Low-intensity, pulsed infrared light provides a novel nerve stimulation modality that avoids the limitations of traditional electrical methods such as necessity of contact, presence of a stimulation artifact, and relatively poor spatial precision. Infrared neural stimulation (INS) is, however, limited by a 2:1 ratio of threshold radiant exposures for damage to that for stimulation. We have shown that this ratio is increased to nearly 6:1 by combining the infrared pulse with a subthreshold electrical stimulus. Our results indicate a nonlinear relationship between the subthreshold depolarizing electrical stimulus and additional optical energy required to reach stimulation threshold. The change in optical threshold decreases linearly as the delay between the electrical and optical pulses is increased. We have shown that the high spatial precision of INS is maintained for this combined stimulation modality. Results of this study will facilitate the development of applications for infrared neural stimulation, as well as target the efforts to uncover the mechanism by which infrared light activates neural tissue.

Accurate, unambiguous detection of molecular interactions in living cells via measurements of Förster (or fluorescence) resonance energy transfer (FRET) events is experimentally challenging. We develop and apply a physiological fluorescence lifetime imaging microscopy (physiological FLIM) system to significantly improve FRET detection in living cells. Multiple positive and negative cellular controls are implemented to validate the experimental method developed. FLIM measurement techniques were found to remove fluorescence intensity-based artifacts, resulting in a seven-fold improvement in fluorescence measurement precision. The addition of cellular environmental controls, including both temperature and CO₂ stabilization, for physiological FLIM eliminates nonspecific FRET in the live-cell system studied. Overall, only physiological FLIM results in statistically significant results that clearly indicated the presence of specific molecular interactions in the live-cell system. This approach can be applied generally to improve the accuracy and precision of FRET measurements in living cells.

We report on the feasibility of rapid, high-resolution, 3-D swept-source optical coherence tomography (SSOCT) to detect early airway injury changes following smoke inhalation exposure in a rabbit model. The SSOCT system obtains 3-D helical scanning using a microelectromechanical system motor-based endoscope. Real-time 2-D data processing and image display at the speed of 20 frames/s are achieved by adopting the technique of parallel computing. Longitudinal images are reconstructed via an image processing algorithm to remove motion artifacts caused by ventilation and pulse. Quantitative analyses of tracheal airway thickness as well as thickness distribution along tracheal circumference are also performed based on the comprehensive 3-D volumetric data.

We describe a wide-field preclinical imaging system optimized for time-gated detection of quantum dot fluorescence emission. As compared to continuous wave measurements, image contrast was substantially improved by suppression of short-lifetime background autofluorescence. Real-time (8 frames/s) biological imaging of subcutaneous quantum dot injections is demonstrated simultaneously in multiple living mice.

The ability to in vivo image deep tissues noninvasively with a high resolution is strongly required for optical virtual biopsy. Higher harmonic generation microscopy, combined with second- and third-harmonic generation microscopies, is applied to 17 Asian volunteers' forearm skin. After continuous observation for 30 min, no visible damage was found. Our study proves that harmonic generation biopsy (HGB) is able to satisfy the safety requirement and to provide high penetrability (~300 μm) and submicron resolution all at the same time and is a promising tool for future virtual biopsy of skin diseases. In contrast to a previous study on fixed human skin specimens, a much improved penetrability and much reduced resolution-degradation versus depth are found in this in vivo examination.

Fourier domain optical coherence tomography (FD-OCT) requires resampling of spectrally resolved depth information from wavelength to wave number, and the subsequent application of the inverse Fourier transform. The display rates of OCT images are much slower than the image acquisition rates due to processing speed limitations on most computers. We demonstrate a real-time display of processed OCT images using a linear-in-wave-number (linear-k) spectrometer and a graphics processing unit (GPU). We use the linear-k spectrometer with the combination of a diffractive grating with 1200 lines/mm and a F2 equilateral prism in the 840-nm spectral region to avoid calculating the resampling process. The calculations of the fast Fourier transform (FFT) are accelerated by the GPU with many stream processors, which realizes highly parallel processing. A display rate of 27.9 frames/sec for processed images (2048 FFT size×1000 lateral A-scans) is achieved in our OCT system using a line scan CCD camera operated at 27.9 kHz.

An abnormal level of red blood cell (RBC) aggregation is a clinical condition associated with many pathologies. Our purpose is to investigate the feasibility of spectral domain phase-resolved optical Doppler tomography incorporated with Doppler variance imaging to characterize RBC aggregation in flowing blood. Variance/standard deviation (SD) of the Doppler frequency spectrum of porcine blood flowing through a glass tube (diameter 300 μm) at 4.7 mm/s is measured. For nonaggregating RBCs with hematocrits of 20, 40, and 60%, the mean centerline SD values were 34.2±1.2, 47.8±1.0, and 47.2±0.5, respectively. After dextran 500 induction, the SD value decreased significantly to 29.9±0.7, 37.9±3.0, and 40.1±0.6 (P<0.01) in the aggregated blood, respectively. The results demonstrate that Doppler variance imaging using the SD value is capable of differentiating normal and aggregated blood.

Finding a way to combine ultrasound and fluorescence optical imaging on an endorectal probe may improve early detection of prostate cancer. The ultrasound provides morphological information about the prostate, while the optical system detects and locates fluorophore-marked tumors. A tissue-mimicking phantom, which is representative of prostate tissues both on its optical (absorption μ_a and diffusion μ^′_s) and its ultrasound properties, has been made by our team. A transrectal probe adapted to fluorescence diffuse optical tomography measurements was also developed. Measurements were taken on the prostate phantom with this probe based on a pulsed laser and a time-resolved detection system. A reconstruction algorithm was then used to help locate and quantify fluorescent inclusions of different concentrations at fixed depths.

