Modulation of the coupling of light into a waveguide via a grating, together with a novel approach to analyzing the data, is used to investigate the attachment of human embryonal carcinoma stem cells to three substrata: silica-titania (representative of artificial implants); poly-lysine (a commonly used laboratory cell culture substrate); and mucin (the coating of the mucosae). By considering both in-coupling peak width and position, the secretion of microexudate by the cells, the formation of filopodia, and the overall change in their shape (spreading) can be distinguished. This cannot be achieved by the conventional microscopic imaging approach. Moreover, we obtain the kinetics of these processes with excellent time resolution.

We present an automated numerical method of compensating for retinal shadows in the choroid. In this method, signal extinction caused by retinal vessels is estimated by subtracting median A-scans obtained from beneath the retinal vessels and A-scans from the surrounding area. Adding the obtained offset vector to A-scans from beneath the retinal vessels allows compensating for shadows in the choroid. In vivo imaging of the human eye was performed by 840-nm-band standard resolution spectral domain optical coherence tomography (SD-OCT), and choroidal vasculature projection images were calculated. Removal of retinal shadows distinctly improved the readability of choroidal images.

As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.

In this study, high resolution backward-mode photoacoustic microscopy (PAM) is used to noninvasively image progressive extravasation and accumulation of nanoshells within a solid tumor in vivo. PAM takes advantage of the strong near-infrared absorption of nanoshells and their extravasation tendency from leaky tumor vasculatures for imaging. Subcutaneous tumors are grown on immunocompetent BALB/c mice. Polyethylene glycol (PEGylated) nanoshells with a peak optical absorption at ~800 nm are intravenously administered. With an 800-nm laser source, a prescan prior to nanoshell injection is taken to determine the background that is free of nanoshell accumulation. After injection, the 3-D nanoshell distribution at the tumor foci is monitored by PAM for 6 h. Experimental results show that accumulated nanoshells delineate the tumor position. Nanoshell accumulation is heterogeneous in tumors: more concentrated within the tumor cortex and largely absent from the tumor core. Because nanoshells have been recently demonstrated to enhance thermal therapy of subcutaneous tumors, we anticipate that PAM will be an important aid before, during, and after nanoshell thermal therapy.

We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 µm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-µm excitation in nonlinear optical microscopy.

The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.

An adaptive pulse shaper controlled by multiphoton intrapulse interference phase scanning (MIIPS) was used, together with a prism-pair, to measure and cancel high-order phase distortions introduced by a high-numerical-aperture objective and other dispersive elements of a two-photon laser-scanning microscope. The delivery of broad-bandwidth (~100 nm), sub-12-fs pulses was confirmed by interferometric autocorrelation measurements at the focal plane. A comparison of two-photon imaging with transform-limited and second-order-dispersion compensated laser pulses of the same energy showed a 6-to-11-fold improvement in the two-photon excitation fluorescence signal when applied to cells and tissue, and up to a 19-fold improvement in the second harmonic generation signal from a rat tendon specimen.

We report the detection of interactions between a photosensitizer, hypericin (HY), and its solvent system prepared with a formulation additive, polyvinylpyrrolidone (PVP), a commonly used pharmaceutical excipient. Fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) were used to study aggregation and binding of HY in the presence of PVP. Digitized fluorescence endoscopic imaging (DFEI) was used to study the effect of the pharmaceutical formulation in the in vivo tumor implanted chick chorioallantoic membrane (CAM) model. The results presented reveal the coordination of HY-PVP binding, HY disaggregation in the presence of PVP, and strengthened HY tumor uptake selectivity. PVP is thus suggested as a potential adjuvant to previously investigated N-methyl pyrrolidone (NMP) in the HY delivery system as well as a replacement for the conventionally used albumin in the HY bladder instillation fluids preparation for clinical use.

