To develop an accurate and convenient method for measuring the heart rate of zebra fish in vivo, a system combining fast differential interference contrast (DIC) imaging with an autocorrelation technique is established. The imaging correlation coefficient corr(i,j) between frame i, selected from the obtained time-lapse imaging series as the reference image, and any other frame j, is calculated as the time-dependent cycle course. Heat rate is determined by the cycle period of the corr with a high temporal resolution of 4 ms, achieved by fast charge-coupled device (CCD) imaging of 250 frames per second. With this high-resolution system, we find that 1-mg/L cadmium not only induces the slowing of the heart rate, but also caused signs of arrhythmia in treated fish.

We report the narrowest to-date (21 gauge, 820-μm-diam) handheld forward-imaging optical coherence tomography (OCT) needle endoscope and demonstrate its feasibility for ophthalmic OCT inspection. The probe design is based on paired-angle-rotation scanning (PARS), which enables a linear B-scan pattern in front of the probe tip by using two counterrotating angle polished gradient-index (GRIN) lenses. Despite its small size, the probe can provide a numerical apertune (NA) of 0.22 and an experimental sensitivity of 92 dB at 0.5 frame/s. The feasibility of retinal imaging is tested on enucleated ex vivo porcine eyes, where structural features including remnant vitreous humor, retina, and choroid can be clearly distinguished. Due to its imaging quality comparable to a commercial OCT system and compatibility with the current ophthalmic surgery standard, the probe can potentially serve as a better alternative to traditional visual inspection by white light illumination during vitreoretinal surgery (e.g., vitrectomy).

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images.

We apply ultrasound-modulated optical tomography (UOT) to image ex-vivo methylene-blue-dyed sentinel lymph nodes embedded in 3.2-cm-thick chicken breast tissues. The UOT system is implemented for the first time using ring-shaped light illumination, intense acoustic bursts, and charge-coupled device (CCD) camera-based speckle contrast detection. Since the system is noninvasive, nonionizing, portable, relatively cost effective, and easy to combine with photoacoustic imaging and single element ultrasonic pulse-echo imaging, UOT can potentially be a good imaging modality for the detection of sentinel lymph nodes in breast cancer staging in vivo.

This PDF file contains the editorial “Special Section Guest Editorial: Optical Clearing of Tissues and Cells” for JBO Vol. 13 Issue 02

Accelerated diffusion of water and hyperosmotic optical clearing agents is studied as a result of enhanced epidermal permeability. A lattice of microzones (islets) of damage in stratum corneum is induced using a flash-lamp appliqué system. An optical clearing agent composed of 88% glycerol in aqueous solution is used for all experiments. Research of skin dehydration and glycerol delivery through epidermis at both intact and perforated stratum corneum is presented. The dehydration process induced by both stimuli of evaporation and osmotic agent action is studied by weight measurements. Dynamics of refractive index alteration of both glycerol solution and water during their interaction with skin samples is monitored. The amounts of water escaping from skin through the stratum corneum, due to hyperosmotic-agent action, and glycerol penetrating through the skin sample, are estimated. The results show that the proposed method allows for effective transepidermal water loss and delivery of optical clearing agents.

Light scattering in biological tissues can be reduced by using optical clearing agents. Various physical methods in conjunction with agents have been studied to enhance the optical clearing efficacy of skin for diagnostic and therapeutic applications. In this study, we propose a new physical method to enhance the optical clearing potential of topically applied glycerol. A microneedle roller is used to easily create numerous transdermal microchannels prior to glycerol application. The optical clearing efficacy of skin is quantitatively evaluated with the use of a modulation transfer function target placed underneath ex vivo porcine skin samples. From cross-polarized images acquired at various time points after glycerol application, we find that samples treated with the microneedle roller resulted in an approximately two-fold increase in contrast compared to control samples 30 min after glycerol application. In conclusion, our data suggest that the microneedle roller can be a good physical method to enhance transdermal delivery of optical clearing agents, and hence their optical clearing potential over large regions of skin.