We present for the first time in vivo full three-dimensional (3-D) photoacoustic tomography (PAT) of the distal interphalangeal joint in a human subject. Both absorbed energy density and absorption coefficient images of the joint are quantitatively obtained using our finite-element-based photoacoustic image reconstruction algorithm coupled with the photon diffusion equation. The results show that major anatomical features in the joint along with the side arteries can be imaged with a 1-MHz transducer in a spherical scanning geometry. In addition, the cartilages associated with the joint can be quantitatively differentiated from the phalanx. This in vivo study suggests that the 3-D PAT method described has the potential to be used for early diagnosis of joint diseases such as osteoarthritis and rheumatoid arthritis.

It is well known that the inverse problem in optical tomography is highly ill-posed. The image reconstruction process is often unstable and nonunique, because the number of the boundary measurements data is far fewer than the number of the unknown parameters to be reconstructed. To overcome this problem, one can either increase the number of measurement data (e.g., multispectral or multifrequency methods), or reduce the number of unknowns (e.g., using prior structural information from other imaging modalities). We introduce a novel approach for reducing the unknown parameters in the reconstruction process. The discrete cosine transform (DCT), which has long been used in image compression, is here employed to parameterize the reconstructed image. In general, only a few DCT coefficients are needed to describe the main features in an optical tomographic image. Thus, the number of unknowns in the image reconstruction process can be drastically reduced. We show numerical and experimental examples that illustrate the performance of the new algorithm as compared to a standard model-based iterative image reconstructions scheme. We especially focus on the influence of initial guesses and noise levels on the reconstruction results.

We report the quantitative measurements of optical and physiological parameters of normal breasts from 19 Singapore women by using time-resolved diffuse optical spectroscopy. Intrinsic absorption coefficient (μ_a) and reduced scattering coefficients (μ^′_s) of breasts were calculated from the time-resolved photon migration data. Physiology of breasts was characterized using the concentrations of oxyhemoglobin, deoxyhemoglobin, total hemoglobin (THC), and oxygenation saturation. On average, the experiment results showed that the μ_a of young women (below 40 years old) was 36 to 38% greater than that of older women (above 40 years old) and that parameter THC was approximately 42% greater. Results also showed that the THC of premenopausal women was 24.3 μMol/L, which was approximately 69% larger than that of postmenopausal women at 14.1 μMol/L. Meanwhile, the µa of premenopausal women was approximately 60% larger than that of postmenopausal women. Correlation analysis further showed that the optical and physiological parameters of breasts were strongly influenced by changes in the women's age, menopausal states, and body mass index. These in vivo experiment results will contribute to the breast tissue diagnosis between healthy and diseased breast tissues.

We studied the dynamics of two types of intracellular probe particles, ballistically injected latex spheres and endogenous granules, in tumor cell lines of differerent metastatic potential: breast tumor cells (MCF-7 malignant, MCF-10A benign) and pancreas adenocarcinoma (PaTu8988T malignant, PaTu8988S benign). For both tissue types and for both probes, the mean squared displacement (MSD) function measured in the malignant cells was substantially larger than in the benign cells. Only a few cells were needed to characterize the tissue as malignant or benign based on their MSD, since variations in MSD within the same cell line were relatively small. These findings suggest that intracellular particle tracking (IPT) can serve as a simple and reliable method for characterization of cell states obtained from a small amount of cell sample. Mechanical analysis of the same cell lines with atomic force microscopy (AFM) in force-distance mode revealed that AFM could distinguish between the benign and malignant breast cancer cells but not the pancreatic tumor cell lines. This underlines the potential value of IPT as a complementary nanomechanical tool for studying cell-state-dependent mechanical properties.

As part of an ongoing program to develop two-photon (2-γ)photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-γ and 2-γ activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-γ cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-γ PDT of AMD.

We develop a system for three-dimensional whole-body optoacoustic tomography of small animals for applications in preclinical research. The tomographic images are obtained while the objects of study (phantoms or mice) are rotated within a sphere outlined by a concave arc-shaped array of 64 piezocomposite transducers. Two pulsed lasers operating in the near-IR spectral range (755 and 1064 nm) with an average pulsed energy of about 100 mJ, a repetition rate of 10 Hz, and a pulse duration of 15 to 75 ns are used as optical illumination sources. During the scan, the mouse is illuminated orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. The system is capable of generating images of individual organs and blood vessels through the entire body of a mouse with spatial resolution of ~0.5 mm.

Measurements of oxygen saturation and flow in the retina can yield information about eye health and the onset of eye pathologies such as diabetic retinopathy. Recently, we developed a multiaperture camera that uses the division of the retinal image into several wavelength-sensitive subimages to compute retinal oxygen saturation. The calibration of such instruments is particularly difficult due to the layered structure of the eye and the lack of alternative measurement techniques. For this purpose, we realize an in vitro model of the human eye composed of a lens, the retina vessel, and three layers: the choroid, the retinal pigmented epithelium, and the sclera. The retinal vessel is modeled with a microtube connected to a micropump and a hemoglobin reservoir in a closed circulatory system. Hemoglobin oxygenation in the vessel could be altered using a reversible fuel cell. The sclera is represented by a Spectralon slab. The optical properties of the other layers are mimicked using titanium dioxide as a scatterer, ink as an absorber, and epoxy as a supporting structure. The optical thickness of each layer of the eye phantom is matched to each respective eye layer.