Highly localized reflectance measurements can be used to directly quantify scatter changes in tissues. We present a microsampling approach that is used to raster scan tumors to extract parameters believed to be related to the tissue ultrastructure. A confocal reflectance imager was developed to examine scatter changes across pathologically distinct regions within tumor tissues. Tissue sections from two murine tumors, AsPC-1 pancreas tumor and the Mat-LyLu Dunning prostate tumor, were imaged. After imaging, histopathology-guided region-of-interest studies of the images allowed analysis of the variations in scattering resulting from differences in tissue ultra-structure. On average, the median scatter power of tumor cells with high proliferation index (HPI) was about 26% less compared to tumor cells with low proliferation index (LPI). Necrosis exhibited the lowest scatter power signature across all the tissue types considered, with about 55% lower median scatter power than LPI tumor cells. Additionally, the level and maturity of the tumor's fibroplastic response was found to influence the scatter signal. This approach to scatter visualization of tissue ultrastructure in situ could provide a unique tool for guiding surgical resection, but this kind of interpretation into what the signal means relative to the pathology is required before proceeding to clinical studies.

Objective quantification of epidermal melanin concentration (EMC) should be useful in laser dermatology to determine the individual maximum safe radiant exposure (IMSRE). We propose a single-wavelength remittance measurement at 390 nm as an alternative optical method to determine EMC and IMSRE. Remittance spectra (360 to 740 nm), melanin index (MI) measurements and the transient radiometric temperature increase, T(t), upon skin irradiation with an Alexandrite laser (755 nm, 3-ms pulse duration, 6 J/cm2) were measured on 749 skin spots (arm and calf) on 23 volunteers (skin phototypes I to IV). Due to the shallow penetration depth and independence of blood oxygen saturation (isosbestic point), remittance at 390 nm appears to provide better estimates for EMC and IMSRE than MI.

This work investigates the use of optical coherence tomography (OCT) to identify virus infection in orchid plants. Besides revealing the cross-sectional structure of orchid leaves, highly scattering upper leaf epidermides are detected with OCT for virus-infected plants. This distinct feature is not observable under histological examination of the leaf samples. Furthermore, the leaf epidermides of stressed but healthy plants, which exhibit similar visual symptoms as virus-infected plants, are not highly scattering and are similar to those of healthy plants. The results suggest that virus-infected orchid plants can be accurately identified by imaging the epidermal layers of their leaves with OCT. The OCT modality is suitable for fast, nondestructive diagnosis of orchid virus infection, which may potentially lead to significant cost savings and better control of the spread of viruses in the orchid industry.

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups.

Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. A noninvasive in vivo tool is essential to evaluate the histopathological changes during the clinical course of AD. We used Cr:forsterite laser-based multimodality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC), and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively), are clearly demonstrated in in vitro studies. Increased TPF levels are positively associated with the levels of the THG signals from the SC. Our in vitro observations of histological changes are replicated in the in vivo studies. These findings were reproducible in skin lesions from human AD. For the first time, we demonstrate the feasibility of preclinical applications of Cr:forsterite laser-based nonlinear microscopy. Our findings suggest that the optical signatures of THG, TPF, and SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.

A Monte Carlo model with site-specific input is used to predict depth-resolved fluorescence spectra from individual normal, inflammatory, and neoplastic oral sites. Our goal in developing this model is to provide a computational tool to study how the morphological characteristics of the tissue affect clinically measured spectra. Tissue samples from the measured sites are imaged using fluorescence confocal microscopy; autofluorescence patterns are measured as a function of depth and tissue sublayer for each individual site. These fluorescence distributions are used as input to the Monte Carlo model to generate predictions of fluorescence spectra, which are compared to clinically measured spectra on a site-by-site basis. A lower fluorescence intensity and longer peak emission wavelength observed in clinical spectra from dysplastic and cancerous sites are found to be associated with a decrease in measured fluorescence originating from the stroma or deeper fibrous regions, and an increase in the measured fraction of photons originating from the epithelium or superficial tissue layers. The simulation approach described here can be used to suggest an optical probe design that samples fluorescence at a depth that gives optimal separation in the spectral signal measured for benign, dysplastic, and cancerous oral mucosa.

