A method for noninvasively determining blood oxygenation in individual vessels inside bulk tissue would provide a powerful tool for biomedical research. We explore the potential of coherent anti-Stokes Raman scattering (CARS) spectroscopy to provide this capability. Using the multiplex CARS approach, we measure the vibrational spectrum in hemoglobin solutions as a function of the oxygenation state and observe a clear dependence of the spectral shape on oxygenation. The direct extraction of the Raman line shape from the CARS data using a maximum entropy method phase retrieval algorithm enables quantitative analysis. The CARS spectra associated with intermediate oxygenation saturation levels can be accurately described by a weighted sum of the fully oxygenated and fully deoxygenated spectra. We find that the degree of oxygenation determined from the CARS data agrees well with that determined by optical absorption. As a nonlinear optical technique, CARS inherently provides the 3-D imaging capability and tolerance to scattering necessary for biomedical applications. We discuss the challenges in extending the proof of principle demonstrated to in vivo applications.

For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly (dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (≥600 ps), high spatial resolution (1.4 µm), calibrated detection (0 to 300±8 µM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.

We develop an optical whole-body imaging technique for monitoring normal and leukemic hematopoietic cell homing in vivo. A recently developed near-infrared (NIR) lipophilic carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) is used to safely and directly label the membranes of human leukemic Pre-B ALL G2 cell lines as well as primary murine lymphocytes and erythrocytes. DiR has absorption and fluorescence maxima at 750 and 782 nm, respectively, which corresponds to low light absorption and autofluorescence in living tissues. This allows us to obtain a significant signal with very low background level. A charge-coupled device (CCD)-based imager is used for noninvasive whole-body imaging of DiR-labeled cell homing in intact animals. This powerful technique can potentially visualize any cell type without use of specific antibodies conjugated with NIR fluorescent tag or loading cells with transporter-delivered NIR fluorophores. Thus, in vivo imaging based on NIR lipophilic carbocyanine dyes in combination with advanced optical techniques may serve as a powerful alternative or complementation to other small animal imaging methods.

We report the first realization of interstitial surgery by ultrabright lamp light, on the kidneys and livers of live animals. A high-flux optic concentrates lamp emissions into an optical fiber for power delivery inside the body. The trials reveal surgical efficacy comparable to corresponding laser fiber optic treatments, as well as pronounced delayed tissue death.

We employ Fourier–transform Raman spectroscopy to study normal and tumoral human breast tissues, including several subtypes of cancers. We analyzed 194 Raman spectra from breast tissues that were separated into 9 groups according to their corresponding histopathological diagnosis. The assignment of the relevant Raman bands enabled us to connect the several kinds of breast tissues (normal and pathological) to their corresponding biochemical moieties alterations and distinguish among 7 groups: normal breast, fibrocystic condition, duct carcinoma in situ, duct carcinoma in situ with necrosis, infiltrating duct carcinoma not otherwise specified, colloid infiltrating duct carcinoma, and invasive lobular carcinomas. We were able to establish the biochemical basis for each spectrum, relating the observed peaks to specific biomolecules that play a special role in the carcinogenesis process. This work is very useful for the premature optical diagnosis of a broad range of breast pathologies. We noticed that we were not able to differentiate inflammatory and medullary duct carcinomas from infiltrating duct carcinoma not otherwise specified.