The optical clearing technique has shown great potential in improving light penetration into biotissues. Among various optical clearing agents (OCAs) under study, the hydroxyl-terminated agents induce the highest optical clearing effect of skin, but the exact mechanism of optical clearing is still unclear. In consideration of several probable factors, such as the number of hydroxyl groups, the refractive index, and the molecular weight, we investigate the optical clearing effect of porcine skin after applying six alcohols to the epidermis and saline to the dermis. The dynamical transmission intensity of porcine skin is monitored by an integrating sphere system, and the thickness of skin samples is measured before and after experiments. The results show that the transmittance of skin increases significantly, but there is no significant change in thickness after the treatment of OCAs. The optical clearing effect of skin induced by alcohols is related to the number of hydroxyl groups. The refractive index or molecular weight of optical clearing agents does not correlate with the degree of optical clearing effect for a 60-min time interval of measurement. However, the behavior of skin transmittance after 60 min needs to be further investigated.

By use of a Fourier transform infrared (FTIR) spectroscopic imaging technique, we examine the dynamic optical clearing processes occurring in hyperosmotically biocompatible agents penetrating into skin tissue in vitro. The sequential collection of images in a time series provides an opportunity to assess penetration kinetics of dimethyl sulphoxide (DMSO) and glycerol beneath the surface of skin tissue over time. From 2-D IR spectroscopic images and 3-D false color diagrams, we show that glycerol takes at least 30 min to finally penetrate the layer of epidermis, while DMSO can be detected in epidermis after only 4 min of being topically applied over stratum corneum sides of porcine skin. The results demonstrate the potential of a FTIR spectroscopic imaging technique as an analytical tool for the study of dynamic optical clearing effects when the bio-tissue is impregnated by hyperosmotically biocompatible agents such as glycerol and DMSO.

The tissue optical clearing technique shows great potential in optical diagnosis and therapy. However, short-term and long-term effects of optical clearing agents on blood vessels, which are relevant to the safety of clinical applications, have not been clarified. We used laser speckle contrast imaging to monitor the changes in blood vessels in chick chorioallantoic membrane (CAM) after application of glycerol or glucose. The changes in morphology of vessels and blood flow velocity were measured. Long-term effects on blood vessels were investigated by observing the function and the development of blood vessels. The results show that glycerol reduces the local blood flow velocity and constricts and even blocks vessels quickly. At 2 days, the blood flow velocity is recovered to different extents, and new blood vessels develop but are fewer in number. Glucose induces slow changes in blood flow or vessels. However, most blood vessels are blocked, and no new blood vessel develops at 2 days. The effects depend on the dosage of agents, including volume and concentration, and decrease with the dosage of agents. Therefore, short-term effects of glucose on blood vessels are slighter than those of glycerol, but long-term effects of glucose are greater.

Spectral domain optical coherence tomography (SDOCT) images have been used to investigate the mechanism of optical clearing in flowing blood using dextrans. The depth reflectivity profiles from SDOCT indicate that dextrans become increasingly more effective in reducing scattering in flowing blood, except for 5 mgdl^-1 of D×500, with increasing molecular weights (MW 70,000 and 500,000) and concentrations (0.6, 2, and 5 mgdl^-1). Among the tested dextrans, Dx500 at 2 mgdl^-1 had the most significant effect on light scattering reduction with the strongest capability to induce erythrocyte aggregation. D×500 at 5 mgdl^-1 contributes more refractive index matching but induces a decrease in aggregation that leads to the same level as 0.6 mgdl^-1 D×500. Previous studies identified various mechanisms of light scattering reduction in stationary blood induced by optical clearing agents. Our results suggest that erythrocyte aggregation is a more important mechanism for optical clearing in flowing blood using dextrans, providing a rational design basis for effective flowing blood optical clearing, which is essential for improving OCT imaging capability through flowing blood.

The effect of optical clearing with glycerol on the Raman spectra of bone tissue acquired transcutaneously on right and left tibiae from four mice is studied. Multiple transcutaneous measurements are obtained from each limb; glycerol is then applied as an optical clearing agent, and additional transcutaneous measurements are taken. Glycerol reduces the noise in the raw spectra (p=0.0037) and significantly improves the cross-correlation between the recovered bone factor and the exposed bone measurement in a low signal-to-noise region of the bone spectra (p=0.0245).