In lung transplant recipients, the submucosal vascular plexus of the airway wall potentially represents one of the key structures of graft injury. Narrow band imaging is a novel endoscope technique that allows visual enhancement of the mucosa vasculature. It was our aim to investigate the ability of narrow-band imaging in combination with computerized image analysis to quantitatively assess airway vascularity in lung transplant recipients. In consecutive lung transplant recipients, in addition to the routine procedures, optical analysis of the main carina (autologous tissue) and the upper lobe carina (allogeneic tissue) were performed. From every site, three representative pictures were chosen. A total of 63 bronchoscopies were analyzed. The intraclass correlation coefficient (measure for test–retest reliability) of the three measurements were 0.69 and 0.74 for the main carina and the upper lobe carina, respectively. A mixed linear regression revealed increased vascularity in autologous tissue of patients with cystic fibrosis (p=0.06) and decreased vascularity in allogeneic tissue with time after transplantation (p=0.09). Endoscopic narrow-band imaging (NBI) in combination with computerized image analysis allows consistent assessment of airway vascularity in vivo. In lung transplant recipients, there might be differences in airway vascularity in both autologous and allogeneic large airways.

Histopathological analysis and in vivo optical spectroscopy were used to discriminate several histological stages of UV-irradiated mouse skin. At different times throughout the 30-week irradiation, autofluorescence (AF) and diffuse reflectance (DR) spectra were acquired in a bimodal approach. Then skin was sampled and processed to be classified, according to morphological criteria, into four histological categories: normal, and three types of hyperplasia (compensatory, atypical, and dysplastic). After extracting spectral characteristics, principal component analysis (data reduction) and the k-nearest neighbor classifying method were applied to compare diagnostic performances of monoexcitation AF (based on each of the seven excitation wavelengths: 360, 368, 390, 400, 410, 420, and 430 nm), multiexcitation AF (combining the seven excitation wavelengths), DR, and bimodal spectroscopies. Visible wavelengths are the most sensitive ones to discriminate compensatory from precancerous (atypical and dysplastic) states. Multiexcitation AF provides an average 6-percentage-point increased sensitivity compared to the best scores obtained with monoexcitation AF for all pairs of tissue categories. Bimodality results in a 4-percentage-point increase of specificity when discriminating the three types of hyperplasia. Thus, bimodal spectroscopy appears to be a promising tool to discriminate benign from precancerous stages; clinical investigations should be carried out to confirm these results.

Protoporphyrin IX (PpIX)-encapsulated mesoporous silica nanoparticles were synthesized, characterized, and utilized for photodynamic therapy (PDT) of cancer. Silica encapsulation is relatively transparent for activated light and can protect the PpIX against denaturation induced by the extreme bioenvironment. The mesoporous silica can also ensure that the encapsulated PpIX can be well-contacted with oxygen, stimulated, and released. PpIX-encapsulated colloidal mesoporous silica nanoparticles were uptaken by tumor cells in vitro, and the effect of photon-induced toxicity was demonstrated after comparison with some control experiments. The surface of PpIX-encapsulated silica nanoparticles can be grafted with appropriate functionalized groups and conjugated with certain biomolecules for specific targeting.

Fixation disparity, that is, misalignment of the gaze direction of both eyes, may be observed in static conditions (through standard optometric evaluation) and dynamic conditions (through eye movement recording). A computation method is presented to determine vergence angles and fixation disparity from gaze positions as commonly recorded by infrared eye-trackers when a participant looks at a personal computer (PC) screen. Eye-tracking devices provide gaze position in coordinates relative to the bidimensional screen surface. From these data, vergence angles can be calculated by trigonometric triangulations; fixation disparity is then calculated from the vergence angles. The application of the procedure to the recordings of one participant is described. To control for the effective alignment of the two eyes on the target during binocular calibration, a procedure based on the dichoptic presentation of nonius lines was used. The recordings confirm that computation and the dichoptic calibration procedures ensure reliable measures of vergence and fixation disparity. The usefulness of this approach with infrared recording of eye position is discussed.