Early surgical management of those burn injuries that will not heal spontaneously is critical. The decision to excise and graft is based on a visual assessment that is often inaccurate but yet continues to be the primary means of grading the injury. Superficial and intermediate partial-thickness injuries generally heal with appropriate wound care while deep partial- and full-thickness injuries generally require surgery. This study explores the possibility of using near-infrared spectroscopy to provide an objective and accurate means of distinguishing shallow injuries from deeper burns that require surgery. Twenty burn injuries are studied in five animals, with burns covering <1% of the total body surface area. Carefully controlled superficial, intermediate, and deep partial-thickness injuries as well as full-thickness injuries could be studied with this model. Near-infrared reflectance spectroscopy was used to evaluate these injuries 1 to 3 hours after the insult. A probabilistic model employing partial least-squares logistic regression was used to determine the degree of injury, shallow (superficial or intermediate partial) from deep (deep partial and full thickness), based on the reflectance spectrum of the wound. A leave-animal-out cross-validation strategy was used to test the predictive ability of a 2–latent variable, partial least-squares logistic regression model to distinguish deep burn injuries from shallow injuries. The model displayed reasonable ranking quality as summarized by the area under the receiver operator characteristics curve, AUC=0.879. Fixing the threshold for the class boundaries at 0.5 probability, the model sensitivity (true positive fraction) to separate deep from shallow burns was 0.90, while model specificity (true negative fraction) was 0.83. Using an acute porcine model of thermal burn injuries, the potential of near-infrared spectroscopy to distinguish between shallow healing burns and deeper burn injuries was demonstrated. While these results should be considered as preliminary and require clinical validation, a probabilistic model capable of differentiating these classes of burns would be a significant aid to the burn specialist.

Visible and near infrared transmission and diffuse reflection spectroscopy were used to monitor changes in whole blood resulting from hemodialysis treatment for end-stage renal disease. Blood samples from 8 patients on chronic hemodialysis therapy were measured in the 500- to 1700-nm wavelength range immediately before and after a single treatment. Principal component scores characteristic of each spectrum were derived, and mean pre- and posttreatment scores of the first principal component indicated a significant treatment-dependent change in both optical transmission (P=0.004) and diffuse reflection (P<0.001). Significant treatment-induced change persisted (P<0.05) when the first four principal components were used to account for >97% of the treatment-dependent spectral variation. Some blood spectral changes expressed in terms of difference spectra (posttreatment – pretreatment) were consisent with standard clinical indicators of weight reduction, urea reduction, and potassium change, with probable origins at a molecular level. The results indicate the feasibility of using optical transmission and diffuse reflection spectroscopy to characterize clinically relevant blood changes for the future development of more comprehensive indicators of hemodialysis efficacy and long-term clinical outcomes. Moreover, the optical techniques employed are adaptable for potential online monitoring of blood changes during the hemodialysis treatment.

Near-infrared spectroscopic imaging (NIRSI) is useful to assess cardiac tissue oxygenation in arrested and beating hearts, and it shows potential as an intraoperative gauge of the effectiveness of bypass grafting. The purpose of this study was to determine whether NIRSI can reliably differentiate among a range of cardiac oxygenation states, using ischemia and hypoxia models independently. An ischemia-reperfusion model was applied to isolated, beating, blood-perfused porcine hearts, in which the left anterior descending (LAD) artery was cannulated. LAD flow was decreased stepwise to approximately 50, 20, and 0% of normal flow and was completely restored between ischemic episodes. Upon completion of the ischemia-reperfusion protocol, the hearts were further subjected to periods of increasingly severe global hypoxia. Regional oxy- and deoxy-hemoglobin (myoglobin) levels were derived from spectroscopic images (650 to 1050 nm) acquired at each step. Oxygenation maps vividly highlighted the area at risk for all degrees of ischemia. Oxygenation values differed significantly for different LAD flow rates, regardless of whether intermediate reperfusion was applied, and oxygenation values during progressive hypoxia correlated well with blood oxygen saturation. These results suggest that NIRSI is well suited, not only to identify ischemic or hypoxic regions of cardiac tissue, but also to assess the severity of deoxygenation.

The use of cerebral tissue oxygenation index (c-TOI) in a clinical setting is limited by doubts concerning the accuracy of the measurements. Since there is no gold standard, validation is difficult. Our modest aim was to quantify the precision of c-TOI doing repeated measurements by reapplying the optode several times presuming no regional differences in cerebral oxygenation. Thirty-seven premature infants were examined with several measurements of c-TOI using the NIRO 300 oximeter. Three to eight measurements were done on each infant over a period of 15 to 25 min. One-way analysis of variance was used to estimate within- and between-infant variation. The median gestational age was 27.6 weeks (23.9 to 33.0). Mean c-TOI (n=253) was 74.6±8.5%. The within-infant variation was 5.2% when resiting the optode. For comparison, the between-infants variation was 6.9%, while the spontaneous 2-sec variation was 2.9%. The precision of a single measurement of c-TOI was not good. By measuring five times instead of one on each subject, the precision of the mean can be assumed to be comparable to pulse oximetry. This may be too cumbersome for clinical use, but may reduce sample size in research.