We have investigated the mechanisms and capabilities of optical clearing in conjunction with second harmonic generation (SHG) imaging in tendon and striated muscle. Our approach combines three-dimensional (3-D) SHG imaging of the axial attenuation and directional response with Monte Carlo simulation (based on measured bulk optical properties) of the creation intensity and propagation through the tissues. Through these experiments and simulations, we show that reduction of the primary filter following glycerol treatment dominates the axial attenuation response in both muscle and tendon. However, these disparate tissue types are shown to clear through different mechanisms of the glycerol-tissue interaction. In the acellular tendon, glycerol application reduces scattering by both index matching as well as increasing the interfibril separation. This results in an overall enhancement of the 3-D SHG intensity, where good agreement is found between experiment and simulation. Through analysis of the axial response as a function of glycerol concentration in striated muscle, we conclude that the mechanism in this tissue arises from matching of the refractive index of the cytoplasm of the muscle cells with that of the surrounding higher-index collagenous perimysium. We further show that the proportional decrease in the scattering coefficient μs with increasing glycerol fraction can be well-approximated by Mie theory.

Imaging of biological tissues with optical coherence tomography (OCT) poses a great interest for its capability to noninvasively outline subsurface microstructures within tissues. However, a major limitation for many optical imaging techniques is inadequate depth penetration of light in turbid media, which is bounded to just a few millimeters. There have been several attempts to improve light penetration depth in biological tissues, including application of different tissue optical clearing methods. In this study, an aqueous solution of glucose (40%) is added to rabbit sclera in vitro, where depth-resolved permeability coefficients and optical clearing are calculated with OCT. The permeability rate in regions in the upper 80- to 100-μm region is found to be different from that of regions in the deeper 100-μm region: (6.01±0.37)×10^-6 cm/sec and (2.84±0.68)×10^-5 cm/sec, respectively. A difference in percent clearing is also noted. Optical clearing of the upper region is about 10% and increased to 17 to 22% in the one beneath. These results demonstrate the capability of OCT-based methods to not only measure the diffusion rate and optical clearing of a tissue, but also its ability of functional differentiation between layers of epithelial tissues.

Optical clearing is a well-known phenomenon. It is based on the matching of refractive indices of a bulk material and scattering particles. The same principle is also used in scattering-based optical measurements of different constituents, such as glucose. By registering changes in scattering, it is possible to evaluate changes in the concentration of a solvent. This work describes the use of optical coherence tomography (OCT) to monitor glucose-induced changes in the optical properties of samples. Intralipid^TM, mouse skin tissue, and mice (C57BL) are used as samples. Differences between in vitro and in vivo measurement conditions, the effect of glucose on the samples' optical properties, as well as possible problems in OCT experiments are discussed. A comparison of OCT signals from mouse skin samples and mice in vivo shows that the intensity of backscattered radiation is stronger in a living animal than in cultured tissue. Moreover, the effect of glucose on the scattering properties is larger in an in vivo case than in an in vitro case. In comparison with tissue, the effect of glucose is the smallest in Intralipid. The results increase the value of using cultured tissue in developing optical sensing techniques.

We proposed to use optical coherence tomography (OCT) for continuous noninvasive blood glucose monitoring, and recently we significantly improved the sensitivity of this technique. The accuracy of OCT glucose monitoring is limited by several factors, including variation of tissue pressure exerted by the OCT probe. We studied the influence of high (>10 kPa) and low (<1 kPa) pressure levels on OCT blood glucose monitoring. We showed that controlling external pressure to <1 kPa substantially improved the accuracy and reproducibility of OCT-based glucose monitoring.

Optical computed tomography (optical-CT) and optical emission computed tomography (optical-ECT) are new techniques that enable unprecedented high-resolution 3-D multimodal imaging of tissue structure and function. Applications include imaging macroscopic gene expression and microvasculature structure in unsectioned biological specimens up to 8 cm³. A key requisite for these imaging techniques is effective sample preparation including optical clearing, which enables light transport through the sample while preserving the signal (either light absorbing stain or fluorescent proteins) in representative form. We review recent developments in optical-CT and optical-ECT, and compatible "fluorescence-friendly" optical clearing protocols.

A method is presented for decreasing radiant exposures required for photocoagulation of cutaneous blood vessels using a hyperosmotic agent applied to skin prior to laser irradiation. The 50% probability for a given radiant exposure (RE50) to result in photocoagulation of vessels classified by type (arteriole, venule) and diameter was determined following direct (subcutaneous) laser irradiation of 84 vessels in a dorsal skin preparation pretreated with glycerol. Values were compared against results without glycerol pretreatment. A second set of experiments involved irradiation of blood vessels through the skin from the epidermal surface after application of glycerol. Subcutaneous RE50s for vessels treated with glycerol were typically several factors lower than untreated vessels. For example, arterioles in the 80- to 110-μm-diam range in untreated skin had RE50 values ~12 J/cm², compared to ~2 J/cm² in glycerol-treated cases. Results from epidermal irradiations also indicate that pretreatment with glycerol decreases radiant exposures required for photocoagulation. Vessels were successfully coagulated from the epidermal side in glycerol-treated samples using radiant exposures ranging from 1.6 J/cm² to 5 J/cm², compared to the 12 to >16 J/cm² range for control cases. We believe that this method could be a powerful technique for reducing the radiant exposures required for vessel photocoagulation.