One of the problems of near-infrared-spectroscopy (NIRS) measurements is low reproducibility. The aim of the present study was to introduce quality criteria to increase reproducibility of peripheral NIRS measurements. In a prospective cohort study in 40 neonates, repeated NIRS measurements were performed on the calf. During five “reapplication” periods (of NIRS optodes), five “measurements” (venous occlusions) were performed. Tissue oxygenation index (TOI), mixed venous oxygenation (SvO2), fractional oxygen extraction (FOE), hemoglobin flow (Hbflow), oxygen delivery (DO2), and oxygen consumption (VO2) were assessed. Measurements with linear changes during venous occlusions were included for further analysis (first quality criterion: R2>0.95). The second quality criterion was the equation 0TOI−SvO2(SaO2−SvO2)×0.2. Variance components and mean standard deviations were analyzed after introduction of the quality criteria. Variance components of reapplication and measurement decreased after introduction of the second quality criterion (TOI: 46.6–35.0%, SvO2: 76.8–38.2%, FOE: 73.1–37.5%, Hbflow: 70.3–51.9%, DO2: 71.5–52.7%, and VO2: 70.9–63.8%). Mean standard deviations of TOI (6.6±3.0 to 4.7±3.2%), SvO2 (11.1±4.8 to 5.7±3.9%), FOE (11.3±4.8 to 5.9±4.0%), Hbflow (4.3±2.0 to 2.9±1.6 µmol/100 mL/min), and DO2 (17.8±7.6 to 11.4±6.2 µmol/100 mL/min) decreased significantly, too. Only 12% of measurements fulfilled both quality criteria. With the introduction of two quality criteria, test–retest variability of peripheral NIRS measurements decreased significantly and reproducibility increased significantly.

Recently we developed ultrasound-modulated optical microscopy (UOM) based on a long-cavity confocal Fabry-Perot interferometer (CFPI). This interferometer is used for real-time detection of multiply scattered light modulated by high frequency (30 to 75 MHz) ultrasound pulses propagating in an optically, strongly scattering medium. In this work, we use this microscope to study the dependence of ultrasound-modulated optical signals on the optical absorption and scattering properties of objects embedded about 3 mm deep in tissue mimicking phantoms. These results demonstrate that UOM has the potential to map both optical absorption and scattering contrast. Most importantly, for the first time in the field of ultrasound-modulated optical imaging, we image blood vasculature in highly scattering tissue samples from a mouse and a rat. Therefore, UOM could be a promising tool to study the morphology of blood vasculature and blood-associated functional parameters, such as oxygen saturation.

Raman spectroscopy can provide information about the molecular composition of tissues, with potential to be applied as a diagnostic tool in lieu of histopathology. Our objectives are to determine if laser Raman spectra (RS) can be acquired reliably from the oral mucosa of patients, and to determine if the RS signature of normal oral mucosa is reproducible among anatomic oral sites and among subjects of different races and gender. 25 Caucasian and 26 Asian subjects are studied using RS with a signal acquisition time of 1 s at seven specified sites within the mouth. Multivariate analysis is used to determine the variability between tissue types and between races and gender. Unique spectra are defined for various sites in the mouth and are likely related to the degree of keratinization. However, spectral concordance by site is not greatly influenced by subject ethnicity or gender. We demonstrate, for the first time, the potential in-vivo application of RS for oral mucosal disease and demonstrate its specificity for particular mucosal types in the mouth. RS offers the potential to provide a diagnosis of disease using a noninvasive, convenient, sensitive technology that provides immediate results.

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 lens-based probe capable of capturing images of the human larynx by use of spectral domain OCT during a typical office-based laryngoscopy examination is presented. An optical-ballast-based 4f optical relay system is proposed to realize variable working distance with a constant optical delay. In-vivo OCT imaging of the human larynx is demonstrated. Office-based OCT is a promising imaging modality for early laryngeal cancer diagnosis.