The fluorescence lifetime technique offers an effective way to resolve fluorescent components with overlapping emission spectra. The presence of multiple fluorescent components in biological compounds can hamper their discrimination. The conventional method based on the nonlinear least-squares technique is unable to consistently determine the correct number of fluorescent components in a fluorescence decay profile. This can limit the applications of the fluorescence lifetime technique in biological assays and diagnoses where more than one fluorescent component is typically encountered. We describe the use of an expectation-maximization (EM) method with joint deconvolution to estimate the fluorescence decay parameters, and the Bayesian information criterion (BIC) to accurately determine the number of fluorescent components. A comprehensive simulation and experimental study is carried out to compare the performance and accuracy of the proposed method. The results show that the EM-BIC method is able to accurately identify the correct number of fluorescent components in samples with weakly fluorescing components.

We present a 3-D image reconstruction method for free-space fluorescence tomography of mice using hybrid anatomical prior information. Specifically, we use an optically reconstructed surface of the experimental animal and a digital mouse atlas to approximate the anatomy of the animal as structural priors to assist image reconstruction. Experiments are carried out on a cadaver of a nude mouse with a fluorescent inclusion (2.4-mm-diam cylinder) implanted in the chest cavity. Tomographic fluorescence images are reconstructed using an iterative algorithm based on a finite element method. Coregistration of the fluorescence reconstruction and micro-CT (computed tomography) data acquired afterward show good localization accuracy (localization error 1.2±0.6 mm). Using the optically reconstructed surface, but without the atlas anatomy, image reconstruction fails to show the fluorescent inclusion correctly. The method demonstrates the utility of anatomical priors in support of free-space fluorescence tomography.

Spectral unmixing is a useful technique in fluorescence imaging for reducing the effects of native tissue autofluorescence and separating multiple fluorescence probes. While spectral unmixing methods are well established in fluorescence microscopy, they typically rely on precharacterized in-vitro spectra for each fluorophore. However, there are unique challenges for in-vivo applications, since the tissue absorption and scattering can have a significant impact on the measured spectrum of the fluorophore, and therefore make the in-vivo spectra substantially different to that of in vitro. In this work, we introduce a spectral unmixing algorithm tailored for in-vivo optical imaging that does not rely on precharacterized spectral libraries. It is derived from a multivariate curve resolution (MCR) method, which has been widely used in studies of chemometrics and gene expression. Given multispectral images and a few straightforward constraints such as non-negativity, the algorithm automatically finds the signal distribution and the pure spectrum of each component. Signal distribution maps help separate autofluorescence from other probes in the raw images and hence provide better quantification and localization for each probe. The algorithm is demonstrated with an extensive set of in-vivo experiments using near-infrared dyes and quantum dots in both epi-illumination and transillumination geometries.

We present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.

Apoptosis-programmed cell death-is a cellular process exhibiting distinct biochemical and morphological changes. An understanding of the early morphological changes that a cell undergoes during apoptosis can provide the opportunity to monitor apoptosis in tissue, yielding diagnostic and prognostic information. There is avid interest regarding the involvement of apoptosis in cancer. The initial response of a tumor to successful cancer treatment is often massive apoptosis. Current apoptosis detection methods require cell culture disruption. Our aim is to develop a nondisruptive optical method to monitor apoptosis in living cells and tissues. This would allow for real-time evaluation of apoptotic progression of the same cell culture over time without alteration. Elastic scattering spectroscopy (ESS) is used to monitor changes in light-scattering properties of cells in vitro due to apoptotic morphology changes. We develop a simple instrument capable of wavelength-resolved ESS measurements from cell cultures in the backward direction. Using Mie theory, we also develop an algorithm that extracts the size distribution of scatterers in the sample. The instrument and algorithm are validated with microsphere suspensions. For cell studies, Chinese hamster ovary (CHO) cells are cultured to confluence on plates and are rendered apoptotic with staurosporine. Backscattering measurements are performed on pairs of treated and control samples at a sequence of times up to 6-h post-treatment. Initial results indicate that ESS is capable of discriminating between treated and control samples as early as 10- to 15-min post-treatment, much earlier than is sensed by standard assays for apoptosis. Extracted size distributions from treated and control samples show a decrease in Rayleigh and 150-nm scatterers, relative to control samples, with a corresponding increase in 200-nm particles. Work continues to correlate these size distributions with underlying morphology. (Partial Abstract)

A fluorescent analog to 2-deoxy-2 [¹⁸F] fluoro-D-glucose position emission tomography (FDG-PET) would allow for the introduction of metabolic imaging into intraoperative and minimally invasive settings. We present through in vitro and in vivo experimentation an evaluation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescently labeled glucose molecule, as a molecular beacon of glucose utilization. The competitive inhibition of 2-NBDG uptake by excess free glucose is directly compared against FDG uptake inhibition in cultured cells. 2-NBDG uptake in the brain of a mouse experiencing a generalized seizure is measured, as well as in subcutaneously implanted tumors in mice during fed and fasting states. Localization of 2-NBDG into malignant tissues is studied by laser scanning microscopy. The clinical relevance of 2-NBDG imaging is examined by performing fluorescence colonoscopy, and by correlating preoperative FDG-PET with intraoperative fluorescence imaging. 2-NBDG exhibits a similar uptake inhibition to FDG by excess glucose in the growth media. Uptake is significantly increased in the brain of an animal experiencing seizures versus control, and in subcutaneous tumors after the animals are kept nil per os (NPO) for 24 h versus ad libidum feeding. The clinical utility of 2-NBDG is confirmed by the demonstration of very high target-to-background ratios in minimally invasive and intraoperative imaging of malignant lesions. We present an optical analog of FDG-PET to extend the applicability of metabolic imaging to minimally invasive and intraoperative settings.