The influence of tissue optical properties on laser Doppler perfusion imaging (LDPI) is not well understood. We address this problem by quantifying the dependence of the signal response to tissue optical properties based on speckles or coherence areas and on photon statistics. We investigate the effect in vivo, showing the amplitude of photocurrent fluctuations in normal skin and port-wine stain with a range of beam diameters, and its relation to the speckle size variation difference between these two tissues. For the case of a low concentration of moving particles moving within a static turbid medium, a model is described and applied to predict the influence of speckles on the overall and depth sensitivity of LDPI, for a range of scattering levels and absorption levels. The results show that the speckle related effects on overall and depth sensitivity are large and that the depth sensitivity is highly likely to be misinterpreted without taking the speckle phenomenon into account.

Corneal and scleral thickness and anterior chamber dimensions are required for understanding developmental and pathological processes. Parameters of the eyeball are also required to calculate optical and material properties. As the eyeball resembles a pressure vessel, it has been suggested that elasticity of the cornea and sclera could be calculated from the measurements of thickness. Baseline corneal and scleral thicknesses and anterior chamber dimensions and how these change with incremental increases of intraocular fluid are measured in fresh porcine eyes using the Visante OCT (optical coherence tomography). At baseline, corneal thickness is almost constant. Anterior scleral thickness is variable, decreasing from 0.91±0.07 mm near the limbus to a minimum of 0.58±0.13. Posterior scleral thickness is more constant with an average of 0.78±0.09 mm. Near the optic nerve the thickness increases to 1.00±0.09 mm. Average baseline anterior chamber angle, diameter, and depth were found to be 33.15±4.91 deg, 13.60±0.38 mm, and 2.13±0.22 mm, respectively. After fluid injections, maximum changes in corneal and scleral thicknesses were 9 to 10 and 1 to 3%, respectively. Anterior chamber angle and depth decreased slightly but significantly. Changes in the eyeball coats with fluid injections, indicate that the pressure vessel model can be applied to the eye to calculate corneal and scleral elasticities.

The vast majority of bladder cancers originate within 600 µm of the tissue surface, making optical coherence tomography (OCT) a potentially powerful tool for recognizing cancers that are not easily visible with current techniques. OCT is a new technology, however, and surgeons are not familiar with the resulting images. Technology able to analyze and provide diagnoses based on OCT images would improve the clinical utility of OCT systems. We present an automated algorithm that uses texture analysis to detect bladder cancer from OCT images. Our algorithm was applied to 182 OCT images of bladder tissue, taken from 68 distinct areas and 21 patients, to classify the images as noncancerous, dysplasia, carcinoma in situ (CIS), or papillary lesions, and to determine tumor invasion. The results, when compared with the corresponding pathology, indicate that the algorithm is effective at differentiating cancerous from noncancerous tissue with a sensitivity of 92% and a specificity of 62%. With further research to improve discrimination between cancer types and recognition of false positives, it may be possible to use OCT to guide endoscopic biopsies toward tissue likely to contain cancer and to avoid unnecessary biopsies of normal tissue.

We demonstrate the ability of multiple forms of optical coherence tomography (OCT) in the frequency domain to quantitatively size scatterers. Combined with a variety of distinct phantoms, we gain insight into the measurement uncertainties associated with using scattering spectra to size scatterers. We size spherical scatterers on a surface using swept-source OCT with an analysis based on a simple slab-mode resonance model. Automating this technique, a two-dimensional (2-D) image is created by raster scanning across a surface phantom designed to have a distinct size transition to demonstrate accuracy and repeatability. We also investigate the potential of a novel sphere-nanotube structure as a quantitative calibration artifact for use in comparing measured intensity and phase scattering spectra directly to Mie theory predictions. In another experiment, we demonstrate tissue-relevant sizing of scatterers as small as 5 μm on a surface by use of a Fourier domain OCT system with 280 nm of bandwidth from a supercontinuum source. We perform an uncertainty analysis for our high-resolution sizing system, estimating a sizing error of 9% for measurements of spheres with a diameter of 15 μm. With appropriate modifications, our uncertainty analysis has general applicability to other sizing techniques utilizing scattering spectra.

