging provides the unique opportunity to investigate the dynamics of propagating electrical waves during ventricular arrhythmias and the termination of arrhythmias by strong electric shocks. Panoramic imaging systems using charge-coupled device (CCD) cameras as the photodetector have been developed to overcome the inability to monitor electrical activity from the entire cardiac surface. Photodiode arrays (PDAs) are known to have higher temporal resolution and signal quality, but lower spatial resolution compared to CCD cameras. We construct a panoramic imaging system with three PDAs and image Langendorff perfused rabbit hearts (n=18) during normal sinus rhythm, epicardial pacing, and arrhythmias. The recorded spatiotemporal dynamics of electrical activity is texture mapped onto a reconstructed 3-D geometrical heart model specific to each heart studied. The PDA-based system provides sufficient spatial resolution (1.72 mm without interpolation) for the study of wavefront propagation in the rabbit heart. The reconstructed 3-D electrical activity provides us with a powerful tool to investigate the fundamental mechanisms of arrhythmia maintenance and termination.

Nanoparticles 100 nm in diameter containing indocyanine green (ICG) have been developed as a contrast agent for photoacoustic (PA) imaging based on (photonic explorers for biomedical use by biologically localized embedding PEBBLE) technology using organically modified silicate (ormosil) as a matrix. ICG is an FDA-approved dye with strong optical absorption in the near-infrared (NIR) region, where light can penetrate deepest into biological tissue. A photoacoustic imaging system was used to study image contrast as a function of PEBBLE concentration in phantom objects. ICG-embedded ormosil PEBBLEs showed improved stability in aqueous solution compared with free ICG dye. The particles were conjugated with HER-2 antibody for breast cancer and prostate cancer cell targeting. Initial in vitro characterization shows high contrast and high efficiency for binding to prostate cancer cells. ICG can also be used as a photosensitizer (generating toxic oxygen by illumination) for photodynamic therapy. We have measured the photosensitization capability of ICG-embedded ormosil nanoparticles. This feature can be utilized to combine detection and therapeutic functions in a single agent.

The intraoperative diagnosis of brain tumors and the timely evaluation of biomarkers that can guide therapy are hindered by the paucity of rapid adjunctive studies. This study evaluates the feasibility and specificity of using quantum dot-labeled antibodies for rapid visualization of epidermal growth factor receptor (EGFR) expression in human brain tumor cells and in surgical frozen section slides of glioma tissue. Streptavidin-coated quantum dots (QDs) were conjugated to anti-EGFR antibodies and incubated with target cultured tumor cells and tissues. The experiments were conducted first in human glioma tumor cell lines with elevated levels of EGFR expression (SKMG-3, U87) and then in frozen tissue sections of glioblastoma multiforme and of oligodendroglioma. The bioconjugated QDs used in the study were found to bind selectively to brain tumor cells expressing EGFR. QD complexed quickly to the cell membrane (less than 15 min), and binding was highly specific and depended on the expression level of EGFR on the cell membrane. Tissue experiments showed that only tumor specimens expressing EGFR were labeled in less than 30 min by QD complexes. These findings demonstrate that QD-labeled antibodies can provide a quick and accurate method for characterizing the presence or absence of a specific predictive biomarker.

The use of photonic bandgap fibers (PBGF) for biomedical sensing has been demonstrated. The demonstrated PBGF has a blue wavelength shift of 280 nm in the falling photonic bandgap edge (PBE) when the ambient refractive indices inside the holey region change from 1.333 to 1.39, which agrees well with the analytical prediction. Combining this with the knowledge of immobilization techniques and biorecognition elements could open up a new class of PBGF-based label-free biosensors. A sensitivity on the order of 0.1 nmol/L could be achieved by consuming less than 1 μL of sample.

Chip-based biosensor arrays for label-free and high-throughput detection were fabricated and tested. The sensor array was composed of a 150-nm-thick, 50-nm-gap, and 600-nm-period gold nanoslits. Each array size was 100 μm×100 μm. A transverse-magnetic polarized wave in these metallic nanostructures generated resonant surface plasmons at a wavelength of about 800 nm in a water environment. Using the resonant wavelength shift in the nanoslit array, we achieved detection sensitivity up to 668 nm per refractive index unit, about 1.7 times larger than that reported on an array of nanoholes. An antigen–antibody interaction experiment in an aqueous environment verified the sensitivity in a surface binding event.