To improve the spatial resolution and to obtain the depth information of absorbers buried in highly scattering material, we developed a noncontact backscatter-mode near-infrared time-resolved imaging system (noncontact B-TRIS) that is intended for functional human brain mapping. It consists of mode-locked Ti-sapphire lasers as light sources and a charge-coupled device camera equipped with a time-resolved intensifier as a detector. The system was tested with a white polyacetal phantom as a light-scattering medium and black polyacetal particles as absorbers. Illumination and detection of light through an objective lens system (Φ=150 mm) enabled us to capture images from an area whose diameter is about 70 mm without coming into contact with it. The scattering and absorption coefficients of the white phantom obtained by B-TRIS were similar to those obtained by a conventional time-resolved spectroscopy. Although the imaged diameter of an absorber buried within a phantom was considerably larger than the actual diameter, the center position of the absorber coincided with the actual position with accuracy <2 mm. Furthermore, the depth information can be also detected by the noncontact B-TRIS. These results suggest a potential of noncontact B-TRIS for imaging cognitive human brain function.

Near-Infrared Spectroscopy (NIRS) and diffuse optical imaging (DOI) are increasingly used to detect hemodynamic changes in the cerebral cortex induced by brain activity. Until recently, the small number of optodes in NIRS instruments has hampered measurement of optical signals from diverse brain regions. Our new DOI system has 32 detectors and 32 sources; by arranging them in a specific pattern, we can cover most of the adult head. With the increased number of optodes, we can collect optical data from prefrontal, sensorimotor, and visual cortices in both hemispheres simultaneously. We describe the system and report system characterization measurements on phantoms as well as on human subjects at rest and during visual, motor, and cognitive stimulation. Taking advantage of the system's larger number of sources and detectors, we explored the spatiotemporal patterns of physiological signals during rest. These physiological signals, arising from cardiac, respiratory, and blood-pressure modulations, interfere with measurement of the hemodynamic response to brain stimulation. Whole-head optical measurements, in addition to providing maps of multiple brain regions' responses to brain activation, will enable better understandings of the physiological signals, ultimately leading to better signal processing algorithms to distinguish physiological signal clutter from brain activation signals.

We investigated the use of multifrequency diffuse optical tomography (MF-DOT) data for the reconstruction of the optical parameters. The experiments were performed in a 63 mm diameter cylindrical phantom containing a 15 mm diameter cylindrical object. Modulation frequencies ranging from 110 MHz to 280 MHz were used in the phantom experiments changing the contrast in absorption of the object with respect to the phantom while keeping the scattering value the same. The diffusion equation was solved using the finite element method. The sensitivity information from each frequency was combined to form a single Jacobian. The inverse problem was solved iteratively by minimizing the difference between the measurements and forward problem using single and multiple modulation frequency data. A multiparameter Tikhonov scheme was used for regularization. The phantom results show that the peak absorption coefficient in a region of interest was obtained with an error less then 5% using two-frequency reconstruction for absorption contrast values up to 2.2 times higher than background and 10% for the absorption contrast values larger than 2.2. The use of two-frequency data is sufficient to improve the quantitative accuracy compared with the single frequency reconstruction with appropriate selection of these frequencies.

This paper describes a robust single sperm tracking algorithm (SSTA) that can be used in laser optical trapping and sperm motility studies. The algorithm creates a region of interest (ROI) centered about a sperm selected by the user. SSTA contrast enhances the ROI image and implements a modified four-class thresholding method to extract the tracked sperm as it transitions in and out of focus. The nearest neighbor method is complemented with a speed-check feature to aid tracking in the presence of additional sperm or other particles. SSTA has a collision-detection feature for real or perceived collision or near-miss cases between two sperm. Subsequent postcollision analysis employs three criteria to distinguish the tracked sperm in the image. The efficacy of SSTA is validated through examples and comparisons to commercially available computer-aided sperm tracking systems.