Chemotaxis has a meaningful role in several fields, such as microbial physiology, medicine and biotechnology. We present a new application of dynamic laser speckle (or biospeckle) to detect different degrees of bacterial motility during chemotactic response experiments. Encouraging results showed different bacterial dynamic responses due to differences in the hardness of the support in the swarming plates. We compare this method to a conventional technique that uses white light. Both methods showed to be analogous and, in some cases, complementary. The results suggest that biospeckle processed images can be used as an alternative method to evaluate bacterial chemotactic response and can supply additional information about the bacterial motility in different areas of the swarm plate assay that might be useful for biological analysis.

The common method of closing conjunctival incisions is by suturing, which is associated with several disadvantages. It requires skill to apply and does not always provide a watertight closure, which is required in some operations (e.g., glaucoma filtration). The purpose of the present study was to evaluate laser soldering as an alternative method for closing conjunctival incisions. Conjunctival incisions of 20 ex vivo porcine eyes were laser soldered using a temperature-controlled fiberoptic laser system and an albumin mixed with indocyanine green as a solder. The control group consisted of five repaired incisions by a 10-0 nylon running suture. The leak pressure of the repaired incisions was measured. The mean leak pressure in the laser-soldered group was 132 mm Hg compared to 4 mm Hg in the sutured group. There was no statistically significant difference in both the incision's length and distance from the limbus between the groups, before and after the procedure, indicating that there was no severe thermal damage. These preliminary results clearly demonstrate that laser soldering may be a useful method for achieving an immediate watertight conjunctival wound closure. This procedure is faster and easier to apply than suturing.

We demonstrate how the terahertz properties of porcine adipose tissue and skeletal muscle are affected by formalin fixing. Terahertz radiation is sensitive to covalently cross-linked proteins and can be used to probe unique spectroscopic signatures. We study in detail the changes arising from different fixation times and see that formalin fixing reduces the refractive index and the absorption coefficient of the samples in the terahertz regime. These fundamental properties affect the time-domain terahertz response of the samples and determine the level of image contrast that can be achieved.

In multiphoton fluorescence laser-scanning microscopy, ultrafast laser pulses [i.e., light pulses having pulse width ≤1 ps (1 ps=10^-12 s)] are commonly employed to circumvent the low-multiphoton absorption cross-sections of common fluorophores. Because of the broad overlapping two-photon absorption spectra of fluorophores and the large spectral bandwidth of a short pulse, simultaneous excitation of many fluorophores is common, which justifies a persistent demand for selective excitation of individual fluorophores. We describe the use of pulse-pair excitation with possibilities of controlling molecular fluorescence in laser-scanning microscopy and compare it with coherent control using pulse sequence [De and Goswami, "Coherent control in multiphoton fluorescence imaging," Proc. SPIE 7183, 71832B (2009)].

We report results of a study to evaluate effectiveness of a mechanical tissue optical clearing device (TOCD) using optical coherence tomography (OCT). The TOCD uses a pin array and vacuum pressure source to compress localized regions of the skin surface. OCT images (850 and 1310 nm) of in vivo human skin indicate application of the TOCD provides an up to threefold increased light penetration depth at spatial positions correlated with pin indentations. Increased contrast of the epidermal-dermal junction in OCT images spatially correlates with indented zones. OCT M-scans recorded while applying the TOCD indicate optical penetration depth monotonically increased, with most improvement at early times (5 to 10 s) of TOCD vacuum application. OCT M-scans of ex vivo porcine skin compressed using the TOCD suggest average group refractive index of the tissue increased, corresponding to a decrease in water concentration. Results of our study indicate that mechanical optical clearing of skin may provide an effective and efficient means to deliver increased light fluence to dermal and subdermal regions.

Optical coherence tomography (OCT) is an evolving noninvasive imaging modality that has been used to image the human larynx during surgical endoscopy. The design of a long gradient index (GRIN) lens-based probe capable of capturing images of the human larynx by use of swept-source OCT during a typical office-based laryngoscopy examination is presented. In vivo OCT imaging of the human larynx is demonstrated with a rate of 40 frames per second. Dynamic vibration of the vocal folds is recorded to provide not only high-resolution cross-sectional tissue structures but also vibration parameters, such as the vibration frequency and magnitude of the vocal cords, which provides important information for clinical diagnosis and treatment, as well as fundamental research of the voice itself. Office-based OCT is a promising imaging modality to study the larynx for physicians in otolaryngology.

We investigated fabrication of cylindrical micropillars on bovine cortical bone using direct-write femtosecond laser ablation. The ablation threshold of the material was measured by single-pulse ablation tests, and the incubation coefficient was measured from linear scanned ablation tests. A motion system was programmed to apply multiple layers of concentric rings of pulses to machine pillars of various diameters and heights. The diameter of the top surface of the pillar was found to steadily decrease due to incubation of damage from successive layers of pulses during the machining process. Pillar top diameter was predicted based on a paraxial beam fluence approximation and single-pulse ablation threshold and incubation coefficient measurements. Pillar diameters predicted as successive layers of pulses were applied were well-matched to experiments, confirming that femtosecond laser ablation of the cortical bone was well-modeled by single-pulse ablation threshold measurements and an incubation coefficient.