Digital holographic microscopy (DHM) enables a quantitative multifocus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms enables subsequent automated focus realignment by numerical propagation of the digital holographically reconstructed object wave. In combination with a calibrated optical imaging system, the obtained propagation data quantify axial displacements of the investigated sample. The evaluation of quantitative DHM phase contrast images also enables an effective determination of lateral cell displacements. Thus, 3-D displacement data are provided. Results from investigations on sedimenting red blood cells and HT-1080 fibrosarcoma cells in a collagen tissue model demonstrate that DHM enables marker-free automated quantitative dynamic 3-D cell tracking without mechanical focus adjustment.

Monte Carlo (MC) simulations are being used extensively in the field of medical biophysics, particularly for modeling light propagation in tissues. The high computation time for MC limits its use to solving only the forward solutions for a given source geometry, emission profile, and optical interaction coefficients of the tissue. However, applications such as photodynamic therapy treatment planning or image reconstruction in diffuse optical tomography require solving the inverse problem given a desired dose distribution or absorber distribution, respectively. A faster means for performing MC simulations would enable the use of MC-based models for accomplishing such tasks. To explore this possibility, a digital hardware implementation of a MC simulation based on the Monte Carlo for Multi-Layered media (MCML) software was implemented on a development platform with multiple field-programmable gate arrays (FPGAs). The hardware performed the MC simulation on average 80 times faster and was 45 times more energy efficient than the MCML software executed on a 3-GHz Intel Xeon processor. The resulting isofluence lines closely matched those produced by MCML in software, diverging by only less than 0.1 mm for fluence levels as low as 0.00001 cm−2 in a skin model.

Cervical intraepithelial neoplasia (CIN) exhibits certain morphologic features that can be identified during a colposcopic exam. Immature metaplastic and dysplastic cervical squamous epithelia turn white after application of acetic acid during the exam. The whitening process occurs visually over several minutes and subjectively helps to discriminate between dysplastic and normal tissue. Digital imaging technologies enable us to assist the physician in analyzing acetowhite (acetic-acid-induced) lesions in a fully automatic way. We report a study designed to measure multiple parameters of the acetowhitening process from two images captured with a digital colposcope. One image is captured before the acetic acid application, and the other is captured after the acetic acid application. The spatial change of the acetowhitening is extracted using color and texture information in the post-acetic-acid image; the temporal change is extracted from the intensity and color changes between the post-acetic-acid and pre-acetic-acid images with an automatic alignment. In particular, we propose an automatic means to calculate an opacity index that indicates the grades of temporal change. The imaging and data analysis system is evaluated with a total of 99 human subjects. The proposed opacity index demonstrates a sensitivity and specificity of 94 and 87%, respectively, for discriminating high-grade dysplasia (CIN2+) from normal and low-grade subjects, considering histology as the gold standard.

Ocular microtremor (OMT) is a physiological high-frequency (up to 150 Hz) low-amplitude (25-2500 nm peak-to-peak) involuntary motion of the human eye. Recent studies suggest a number of clinical applications for OMT that include monitoring the depth of anesthesia of a patient in surgery, prediction of outcome in coma, and diagnosis of brain stem death. Clinical OMT investigations to date have used mechanical piezoelectric probes or piezoelectric strain gauges that have many drawbacks which arise from the fact that the probe is in contact with the eye. We describe the design of a compact noncontact sensing device to measure OMT that addresses some of the above drawbacks. We evaluate the system performance using a calibrated piezoelectric vibrator that simulates OMT signals under conditions that can occur in practice, i.e., wet eye conditions. We also test the device at low light levels well within the eye safety range.