Intralipid is a well-known emulsion used as a tissue-simulating phantom in developing optical imaging and diagnostic techniques for medical applications. The optical coherence tomography (OCT), pulsed photoacoustic (PA), and time-of-flight (TOF) techniques were used to detect glucose-induced changes in the optical properties of Intralipid. A comparison of the applicability of these techniques to register changes in the scattering properties of Intralipid samples showed that OCT is the most effective method, whereas the sensitivity of the PA technique was less pronounced. Photon migration studies with the TOF technique showed changes in pulse amplitude, pulse width, and arrival time of the pulse maximum as a function of changes in Intralipid concentration. Also the measured signal parameters showed changes when measuring high glucose concentrations.

We build a photoacoustic imaging system using an intensity-modulated continuous-wave laser source, which is an inexpensive, compact, and durable 120-mW laser diode. The goal is to significantly reduce the costs and sizes of photoacoustic imaging systems. By using a bowl-shaped piezoelectric transducer, whose numerical aperture is 0.85 and resonance frequency is 2.45 MHz, we image biological tissues with a lateral resolution of 0.45 mm, an axial resolution of 1 mm, and an SNR as high as 43 dB.

We present systematic characterization of a photoacoustic imaging system optimized for rapid, high-resolution tomographic imaging of small animals. The system is based on a 128-element ultrasonic transducer array with a 5-MHz center frequency and 80% bandwidth shaped to a quarter circle of 25 mm radius. A 16-channel data-acquisition module and dedicated channel detection electronics enable capture of a 90-deg field-of-view image in less than 1 s and a complete 360-deg scan using sample rotation within 15 s. Measurements on cylindrical phantom targets demonstrate a resolution of better than 200 μm and high-sensitivity detection of 580-μm blood tubing to depths greater than 3 cm in a turbid medium with reduced scattering coefficient μ_s^′=7.8 cm^-1. The system is used to systematically investigate the effects of target size, orientation, and geometry on tomographic imaging. As a demonstration of these effects and the system imaging capabilities, we present tomographic photoacoustic images of the brain vasculature of an exvivo mouse with varying measurement aperture. For the first time, according to our knowledge, resolution of sub-200-μm vessels with an overlying turbid medium of greater than 2 cm depth is demonstrated using only intrinsic biological contrast.

Conventional adaptive optics enables correction of high-order aberrations of the eye, but only for a single retinal point. When imaging extended regions of the retina, aberrations increase away from this point and degrade image quality. The zone over which aberrations do not change significantly is called the "isoplanatic patch." Literature concerning the human isoplanatic patch is incomplete. We determine foveal isoplanatic patch characteristics by performing Hartmann-Shack aberrometry in 1 deg increments in 8 directions on 7 human eyes. Using these measurements, we establish the correction quality required to yield at least 80% of the potential patch size for a given eye. Single-point correction systems (conventional adaptive optics) and multiple-point correction systems (multiconjugate adaptive optics) are simulated. Results are compared to a model eye. Using the Maréchal criterion for 555-nm light, average isoplanatic patch diameter for our subjects is 0.80±0.10 deg. The required order of aberration correction depends on desired image quality over the patch. For the more realistically achievable criterion of 0.1 μm root mean square (rms) wavefront error over a 6.0-mm pupil, correction to at least sixth order is recommended for all adaptive optics systems. The most important aberrations to target for a multiconjugate correction are defocus, astigmatism, and coma.

Laser-induced damage is studied in the rat corneal epithelium and stroma using a combination of time-resolved imaging and biological assays. Cavitation bubble interactions with cells are visualized at a higher spatial resolution than previously reported. The shock wave is observed to propagate through the epithelium without cell displacement or deformation. Cavitation bubble expansion is damped in tissue with a reduction in maximum size in the range of 54 to 59%, as compared to 2-D and 3-D cultures. Bubble expansion on nanosecond timescales results in rupture of the epithelial sheet and severe compression of cell layers beyond the bubble rim. In the stroma, the dense collagen lamellae strongly damped bubble expansion, thus resulting in reduced damage. The acute biological response of this tissue to laser pulses is characterized by confocal fluorescence microscopy. A viability assay of the epithelium reveals that only cells around the immediate site of laser focus are killed, while cells seen to undergo large deformations remain alive. Actin morphology in cells facing this mechanical stress is unchanged. Collagen microstructure in the stroma as revealed by second-harmonic imaging around the ablation site shows minimal disruption. These cellular responses are also compared to in vitro 2-D and 3-D cell cultures.