Recently the psychophysical compensation comparison method was developed for routine measurement of retinal stray light. The subject's responses to a series of two-alternative-forced-choice trials are analyzed using a maximum-likelihood (ML) approach assuming some fixed shape for the psychometric function (PF). This study evaluates the reliability of the method using Monte-Carlo simulations. Various sampling strategies were investigated, including the two-phase sampling strategy that is used in a commercially available instrument. Results are given for the effective dynamic range and measurement accuracy. The effect of a mismatch of the shape of the PF of an observer and the fixed shape used in the ML analysis was analyzed. Main outcomes are that the two-phase sampling scheme gives good precision (Standard deviation=0.07 logarithmic units on average) for estimation of the stray light value. Bias is virtually zero. Furthermore, a reliability index was derived from the responses and found to be effective.

We describe a computer eye model that allows for aspheric surfaces and a three-dimensional computer-based ray-tracing technique to simulate optical properties of the human eye and visual perception under various eye defects. Eye surfaces, such as the cornea, eye lens, and retina, are modeled or approximated by a set of Zernike polynomials that are fitted to input data for the respective surfaces. A ray-tracing procedure propagates light rays using Snell's law of refraction from an input object (e.g., digital image) through the eye under investigation (i.e., eye with defects to be modeled) to form a retinal image that is upside down and left-right inverted. To obtain a first-order realistic visual perception without having to model or simulate the retina and the visual cortex, this retinal image is then back-propagated through an emmetropic eye (e.g., Gullstrand exact schematic eye model with no additional eye defects) to an output screen of the same dimensions and at the same distance from the eye as the input object. Visual perception under instances of emmetropia, regular astigmatism, irregular astigmatism, and (central symmetric) keratoconus is simulated and depicted. In addition to still images, the computer ray-tracing tool presented here (simEye) permits the production of animated movies. These developments may have scientific and educational value. This tool may facilitate the education and training of both the public, for example, patients before undergoing eye surgery, and those in the medical field, such as students and professionals. Moreover, simEye may be used as a scientific research tool to investigate optical lens systems in general and the visual perception under a variety of eye conditions and surgical procedures such as cataract surgery and laser assisted in situ keratomileusis (LASIK) in particular.

The influence of changes of both crystalline lens and intraocular lens (IOL) misalignment on the retinal image quality was investigated. The optical model of the eye used in investigations was the Liou-Brennan model, which is commonly considered as one of the most anatomically accurate. The original crystalline lens from this model was replaced with an IOL, made of rigid polymethylmethacrylate, in a way that recommend obligatory procedures. The modifications that were made both for crystalline lens and IOL were the longitudinal, the transversal, and the angular displacement.

Imaging the human retina still represents the main field of application of optical coherence tomography (OCT). A major advantage of OCT is the decoupling of depth resolution (given by the coherence length of the light source) and transverse resolution (given by the focal spot size). This enables images of the retina with high depth resolution. On the other hand, in most OCT systems, a limited transverse resolution has been accepted to provide a sufficiently large (~1 mm) depth of focus. However, to obtain images with high transverse resolution throughout the whole depth of the retina (especially in the nerve head region) a tracking of coherence gate and focus position (dynamic focus) is essential. This study realizes a dynamic focus in a time domain transversal (en face) scanning system for retinal imaging. We show that maintenance of a transverse resolution of ~4.4 µm can be achieved over an optical depth of 1 mm in a model eye and apply our method to imaging the human retina in vivo.

The possibility of measuring subnanometer motions with micron scale spatial resolution in the intact mammalian cochlea using Doppler optical coherence microscopy (DOCM) is demonstrated. A novel DOCM system is described that uses two acousto-optic modulators to generate a stable 500-kHz heterodyne frequency. Images and motion measurements are obtained using phase-resolved analysis of the interference signal. The DOCM system permits imaging with micron-scale resolution and 85-dB sensitivity and motion measurements with 100-kHz bandwidth, directional discrimination, and 30-pm/Hz0.5 noise floor. Images and motion measurements are presented that demonstrate the ability to resolve motions of structures of interest in a mammalian cochlea in vitro including the basilar membrane, reticular lamina, tectorial membrane, and outer hair cells.