The essential feature of the confocal laser scanning microscope (cLSM) is the generation of optical sections by the removal of out-of-focus light. About ten years ago, structured illumination microscopy (SIM) was introduced as an alternative method for obtaining optical sections from biological specimens. Here we compare the resolution of the ApoTome (commercial SIM by Zeiss) to that achieved by a cLSM (Zeiss LSM 510). If fluorescent beads are used as test objects, then the ApoTome will achieve a lower axial resolution than the cLSM. In contrast to that, its lateral resolution scores slightly better. If subresolution homogeneous fluorescent layers are used as test objects, then the ApoTome will achieve a higher axial resolution than the cLSM. The ApoTome's axial resolution is homogeneous over the field-of-view while that of the cLSM changes markedly. Finally, the anisotropy of the ApoTome's resolution was found to be negligible for standard applications while its capability to resolve fine structures within stained tissue slices is limited to one or two cell layers and thus worse than in the cLSM.

Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both overexpress D-galactose receptor (D-galR), but only SKOV3 overexpresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were evaluated. Both galactosyl serum albumin-rhodamine green (GSA-RhodG), which binds D-galR, and trastuzumab-Alexa680, which binds HER2/neu, were administered to tumor-bearing mice for in vivo fluorescence imaging and in situ fluorescence microscopy. In vivo fluorescence imaging depicted 64 of 69 SKOV3 tumors (94.2%) based on their dual spectra corresponding to both RhodG and Alexa680, while all 71 SHIN3 tumors (100%) were detected based on their single spectrum corresponding only to RhodG. All 59 SHIN3 and 36 SKOV3 tumors were correctly diagnosed with in situ microscopy. Additionally, in the mixed tumor model, all tumors could be depicted using the RhodG spectrum, but only SKOV3 components also showed the Alexa680 spectrum. In conclusion, multitargeted multicolor optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy.

Diffuse optical spectroscopy (DOS) has been used to monitor and predict the effects of neoadjuvant (i.e., presurgical) chemotherapy in breast cancer patients in several pilot studies. Because patients with suspected breast cancers undergo biopsy prior to treatment, it is important to understand how biopsy trauma influences DOS measurements in the breast. The goal of this study was to measure the effects of a standard core breast biopsy on DOS measurements of tissue deoxyhemoglobin, hemoglobin, water, and bulk lipid concentrations. We serially monitored postbiopsy effects in the breast tissue in a single subject (31-year-old premenopausal female) with a 37×18×20 mm fibroadenoma. A baseline measurement and eight weekly postbiopsy measurements were taken with a handheld DOS imaging instrument. Our instrument used frequency domain photon migration combined with broadband steady-state spectroscopy to characterize tissues via quantitative measurements of tissue absorption and reduced scattering coefficients from 650 to 1000 nm. The concentrations of significant near-infrared (NIR) absorbers were mapped within a 50 cm2 area over the biopsied region. A 2-D image of a contrast function called the tissue optical index (TOI=deoxyhemoglobin×water/bulk lipid) was generated and revealed that a minimum of 14 days postbiopsy was required to return TOI levels in the biopsied area to their prebiopsy levels. Changes in the TOI images of the fibroadenoma also reflected the progression of the patient's menstrual cycle. DOS could therefore be useful in evaluating both wound-healing response and the effects of hormone and hormonal therapies in vivo.

We seek to analyze the geometry and explain how bounds and intervals of nonzero purely cylindrical powers are obtained and applied in symmetric dioptric power space and envisaged in the clinic. The principal powers at zero and at the focus at the cylinder power of a lens are subject to the same uncertainty when measured. Accompanying these uncertainties is an error in axis position. Error cells are constructed for typical cylinder axes and an associated power. The geometry contains an elegant clinical determination for cross-cylinder compensation of astigmatism in terms of calculation friendly quantities. The extreme positions in the error cells define bounds for the cross-cylinder powers and their meridians. When clinical powers in a chosen error cell are transposed, the new powers are within a different cell. This ambiguous cell pair maps to a single cell in an antistigmatic plane around cross-cylinder powers.