A model of the directional and nondirectional reflection spectrum of the human fovea is developed, incorporating reflectors, absorbers, and a wavelength-dependent optical Stiles-Crawford effect (OSCE). Data from 102 healthy subjects between 18 and 75 years obtained with the fundus reflection analyzer (FRA), an imaging spectrograph that measures the directional reflection profile of the human fovea in the pupil plane from 400 to 950 nm, were analyzed. Subgroups of young (<40 years) and old (>50 years) observers were defined. Mean results of the young group defined a template for directionality versus wavelength. For the whole group, mean reflection at 550 nm from the cones was 2.12%, from the retinal pigment epithelium 0.56%, and from the choroid 7.92%. Lens density, cone disc reflection, and blood layer thickness showed significant trends versus age. The model for the first time simultaneously describes the spectra of the directional and nondirectional reflection of the human fovea. Rayleigh scatter losses of the media and in preretinal layers were assumed zero in the nondirectional pathway. Mean density of the macular pigment of a subgroup (53 subjects, 19 to 75 years) correlated significantly with independent data from reflectance and autoflourescence images obtained by scanning laser ophthalmoscope (SLO) and data from flicker photometry.

We report the results of an oral cavity pilot clinical trial to detect early precancer and cancer using a fiber optic probe with obliquely oriented collection fibers that preferentially probe local tissue morphology and heterogeneity using oblique polarized reflectance spectroscopy (OPRS). We extract epithelial cell nuclear sizes and 10 spectral features. These features are analyzed independently and in combination to assess the best metrics for separation of diagnostic classes. Without stratifying the data according to anatomical location or level of keratinization, OPRS is found to be sensitive to four diagnostic categories: normal, benign, mild dysplasia, high-grade dysplasia, and carcinoma. Using linear discriminant analysis, separation of normal from high-grade dysplasia and carcinoma yield a sensitivity and specificity of 90 and 86%, respectively. Discrimination of morphologically similar lesions such as normal from mild dysplasia is achieved with a sensitivity of 75% and specificity of 73%. Separation of visually indistinguishable benign lesions from high-grade dysplasia and carcinoma is achieved with good sensitivity (100%) and specificity (85%), while separation of benign from mild dysplasia gives a sensitivity of 92% and a specificity of 69%. These promising results suggest that OPRS has the potential to aid screening and diagnosis of oral precancer and cancer.

Using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy, we have developed an algorithm that successfully classifies normal breast tissue, fibrocystic change, fibroadenoma, and infiltrating ductal carcinoma in terms of physically meaningful parameters. We acquire 202 spectra from 104 sites in freshly excised breast biopsies from 17 patients within 30 min of surgical excision. The broadband diffuse reflectance and fluorescence spectra are collected via a portable clinical spectrometer and specially designed optical fiber probe. The diffuse reflectance spectra are fit using modified diffusion theory to extract absorption and scattering tissue parameters. Intrinsic fluorescence spectra are extracted from the combined fluorescence and diffuse reflectance spectra and analyzed using multivariate curve resolution. Spectroscopy results are compared to pathology diagnoses, and diagnostic algorithms are developed based on parameters obtained via logistic regression with cross-validation. The sensitivity, specificity, positive predictive value, negative predictive value, and overall diagnostic accuracy (total efficiency) of the algorithm are 100, 96, 69, 100, and 91%, respectively. All invasive breast cancer specimens are correctly diagnosed. The combination of diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy yields promising results for discrimination of breast cancer from benign breast lesions and warrants a prospective clinical study.

We investigate the potential of near-infrared Raman microspectroscopy to differentiate between normal and malignant skin lesions. Thirty-nine skin tissue samples consisting of normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma from 39 patients were investigated. Raman spectra were recorded at the surface and at 20-μm intervals below the surface for each sample, down to a depth of at least 100 μm. Data reduction algorithms based on the nonlinear maximum representation and discrimination feature (MRDF) and discriminant algorithms using sparse multinomial logistic regression (SMLR) were developed for classification of the Raman spectra relative to histopathology. The tissue Raman spectra were classified into pathological states with a maximal overall sensitivity and specificity for disease of 100%. These results indicate the potential of using Raman microspectroscopy for skin cancer detection and provide a clear rationale for future clinical studies.

Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.

A unified Mie and fractal model for light scattering by biological cells is presented. This model is shown to provide an excellent global agreement with the angular dependent elastic light scattering spectroscopy of cells over the whole visible range (400 to 700 nm) and at all scattering angles (1.1 to 165 deg) investigated. Mie scattering from the bare cell and the nucleus is found to dominate light scattering in the forward directions, whereas the random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, is found to dominate light scattering at other angles. Angularly dependent elastic light scattering spectroscopy aided by the unified Mie and fractal model is demonstrated to be an effective noninvasive approach to characterize biological cells and their internal structures. The acetowhitening effect induced by applying acetic acid on epithelial cells is investigated as an example. The changes in morphology and refractive index of epithelial cells, nuclei, and subcellular structures after the application of acetic acid are successfully probed and quantified using the proposed approach. The unified Mie and fractal model may serve as the foundation for optical detection of precancerous and cancerous changes in biological cells and tissues based on light scattering techniques.

We present a generalized Delta-Eddington phase function to simplify the radiative transfer equation to integral equations with respect to both photon fluence rate and flux vector. The photon fluence rate and flux can be solved from the system of integral equations. By comparing to the Monte Carlo simulation results, the solutions of the system of integral equations accurately model the photon propagation in biological tissue over a wide range of optical parameters.

A Monte-Carlo-based model of fluorescence is developed that is capable of extracting the intrinsic fluorescence properties of tissue, which are independent of the absorption and scattering properties of tissue. This model is flexible in its applicability to different illumination-collection geometries and is also valid for a wide range of optical properties, representative of tissue in the UV-visible spectrum. This is potentially useful in a variety of biomedical applications, including cancer diagnostics and monitoring the physiological response to therapy. The model is validated using phantoms composed of hemoglobin (absorber), polystyrene spheres (scatterer), and furan-2 (fluorophore). It is found that this model is able to retrieve the intrinsic fluorescence spectra of the tissue phantoms and recover the intrinsic fluorescence intensity of furan within the phantoms to within a mean error of less than 10%.

We developed a technique for constructing light diffusing devices comprised of a flexible shape memory polymer (SMP) cylindrical diffuser attached to the tip of an optical fiber. The devices are fabricated by casting an SMP rod over the cleaved tip of an optical fiber and media blasting the SMP rod to create a light diffusing surface. The axial and polar emission profiles and circumferential (azimuthal) uniformity are characterized for various blasting pressures, nozzle-to-sample distances, and nozzle translation speeds. The diffusers are generally strongly forward-directed and consistently withstand over 8 W of incident IR laser light without suffering damage when immersed in water. These devices are suitable for various endoluminal and interstitial biomedical applications.

A multispectral digital microscope (MDM) is designed and constructed as a tool to improve detection of oral neoplasia. The MDM acquires in vivo images of oral tissue in fluorescence, narrow-band (NB) reflectance, and orthogonal polarized reflectance (OPR) modes, to enable evaluation of lesions that may not exhibit high contrast under standard white light illumination. The device rapidly captures image sequences so that the diagnostic value of each modality can be qualitatively and quantitatively evaluated alone and in combination. As part of a pilot clinical trial, images are acquired from normal volunteers and patients with precancerous and cancerous lesions. In normal subjects, the visibility of vasculature can be enhanced by tuning the reflectance illumination wavelength and polarization. In patients with histologically confirmed neoplasia, we observe decreased blue/green autofluorescence and increased red autofluorescence in lesions, and increased visibility of vasculature using NB and OPR imaging. The perceived lesion borders change with imaging modality, suggesting that multimodal imaging has the potential to provide additional diagnostic information not available using standard white light illumination or by using a single imaging mode alone.

An extension of Nomarski differential interference contrast microscopy enables isotropic linear phase imaging through the combination of phase shifting, two directions of shear, and Fourier space integration using a modified spiral phase transform. We apply this method to simulated and experimentally acquired images of partially absorptive test objects. A direct comparison of the computationally determined phase to the true object phase demonstrates the capabilities of the method. Simulation results predict and confirm results obtained from experimentally acquired images.