Optical coherence tomography (OCT) has been demonstrated as a promising means of identifying the boundaries between normal and diseased breast tissue. This capability has yielded promise for the development of OCT techniques for biopsy guidance, surgical margin assessment, and minimally invasive evaluation of disease states. We present methods for the assessment of human breast tissue based on spatial and Fourier-domain analysis. Derived from preliminary OCT data, these methods are aimed at the development of automated diagnostic tools that will aid in the translation of this technology into the clinical environment.

A polarization-sensitive optical coherence tomography (PSOCT) system using a femtosecond-laser as the broadband light source is implemented with the axial resolution of 5 µm in free space. Through the design of path-length difference between the two polarization inputs and the modulation of one of the polarization inputs, the PSOCT images of various input and output polarization combinations can be distinguished and simultaneously collected. The PSOCT system is then used for in vitro scanning of the myocardium tissues of normal and infarcted rat hearts. The destruction of the birefringence nature of the fiber muscle in the infarcted heart can be clearly observed.

The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (~190 kJ/cm²) is then used to permanently photobleach the ICG molecules — resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, Φ_B,cum, defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that Φ_B,cum decreases with fluence as well as with increasing dye concentration. The quantity Φ_B,cum is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.

The purpose of this study was twofold: (1) to investigate the nature and degree of water loss at 21°C, 60% relative humidity (dehydration) and at 105°C (desiccation), and to relate these findings with (2) the strains produced in the dentine structure during dehydration and rehydration processes. In stage 1, digital moiré interferometry (DMI) was used to study the strain distribution pattern during dehydration and rehydration at 21°C. In stage 2, the nature and degree of water loss was determined using gravimetric analysis and nuclear magnetic resonance spectroscopy. DMI showed that dehydration produced strains in the dentine structure after an initial latent period. Gravimetric analysis showed that dentine exhibited an initial rapid water-loss phase followed by a slow and steady water-loss phase. Though the major portion of water loss occurred in the initial 2 h of dehydration (rapid water-loss phase), no obvious strains were produced during this period. Rehydration lead to the major reversal of dehydration-induced water loss and strains in dentine. Heating at 105°C resulted in further substantial loss of water from dentine. These experiments highlighted that the free water in the dentine surface, porosities and tubules are lost rapidly and constitute the major water lost when dehydrated at 21°C.

A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-µm resolution, at tissue depths up to 610 µm. Post-objective scanning enables a large field of view (610×640 µm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.

More than a threefold increase in multiphoton laser scanning microscopy depth penetration using a passive predispersion compensation system is reported. Using dispersion-controlled pulses to counteract the effects of positive group delay dispersion in the imaging platform, optical sectioning of fluorescent samples to depths in excess of 800 µm was observed, compared with only 240 µm using a noncompensated setup. Experimental results obtained from both the predispersion compensated and noncompensated systems are compared with theoretical values of pulse broadening in a laser scanning microscope. The observed improvement in depth profiling potentially widens the applications and user base of nonlinear microscopy techniques.

Type I collagen is a major component of the extracellular matrix as well as many tissue engineered models. To understand changes in collagen related models over time, it is important to evaluate collagen dynamics with noninvasive techniques. Fluorescence spectroscopy provides a method to noninvasively measure endogenous collagen fluorescence. Additionally, second harmonic generation (SHG) imaging of collagen produces high resolution images of the fibrils. In this study, a novel in vitro collagen measurement chamber was developed for measurement in standard spectroscopic cuvette chambers and microscopic imaging. The fluorescence of polymerized collagen was found to be highly variable, primarily depending on incubation time after polymerization. Changes in fluorescence over time were consistent with increases at UVA excitation wavelengths (λex=360 nm) and decreases at UVC excitation wavelengths (λex=270 nm), suggesting changes in nonenzymatic association of the collagen fibrils. SHG imaging of the collagen suggested that a stable network formed during polymerization. Unlike the fluorescence emission, SHG images from the gels varied little with time suggesting that SHG is not as sensitive to cross-linking or fibril-fibril associated changes. The developed measurement system will allow further studies on the effect of enzymatic cleavages and structural alterations on collagen fluorescence and SHG.

Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development.

Autofluorescence of rabbit and human epithelial tissues were studied by using a depth-resolved fluorescence spectroscopy system with multiple excitations. Keratinization was found to be common in the squamous epithelium. Strong keratin fluorescence with excitation and emission characteristics similar to collagen were observed in the topmost layer of the keratinized squamous epithelium. The keratin signal created interference in the assessment of the endogenous fluorescence signals (NADH/FAD fluorescence in epithelium and collagen fluorescence in stroma) associated with the development of epithelial precancer. Furthermore, the keratinized epithelial layer attenuated the excitation light and reduced the fluorescence signals from underlying tissue layers. The autofluorescence of columnar epithelium was found to be dominated by NADH and FAD signals, identical to the autofluorescence measured from nonkeratinized squamous epithelium. The study also demonstrated that a fluorescence signal excited at 355 nm produced sufficient contrast to resolve the layered structure of epithelial tissue, while the signal excited at 405 nm provided the information for a good estimation of epithelial redox ratios that are directly related to tissue metabolism. Overall, the depth-resolved measurements are crucial to isolate the fluorescence signals from different sublayers of the epithelial tissue and provide more accurate information for the tissue diagnosis.

Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non- FRETing populations, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex auto-fluorescent background decay, described using a stretched exponential function (StrEF). We develop a generalized multi-exponential fitting algorithm (GeMEF), which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein–protein interactions and protein conformational assays in a cell-based format. Presenilin 1 (PS1) is known to be important in Amyloid Precursor Protein (APP) processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.

A novel multiplex analysis technology based on quantum dot (QD) optical encoded beads was studied. Carboxyl functionalized polystyrene beads, about 100 µm in size, were precisely encoded by the various ratios of two types of QDs whose emission wavelengths are 576 and 628 nm, respectively. Then the different encoded beads were covalently immobilized with different probes in the existing of sulfo-NHS and 1-[3-(Dimethylamino) propyl]-3-ethylcarbodiimide methiodide, and the probe density could reach to 3.1 mmol/g. These probe-linked encoded beads were used to detect the target DNA sequences in complex DNA solution by hybridization. Hybridization was visualized using fluorescein isothiocynate–labeled DNA sequences. The results show that the QDs and target signals can be obviously identified from a single-bead-level spectrum. This technology can detect DNA targets effectively with a detection limit of 0.2 µg/mL in complex solution.

Tape stripping is a standard measuring method for the investigation of the dermatopharmacokinetics of topically applied substances using adhesive films. These tape strips are successively applied and removed from the skin after application and penetration of topically applied substances. Thus, layers of corneocytes and some amount of topical applied substances are removed. The amount of substances and the amount of stratum corneum removed with a single tape strip has to be determined for the calculation of the penetration profile. The topically applied substances removed from the skin can be determined by classical analytical methods like high-pressure liquid chromatography, mass spectroscopy, and spectroscopic measurements. The amount of corneocytes on the tape strips can be easily detected by their pseudoabsorption. In the present paper, an easy and cheap corneocyte density analyzer is presented that is based on a slide projector. Comparing the results of the measurements obtained by the corneocyte density analyzer and by uv-visible spectrometry, identical results were obtained.