Mental sweating is human sweating that is accelerated via the sympathetic nerve by application of mental or physical stress. In the neurosciences, there is keen interest in this type of sweating, because the amount of sweat in response to a stress applied to a volunteer directly reflects activity of the sympathetic nerve. It is therefore of particular value that optical coherence tomography (OCT) can provide clear in vivo imaging of the spiral lumen of an eccrin sweat gland in the epidermis with a spatial resolution around 10 µm. We demonstrate dynamic OCT of mental sweating of an eccrin sweat gland on a human fingertip, where the sweating dynamics can be tracked by time-sequential OCT images with a frame spacing of one second. An instantaneous amount of sweat stored in the spiral lumen is evaluated quantitatively in each OCT image, resulting in time variation measurements of excess sweat in response to mental or physical stress. In the dynamic OCT of mental sweating, as demonstrated here, we note for the first time internal sweating without ejection of excess sweat from the spiral lumen to the skin surface. Internal sweating has not been previously detected without the availability of our dynamic OCT technique. Until now, it has been commonly accepted that sweating is always accompanied with ejection of excess sweat to the skin surface. On the basis of our findings reported here, this type of sweating should now be referred to as external sweating. In this study, we demonstrate that internal sweating occurs more often in the case where mental stress is applied to a volunteer, and that it is more useful for evaluation of activity of the sympathetic nerve. The dynamic OCT for both external and internal sweating is demonstrated.

The purpose of this study is to determine the role of melanin in the various steps of progression of melanocytic neoplasia. To this aim, we perform a retrospective analysis on 1671 multispectral images of in vivo pigmented skin lesions previously recruited in the framework of a study focused on the computer-assisted diagnosis of melanoma. The series included 288 melanomas in different phases of progression, i.e., in situ, horizontal and vertical growth phase invasive melanomas, 424 dysplastic nevi, and other 957 melanocytic lesions. Analysis of the absorbance spectra in the different groups shows that the levels of eumelanin and pheomelanin increase and decrease, respectively, from dysplastic nevi to invasive melanomas. In both cases, the trend of melanin levels is associated to the progression from dysplastic nevi to vertical growth phase melanomas, reflecting a possible hierarchy in the natural history of the early phases of the disease. Our results suggest that diffuse reflectance spectroscopy used to differentiate eumelanin and pheomelanin in in vivo lesions is a promising technique useful to develop better strategies for the characterization of various melanocytic lesions, for instance, by monitoring melanin in a time-lapse study of a lesion that was supposed to be benign.

The objective of this in vitro tissue study is to investigate the feasibility of using optical spectroscopy to differentiate pediatric neoplastic and epileptogenic brain from normal brain. Specimens are collected from 17 patients with brain tumors, and from 26 patients with intractable epilepsy during surgical resection of epileptogenic cerebral cortex. Fluorescence spectra are measured at excitations of 337, 360, and 440 nm; diffuse reflectance spectra are measured between 400 and 900 nm from each specimen. Pathological analysis is performed to classify abnormalities in brain specimens, and its findings are correlated with spectral data. Statistically significant differences (p<0.01) are found for both raw and normalized diffuse reflectance and fluorescence spectra between 1. neoplastic brain and normal gray matter, 2. epileptogenic brain and normal gray matter, and 3. neoplastic brain and normal white matter. However, no distinct spectral features are identified that effectively separate epileptogenic brain from normal white matter. The outcomes of the study suggest that certain unique compositional and structural characteristics of pediatric neoplastic and epileptogenic brain can be detected using optical spectroscopy in vitro.

We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.

We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-µs time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells.

The dual-tree complex wavelet transform (CDWT) is a relatively recent enhancement to the discrete wavelet transform (DWT), with important additional properties. It is nearly shift-invariant and directionally selective in two and higher dimensions. In this letter, a locally adaptive denoising algorithm is applied to reduce speckle noise in time-domain optical coherence tomography (OCT) images of the prostate. The algorithm is illustrated using DWT and CDWT. Applying the CDWT provides improved results for speckle noise reduction in OCT images. The cavernous nerve and prostate gland can be separated from discontinuities due to noise, and image quality metrics improvements with a signal-to-noise ratio increase of 14 dB are attained.