The confocal microendoscope is an instrument for imaging the surface of the human ovary. Images taken with this instrument from normal and diseased tissue show significant differences in cellular distribution. A real-time computer-aided system to facilitate the identification of ovarian cancer is introduced. The cellular-level structure present in ex vivo confocal microendoscope images is modeled as texture. Features are extracted based on first-order statistics, spatial gray-level-dependence matrices, and spatial-frequency content. Selection of the features is performed using stepwise discriminant analysis, forward sequential search, a nonparametric method, principal component analysis, and a heuristic technique that combines the results of these other methods. The selected features are used for classification, and the performance of various machine classifiers is compared by analyzing areas under their receiver operating characteristic curves. The machine classifiers studied included linear discriminant analysis, quadratic discriminant analysis, and the k-nearest-neighbor algorithm. The results suggest it is possible to automatically identify pathology based on texture features extracted from confocal microendoscope images and that the machine performance is superior to that of a human observer.

We attempt to develop a systematic scheme through adopting high-pass filtering (HPF) to well resolve value-preserved images such as medical images. Our approach is derived from the Poisson maximum a posteriori superresolution algorithm employing the HP filters, where four filters are considered such as two low-pass-filter-combination based filters, wavelet filter, and negative-oriented Laplacian HP filter. The proposed approach is incorporated into the procedure of finite-element-method (FEM)-based image reconstruction for diffuse optical tomography in the direct current domain, posterior to each iteration without altering the original FEM modeling. This approach is justified with various HPF for different cases that breast-like phantoms embedded with two or three inclusions that imitate tumors are employed to examine the resolution performances under certain extreme conditions. The proposed approach to enhancing image resolution is evaluated for all tested cases. A qualitative investigation of reconstruction performance for each case is presented. Following this, we define a set of measures on the quantitative evaluation for a range of resolutions including separation, size, contrast, and location, thereby providing a comparable evaluation to the visual quality. The most satisfactory result is obtained by using the wavelet HP filter, and it successfully justifies our proposed scheme.

Singlet oxygen (¹O₂) is an important factor mediating cell killing in photodynamic therapy (PDT). We previously reported that chemiluminescence (CL) can be used to detect ¹O₂ production in PDT and linked the signal to the PDT-induced cytotoxicity in vitro. We develop a new CL detection apparatus to achieve in vivo measurements. The system utilizes a time-delayed CL signal to overcome the interference from scattered excitation light, thus greatly improving the accuracy of the detection. The system is tested on healthy skin of BALB/ca mouse for its feasibility and reliability. The CL measurement is made during a synchronized gating period of the irradiation light. After each PDT treatment and in situ CL measurement, the skin response is scored over a period of 2 weeks. A remarkable relationship is observed between the score and the CL, regardless of the PDT treatment protocol. Although there are many issues yet to be addressed, our results clearly demonstrate the feasibility of CL measurement during PDT and its potential for in vivo PDT dosimetry. This requires further investigations.

An integrated microfluidic planar optical waveguide system for measuring light scattered from a single scatterer is described. This system is used to obtain 2D side-scatter patterns from single polystyrene microbeads in a fluidic flow. Vertical fringes in the 2D scatter patterns are used to infer the location of the 90-deg scatter (polar angle). The 2D scatter patterns are shown to be symmetrical about the azimuth angle at 90 deg. Wide-angle comparisons between the experimental scatter patterns and Mie theory simulations are shown to be in good agreement. A method based on the Fourier transform analysis of the experimental and Mie simulation scatter patterns is developed for size differentiation.

We present a novel method for estimating the intrinsic fluorescence lifetime of deeply embedded localized fluorophores. It is based on scaling relations, characteristic for turbid media. The approach is experimentally substantiated by successfully reconstructing lifetimes for targets at depths up to 14.5 mm. A derived correction factor was determined from the product of the transport-corrected scattering coefficient μ_s^′ and the index of refraction nr. In addition, data from an array of detectors (≥2) can be used to estimate μ_s^′ n_r. The suggested algorithm is a promising tool for diagnostic fluorescence, since lifetime can be a sensitive indicator of the fluorophore environment.

This PDF file contains the errata for “JBO Vol. 13 Issue 02 Paper 2891153” for JBO Vol. 13 Issue 02

This PDF file contains the editorial “Principles of Fluorescence Spectroscopy, Third Edition” for JBO Vol. 13 Issue 02

This PDF file contains the editorial “Handbook of Biological Confocal Microscopy, Third Edition” for JBO Vol. 13 Issue 02