Optically sectioned detection of fluorescence immunoassays using a confocal microscope enables the creation of both homo- and heterogeneous planar format assays. We report a set assays requiring optically sectioned detection using a model system and analysis procedures for separating signals of a surface layer from an overlying solution. A model sandwich assay with human immunoglobulin G as the target antigen is created on a glass substrate. The prepared surfaces are exposed to antigen and a FITC-labeled secondary antibody. The resulting preparations are either read directly to provide a homogeneous assay or after wash steps, giving a heterogeneous assay. The simplicity of the object shapes arising from the planar format makes the decomposition of analyte signals from the thin film bound to the surface and overlayer straightforward. Measured response functions of the thin film and overlayer fit well to the Cauchy-Lorentz and cumulative Cauchy-Lorentz functions, respectively, enabling the film and overlayer to be separated. Under the conditions used, the detection limits for the homogeneous and heterogeneous forms of the assay are 2.2 and 5.5 ng/ml, respectively. Planar format, confocally read fluorescence assays enable wash-free detection of antigens and should be applicable to a wide range of assays involving surface-bound species.

We determine the reliability and reproducibility of retinal thickness measurements with a custom-built OCT retinal image analysis software (OCTRIMA). Ten eyes of five healthy subjects undergo repeated standard macular thickness map scan sessions by two experienced examiners using a Stratus OCT device. Automatic/semi automatic thickness quantification of the macula and intraretinal layers is performed using OCTRIMA software. Intraobserver, interobserver, and intervisit repeatability and reproducibility coefficients, and intraclass correlation coefficients (ICCs) per scan are calculated. Intraobserver, interobserver, and intervisit variability combined account for less than 5% of total variability for the total retinal thickness measurements and less than 7% for the intraretinal layers except the outer segment/ retinal pigment epithelium (RPE) junction. There is no significant difference between scans acquired by different observers or during different visits. The ICCs obtained for the intraobserver and intervisit variability tests are greater than 0.75 for the total retina and all intraretinal layers, except the inner nuclear layer intraobserver and interobserver test and the outer plexiform layer, intraobserver, interobserver, and intervisit test. Our results indicate that thickness measurements for the total retina and all intraretinal layers (except the outer segment/RPE junction) performed using OCTRIMA are highly repeatable and reproducible.

We present an experimental comparison of pulsed photothermal (PPT) profiling in collagen-based tissue phantoms utilizing different midinfrared spectral bands. Laser-induced temperature profiles are reconstructed using a custom optimization code within the customary monochromatic approximation. Both experimental results and a detailed numerical simulation of the procedure demonstrate that, despite the associated reduction of signal-to-noise ratio, appropriate spectral filtering reduces the broadening of temperature peaks and thus improves the accuracy of temperature profiling. For our experimental system, best performance is obtained when applying a long-pass filter with cut-on wavelength at 3.4-3.8 μm. Because our collagen gel mimics infrared and thermal properties of human skin, we believe that this conclusion is transferrable to PPT radiometric profiling of human skin in vivo.

The performance of near-infrared spectroscopy is sometimes degraded by the systemic physiological interference in the extracerebral layer. There is some systemic interference, which is highly correlated with the functional response evoked by a task execution. This kind of interference is difficult to remove by using ordinary techniques. A multidistance measurement method is one of the possible solutions for this problem. The multidistance measurement method requires estimation parameters derived from partial pathlength values of tissue layers to calculate an absorption coefficient change from a temporal absorbance change. Because partial path lengths are difficult to obtain, experimentally, we estimated them by a Monte Carlo simulation based on a five-layered slab model of a human adult head. Model parameters such as thickness and the transport scattering coefficient of each layer depend on a subject and a measurement position; thus, we assumed that these parameters obey normal distributions around standard parameter values. We determined the estimation parameters that provide a good separation performance in average for the model parameter distribution. The obtained weighting is robust to model parameter deviation and provides smaller errors on average compared to the parameters, which are determined without considering parameter distribution.

We demonstrate a first application, of optical-feedback cavity-enhanced absorption spectroscopy (OF-CEAS) to breath analysis in a medical environment. Noninvasive monitoring of trace species in exhaled air was performed simultaneous to spirometric measurements on patients at Bichat Hospital (Paris). The high selectivity of the OF-CEAS spectrometer and a time response of 0.3 s (limited by sample flow rate) allowed following the evolution of carbon monoxide and methane concentrations during individual respiratory cycles, and resolving variations among different ventilatory patterns. The minimum detectable absorption on this time scale is about 3×10^-10 cm^-1. At the working wavelength of the instrument (2.326 μm), this translates to concentration detection limits of ~1 ppbv (45 picomolar, or ~1.25 μg/m³) for CO and 25 ppbv for CH₄, well below concentration values found in exhaled air. This same instrument is also able to provide measurement of NH₃ concentrations with a detection limit of ~10 ppbv; however, at present, memory effects do not allow its measurement on fast time scales.

Shape-dependent sensitivity of localized surface plasmon-based biosensing was investigated by combining single-particle protein-sensing and multiple multipole program simulation. Significantly higher sensitivity was observed for tetrahedral particles than spherical ones, which was revealed by careful structural analysis of individually measured particles. The simulation of the corresponding particles with layered protein adsorption model showed consistent optical property and sensitivity, which were explained in terms of the field enhancement at the pointing edges.