The objective of this study was to evaluate glycerol (GLY) and GLY + dimethyl sulfoxide (DMSO) to increase photonic detection of transformed Salmonella typhimurium (S. typh-lux) through porcine skin. Skin was placed on 96-well plates containing S. typh-lux, imaged (5 min) using a CCD camera, and then completely immersed in PBS, GLY, DMSO, GLY+DMSO in a dose- and time-dependent manner and re-imaged (5 min). The percent of photonic emissions detected (treated or untreated skin relative to no skin controls) was used for analysis. Treatment for 4 h with 50% GLY-PBS and 50:30:20% GLY:DMSO:PBS increased photonic detection compared to untreated skin, 100% PBS, or 30:70% DMSO:PBS. Treatment with 50% GLY in the presence of 20 and 40% DMSO (v/v with PBS) increased photonic detection compared to 50% GLY alone and in the presence of 10% DMSO: 50% GLY (v/v with PBS). Data indicate that GLY and GLY+DMSO are effective optical clearing agents on porcine skin (2–3 mm thick) when treated for 4 h to increase detection of emitted photons. Clearing agents such as GLY have the potential to minimize effects of porcine skin tissue as one of the photon transmittance barriers (i.e., skin, fat, muscle, and visceral tissues) in vivo.

We describe a new optical method for noninvasive blood glucose (BGL) measurement. Optical methods are confounded by basal optical properties of tissues, especially water and other biochemical species, and by the very small glucose signal. We address these problems by using fast spectrophotometric analysis in a finger, deriving 100 transmittance spectra per second, to resolve optical spectra (900 to 1700 nm) of blood volume pulsations throughout the cardiac cycle. Difference spectra are calculated from the pulsatile signals, thereby eliminating the effects of bone, other tissues, and nonpulsatile blood. A partial least squares (PLS) model is used with the measured spectral data to predict BGL levels. Using glucose tolerance tests in 27 healthy volunteers, periodic optical measurements were made simultaneously with collection of blood samples for in vitro glucose analysis. Altogether, 603 paired data sets were obtained in all subjects and two-thirds of the data or of the subjects randomly selected were used for the PLS calibration model and the rest for the prediction. Bland-Altman and error-grid analyses of the predicted and measured BGL levels indicated clinically acceptable accuracy. We conclude that the new method, named pulse glucometry, has adequate performance for safe, noninvasive estimation of BGL.

We used the effect of temperature on the localized reflectance of human skin to assess the role of noise sources on the correlation between temperature-induced fractional change in optical density of human skin (ODT) and blood glucose concentration [BG]. Two temperature-controlled optical probes at 30°C contacted the skin, one was then cooled by –10°C; the other was heated by +10°C. ODT upon cooling or heating was correlated with capillary [BG] of diabetic volunteers over a period of three days. Calibration models in the first two days were used to predict [BG] in the third day. We examined the conditions where the correlation coefficient (R2) for predicting [BG] in a third day ranked higher than R2 values resulting from fitting permutations of randomized [BG] to the same ODT values. It was possible to establish a four-term linear regression correlation between ODT upon cooling and [BG] with a correlation coefficient higher than that of an established noise threshold in diabetic patients that were mostly females with less than 20 years of diabetes duration. The ability to predict [BG] values with a correlation coefficient above biological and body-interface noise varied between the cases of cooling and heating.

The ways to the polarization singularities of the biological tissues images of various morphological structures have been theoretically analyzed. The coordinate distributions of singly (linear states of polarization) and doubly degenerated (circular states of polarization) polarization singularities of the physiologically normal and pathologically changed biological tissues have been experimentally examined. The statistical criteria of diagnostics of the kidney tissue collagenous disease (the third and the fourth statistical moments of the linear density singularity points) have been determined. It was discovered that the process of the pathological change of the kidney tissue morphology leads to the formation of the self-similar (fractal) coordinate distribution of the polarization singularities of its image.

A need exists for the continued development of diagnostic tools and methods capable of distinguishing and characterizing slight differences in the optical properties of tissues. We present a method to estimate the scattering coefficient contribution as a function of particle size in complex mixtures of polystyrene spheres. The experimental method we used is a Mueller matrix imaging approach. The Mueller matrix encodes the polarization-dependent properties of the sample and describes how a given sample will transform an incident light polarization state. A partial least-squares approach is used to form a model around a set of Mueller matrix image-based measurements to accurately predict the individual scattering coefficient contributions in phantoms containing 0.2, 0.5, 1, and 2 µm-diameter polystyrene spheres. The results show individual scattering coefficient contribution errors as low as 0.1585 cm–1 can be achieved. In addition, it is shown how the scattering type (i.e., Rayleigh and Mie) is encoded within the Mueller matrix. Such methods may eventually lead to the development of improved diagnostic tools capable of characterizing and distinguishing between tissue abnormalities, such as superficial cancerous lesions from their benign counterparts.