Fiber-optic image guides in confocal reflectance endomicroscopes introduce background backscatter that limits the achievable contrast in these devices. We show the dominant source of backscatter from the image guide is due to Rayleigh scattering at short wavelengths and terminal reflections of the fibers at long wavelengths. The effective Rayleigh scattering coefficient and the wavelength-independent reflectivity due terminal reflections are measured experimentally in a commercial image guide. The Rayleigh scattering component of backscatter can be accurately predicted using the fractional refractive-index difference and length of the fibers in the image guide. We also presented a simple model that can be used to predict signal-to-background ratio in a fiber-optic confocal reflectance endomicroscope for biologically relevant tissues and contrast agents that cover a wide range of reflectivity.

A lateral shearing interferometer was used to examine the smoothness of the tear film. The information about the distribution and stability of the precorneal tear film is carried out by the wavefront reflected from the surface of tears and coded in interference fringes. Smooth and regular fringes indicate a smooth tear film surface. On corneae after laser in situ keratomileusis (LASIK) or radial keratotomy (RK) surgery, the interference fringes are seldom regular. The fringes are bent on bright lines, which are interpreted as tear film breakups. The high-intensity pattern seems to appear in similar location on the corneal surface after refractive surgery. Our purpose was to extract information about the pattern existing under the interference fringes and calculate its shape reproducibility over time and following eye blinks. A low-pass filter was applied and correlation coefficient was calculated to compare a selected fragment of the template image to each of the following frames in the recorded sequence. High values of the correlation coefficient suggest that irregularities of the corneal epithelium might influence tear film instability and that tear film breakup may be associated with local irregularities of the corneal topography created after the LASIK and RK surgeries.

We have developed a novel dual-window approach for spectroscopic optical coherence tomography (OCT) measurements and applied it to probe nuclear morphology in tissue samples drawn from the hamster cheek pouch carcinogenesis model. The dual-window approach enables high spectral and depth resolution simultaneously, allowing detection of spectral oscillations, which we isolate to determine the structure of cell nuclei in the basal layer of the epithelium. The measurements were executed with our parallel frequency domain OCT system, which uses light from a thermal source, providing high bandwidth and access to the visible portion of the spectrum. The structural measurements show a highly statistically significant difference between untreated (normal) and treated (hyperplastic/dysplastic) tissues, indicating the potential utility of this approach as a diagnostic method.

Goblet cells are a requirement for the diagnosis of intestinal metaplasia of the stomach. The gastric mucosa is lined by a monolayer of columnar epithelium with some specialization at the crypts, but there are no goblet cells in normal gastric epithelium. The appearance of goblet cells in gastric epithelium is an indicator of potential malignant progression toward adenocarcinoma. Therefore, in vivo three-dimensional imaging of goblet cells is essential for diagnoses of a premalignant stage of gastric cancers called intestinal metaplasia. We used mouse intestine, which has goblet cells, as a model of intestinal metaplasia. One-photon confocal fluorescence endomicroscopy and two-photon fluorescence endomicroscopy are employed for 3-D imaging of goblet cells. The penetration depth, the sectioning ability, and the photobleaching information of imaging are demonstrated. Both endomicroscopy techniques can three-dimensionally observe goblet cells in mouse large intestine and achieve an imaging depth of 176 µm. The two-photon fluorescence endomicroscopy shows higher resolution and contrast of the imaging sections at each depth. In addition, two-photon fluorescence endomicroscopy also has advantages of sectioning ability and less photobleaching. These results prove that two-photon fluorescence endomicroscopy is advantageous in diagnoses of a premalignant stage of gastric cancers.

A pilot human study is conducted to evaluate the potential of using diffuse photon density wave (DPDW) methodology at near-infrared (NIR) wavelengths (685 to 830 nm) to monitor changes in tissue hemoglobin concentration in diabetic foot ulcers. Hemoglobin concentration is measured by DPDW in 12 human wounds for a period ranging from 10 to 61 weeks. In all wounds that healed completely, gradual decreases in optical absorption coefficient, oxygenated hemoglobin concentration, and total hemoglobin concentration are observed between the first and last measurements. In nonhealing wounds, the rates of change of these properties are nearly zero or slightly positive, and a statistically significant difference (p<0.05) is observed in the rates of change between healing and nonhealing wounds. Differences in the variability of DPDW measurements over time are observed between healing and nonhealing wounds, and this variance may also be a useful indicator of nonhealing wounds. Our results demonstrate that DPDW methodology with a frequency domain NIR device can differentiate healing from nonhealing diabetic foot ulcers, and indicate that it may have clinical utility in the evaluation of wound healing potential.

We developed a two-photon microscope optimized for physiologically manipulating single neurons through their postsynaptic receptors. The optical layout fulfills the stringent design criteria required for high-speed, high-resolution imaging in scattering brain tissue with minimal photodamage. We detail the practical compensation of spectral and temporal dispersion inherent in fast laser beam scanning with acousto-optic deflectors, as well as a set of biological protocols for visualizing nearly diffraction-limited structures and delivering physiological synaptic stimuli. The microscope clearly resolves dendritic spines and evokes electrophysiological transients in single neurons that are similar to endogenous responses. This system enables the study of multisynaptic integration and will assist our understanding of single neuron function and dendritic computation.

Functional near-infrared spectroscopy has the potential to easily detect cerebral functional hemodynamics. However, in practical fNIRS measurements, a subject's physical or systemic physiological activities often cause undesirable artifacts. Such activities can be evoked even by task execution. In this case, observed artifacts may correlate strongly with the task sequence, and it is difficult to eliminate them by conventional signal filtering techniques. We present a theoretical analysis and Monte Carlo simulations of layered media in which both scattering and absorption changes occur, and show that a multidistance probe arrangement is effective in removing artifacts and extracting functional hemodynamics. The probe arrangement is determined based on simulation results. Artifacts induced by nonfunctional tasks (body tilting, head nodding, and breath holding) are clearly observed when a conventional method is used; such artifacts are appreciably reduced by the proposed method. Signals evoked by single-sided finger movements are observed at both hemispheres when we use a conventional method. On the other hand, localized signals at the primary motor area are observed by the proposed method. A statistically significant increase in oxygenated hemoglobin and decrease in deoxygenated hemoglobin are simultaneously observed at the contralateral primary motor area

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

We characterize T- and B-lymphocytes from several donors, determining cell diameter, ratio of nucleus to cell diameter, and refractive index of the nucleus and cytoplasm for each individual cell. We measure light-scattering profiles with a scanning flow cytometer and invert the signals using a coated sphere as an optical model of the cell and by relying on a global optimization technique. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. However, the difference is smaller than the natural biological variability of a single cell. We propose nuclear inhomogeneity as a possible reason for the deviation of measured light-scattering profiles from real lymphocytes from those obtained from the coated sphere model.

Three-dimensional Fourier domain optical coherence tomography (3-D FDOCT) is used to demonstrate that perfusion fixation with a mixture of glutaraldehyde and paraformaldehyde does not alter the geometry of subpleural lung parenchyma in isolated and perfused rabbit lungs. This is confirmed by simultaneous imaging of lung parenchyma with intravital microscopy. To eliminate the diffraction index interfaces between alveolar pockets and walls, we fill the fixed lungs with ethanol by perfusing with gradually increasing concentrations. This bottom-up filling process leaves no remaining air bubbles in the alveolar structures, thus drastically improving the resolution and penetration depth of 3-D FDOCT imaging. We observe an approximately 18% increase in alveolar area after ethanol filling, likely due in large part to elimination of the air/tissue interfaces. 3-D OCT datasets acquired from ethanol-filled lungs allow segmentation of the ethanol-filled structures, which were formerly air-filled, and 3-D reconstruction of larger areas of subpleural alveolar structures. Our innovative process of filling the lungs with ethanol postperfusion fixation thus enables more accurate quantification of alveolar geometries, a critical component of modeling lung function.

Wide-field continuous wave fluorescence imaging, fluorescence lifetime imaging, frequency domain photon migration, and spatially modulated imaging have the potential to provide quantitative measurements in vivo. However, most of these techniques have not yet been successfully translated to the clinic due to challenging environmental constraints. In many circumstances, cardiac and respiratory motion greatly impair image quality and/or quantitative processing. To address this fundamental problem, we have developed a low-cost, field-programmable gate array-based, hardware-only gating device that delivers a phase-locked acquisition window of arbitrary delay and width that is derived from an unlimited number of pseudo-periodic and nonperiodic input signals. All device features can be controlled manually or via USB serial commands. The working range of the device spans the extremes of mouse electrocardiogram (1000 beats per minute) to human respiration (4 breaths per minute), with timing resolution ≤0.06%, and jitter ≤0.008%, of the input signal period. We demonstrate the performance of the gating device, including dramatic improvements in quantitative measurements, in vitro using a motion simulator and in vivo using near-infrared fluorescence angiography of beating pig heart. This gating device should help to enable the clinical translation of promising new optical imaging technologies.

Interferometry is a sensitive technique for recording tear film surface irregularities in a noninvasive manner. At the same time, the technique is hindered by natural eye movements resulting in measurement noise. Estimating tear film surface quality from interferograms can be reduced to a spatial-average-localized weighted estimate of the first harmonic of the interference fringes. However, previously reported estimation techniques proved to perform poorly in cases where the pattern fringes were significantly disturbed. This can occur in cases of measuring tear film surface quality on a contact lens on the eye or in a dry eye. We present a new estimation technique for extracting the first harmonic from the interference fringes that combines the traditional spectral estimation techniques with morphological image processing techniques. The proposed technique proves to be more robust to changes in interference fringes caused by natural eye movements and the degree of dryness of the contact lens and corneal surfaces than its predecessors, resulting in tear film surface quality estimates that are less noisy.

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

A fiber-optic surface plasmon resonance (SPR) sensor for the detection of human blood groups is proposed. Previous experimental results describing the wavelength-dependent refractive index variation of multiple samples of different blood groups are considered for theoretical calculations. The spectral interrogation method, along with silica fiber and silver layer, is considered. The sensor's performance is closely analyzed in terms of shift in SPR wavelength and SPR curve width in order to optimize the design parameters for a reliable and accurate blood-group identifier. The sensor design parameters include silver layer thickness, fiber core diameter, sensing region length, and temperature variation. The results are explained in terms of light coupling and plasmon resonance condition. The proposed sensing probe is able to provide high sensitivity and accuracy of blood-group detection, thereby opening an easy and reliable window for medical applications.

We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. The laser technology targets the global mechanical properties of the viral protein shell, making it relatively insensitive to the local genetic mutation in the target viruses. As a result, the approach can inactivate both the wild and mutated strains of viruses. This intriguing advantage is particularly important in the treatment of diseases involving rapidly mutating viral species such as HIV. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.