Cancer cell motility and invasion are critical targets for anticancer therapeutics. Whereas in vitro models could be designed for rapid screening with a view to investigate these targets, careful consideration must be given to the construction of appropriate model systems. Most investigations focus on two-dimensional (2-D) assays despite the fact that increasing evidence suggests that migration across rigid and planar substrates fails to recapitulate in vivo behavior. In contrast, few systems enable three-dimensional (3-D) cell migration to be quantitatively analyzed. We previously developed a digital holographic microscope (DHM) working in transmission with a partially spatial coherence source. This configuration avoids the noise artifacts of laser illumination and makes possible the direct recording of information on the 3-D structure of samples consisting of multiple objects embedded in scattering media, such as cell cultures in matrix gels. The software driving our DHM system is equipped with a time-lapse ability that enables the 3-D trajectories of living cells to be reconstituted and quantitatively analyzed.

The clinical significance of a burn depends on the percentage of total body involved and the depth of the burn. Hence a noninvasive method that is able to evaluate burn depth would be of great help in clinical evaluation. To this end, photoacoustic microscopy is used to determine the depth of acute thermal burns by imaging the total hemoglobin concentration in the blood that accumulates along the boundaries of injuries as a result of thermal damage to the vasculature. We induce acute thermal burns in vivo on pig skin with cautery. Photoacoustic images of the burns are acquired after skin excision. In a burn treated at 175°C for 20 s, the maximum imaged burn depth is 1.73±0.07 mm. In burns treated at 150°C for 5, 10, 20, and 30 s, respectively, the trend of increasing maximum burn depth with longer thermal exposure is demonstrated.

In vivo, label-free, high-speed (up to 10,000 with the potential for 40,000 frames per second), high-resolution (up to 300 nm) real-time continuous imaging with successive framing of circulating individual erythrocytes, leukocytes, and platelets in fast blood flow is developed. This technique, used in an animal model, reveals the extremely high dynamic deformability of erythrocytes in natural flow. Potential applications of this technique are discussed with focus on time-resolved monitoring of the cell deformation dynamics in the native biological environment, which may have diagnostic value for the early diagnosis of diseases.

We demonstrate the feasibility of white-light time-resolved optical mammography. The instrumentation is based on supercontinuum light generated in photonic crystal fiber and 32-channel parallel time-correlated single-photon-counting detection. Total measurement time is of the order of 10 min for typical clinical applications. Preliminary measurements performed on volunteers show the ability of the system to determine tissue constituent concentrations and structure over the entire breast area. Furthermore, measurements on a tissue-like sample demonstrate detection and characterization of inclusions.

We describe the phenomenon of a sudden decrease in the amplitude of photoacoustic signals arising from nanosecond laser pulse irradiation of biological samples, measured in vitro. Several dental enamel and chicken/turkey breast samples are examined. Moderate optical energy densities (i.e., about 300 mJ/cm2) are used, typical of those exploited in photoacoustic investigations. Measurements show a rapid decay of photoacoustic signals within the first few laser pulses absorbed by the sample. This phenomenon indicates that laser irradiation interacts with biological samples, causing long-term physical changes that can be attributed to a reduction of optical absorption within the samples.

This PDF file contains the errata for “JBO Vol. 11 Issue 05 Paper 2348896” for JBO Vol. 11 Issue 05

This PDF file contains the errata for “JBO Vol. 11 Issue 05 Paper 2362857” for JBO Vol. 11 Issue 05

This PDF file contains the errata for “JBO Vol. 11 Issue 05 Paper JBO059803ERR” for JBO Vol. 11 Issue 05