Gene therapy using wound healing-associated growth factor gene has received much attention as a new strategy for improving the outcome of tissue transplantation. We delivered plasmid DNA coding for human hepatocyte growth factor (hHGF) to rat free skin grafts by the use of laser-induced stress waves (LISWs); autografting was performed with the grafts. Systematic analysis was conducted to evaluate the adhesion properties of the grafted tissue; angiogenesis, cell proliferation, and reepithelialization were assessed by immunohistochemistry, and reperfusion was measured by laser Doppler imaging as a function of time after grafting. Both the level of angiogenesis on day 3 after grafting and the increased ratio of blood flow on day 4 to that on day 3 were significantly higher than those in five control groups: grafting with hHGF gene injection alone, grafting with control plasmid vector injection alone, grafting with LISW application alone, grafting with LISW application after control plasmid vector injection, and normal grafting. Reepithelialization was almost completed on day 7 even at the center of the graft with LISW application after hHGF gene injection, while it was not for the grafts of the five control groups. These findings demonstrate the validity of our LISW-based HGF gene transfection to accelerate the adhesion of grafted skins.

Despite growing clinical evidence of ablative fractional photothermolysis (AFP), little is known about the spatiotemporal molecular changes within the targeted compartments. Six subjects received three different single AFP treatments using a scanned 250 µm CO₂-laser beam. Spatiotemporal changes of skin regeneration were estimated by immunohistochemical investigation (HSP70, HSP72, HSP47, TGF^β, procollagen III, CD3, CD20, and CD68) in skin samples 1 h, 3 days, and 14 days postintervention. The remodeling was uniformly started by regrowth of the epidermal compartment followed by partial to complete replacement of the microscopic ablation zones (MAZ) by newly synthesized condensed procollagen III. From day 3 to 14, the number of macrophages as well as giant cells surrounding the MAZ increased. TGF^β expression was highest 1 h to 3 days following AFP. HSP70 and HSP72 expressions were highest 3-14 days postintervention in the spinocellular layer leading to an upregulation of HSP47. AFP performed by a scanned CO₂-laser results in an early epidermal remodeling, which is followed by a dermal remodeling leading to a replacement of the MAZ with newly synthesized (pro)-collagen. During this, an inflammatory infiltrate with CD3⁺ and CD20⁺ cells surrounds the MAZ. The count of macrophages and giant cells involved in the replacement of the necrotic zones seems to be crucial for wound healing.

The purpose of this study was to develop a dynamic tunable focal distance graded-refractive-index lens rod-based high-speed 3-D swept-source (SS) optical coherence tomography (OCT) endoscopic system and demonstrate real-time in vivo, high-resolution (10-μm) imaging of pleural-based malignancies in an animal model. The GRIN lens-based 3-D SS OCT system, which images at 39 fps with 512 A-lines per frame, was able to capture images of and detect abnormalities during thoracoscopy in the thoracic cavity, including the pleura, chest wall, pericardium, and the lungs. The abnormalities were confirmed by histological evaluation and compared to OCT findings. The dynamic tunable focal distance range and rapid speed of the probe and SS prototype OCT system enabled this first-reported application of in vivo 3-D thoracoscopic imaging of pleural-based malignancies. The imaging probe of the system was found to be easily adaptable to various sites within the thoracic cavity and can be readily adapted to other sites, including rigid airway endoscopic examinations.

A comparison of macular pigment optical density (MPOD) spatial profiles determined by an optical and a psychophysical technique is presented. We measured the right eyes of 19 healthy individuals, using fundus reflectometry at 0, 1, 2, 4, 6, and 8 deg eccentricity; and heterochromatic flicker photometry (HFP) at 0, 0.5, 1, 2, 3, 4, 5, 6, and 7 deg, and a reference point at 8 deg eccentricity. We found a strong correlation between the two techniques. However, the absolute estimates obtained by fundus reflectometry data were higher than by HFP. These differences could partly be explained by the fact that at 8 deg eccentricity the MPOD is not zero, as assumed in HFP. Furthermore, when performing HFP for eccentricities of <1 deg, we had to assume that subjects set flicker thresholds at 0.4 deg horizontal translation when using a 1-deg stimulus. MPOD profiles are very similar for both techniques if, on average, 0.05 DU is added to the HFP data at all eccentricities. An additional correction factor, dependent on the steepness of the MPOD spatial distribution, is required for 0 deg.

Near-infrared (NIR) is preferred for caries detection compared to visible light imaging because it exhibits low absorption by stain and deeper penetration into teeth. Hyperspectral images from 1000 to 2500 nm have been obtained for a total of 12 extracted teeth (premolars and molars) with different degrees of natural lesion. Analysis of the reflectance spectra suggests that light scattering by porous enamel and absorption by water in dentin can be used to quantify the lesion severity and generate a NIR caries score. Teeth were ground for histological examination after the measurements. The NIR caries score obtained correlates significantly (Spearman's correlation of 0.89, p<0.01) with the corresponding histological score. Results yield a sensitivity of >99% and a specificity of 87.5% for enamel lesions and a sensitivity of 80% and a specificity >99% for dentine lesions. The nature of the technique offers significant advantages, including the ability to map the lesion distribution rather than obtaining single-point measurements, it is also noninvasive, noncontact, and stain insensitive. These results suggest that NIR spectral imaging is a potential clinical technique for quantitative caries diagnosis and can determine the presence of occlusal enamel and dentin lesions.