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Changes in the pattern and distribution of both melanocytes (pigment producing) and vasculature (hemoglobin containing) are important in distinguishing melanocytic proliferations. The ability to accurately measure melanin distribution at different depths and to distinguish it from hemoglobin is clearly important when assessing pigmented lesions (benign versus malignant). We have developed a multimode hyperspectral dermoscope (SkinSpect™) able to more accurately image both melanin and hemoglobin distribution in skin. SkinSpect uses both hyperspectral and polarization-sensitive measurements. SkinSpect’s higher accuracy has been obtained by correcting for the effect of melanin absorption on hemoglobin absorption in measurements of melanocytic nevi. In vivo human skin pigmented nevi (N=20) were evaluated with the SkinSpect, and measured melanin and hemoglobin concentrations were compared with spatial frequency domain spectroscopy (SFDS) measurements. We confirm that both systems show low correlation of hemoglobin concentrations with regions containing different melanin concentrations (R=0.13 for SFDS, R=0.07 for SkinSpect).

We have developed a multimode dermoscope (SkinSpect™) capable of illuminating human skin samples in-vivo with spectrally-programmable linearly-polarized light at 33 wavelengths between 468nm and 857 nm. Diffusely reflected photons are separated into collinear and cross-polarized image paths and images captured for each illumination wavelength. In vivo human skin nevi (N = 20) were evaluated with the multimode dermoscope and melanin and hemoglobin concentrations were compared with Spatially Modulated Quantitative Spectroscopy (SMoQS) measurements. Both systems show low correlation between their melanin and hemoglobin concentrations, demonstrating the ability of the SkinSpectTM to separate these molecular signatures and thus act as a biologically plausible device capable of early onset melanoma detection.

A polarization-sensitive hyperspectral imaging system (SkinSpect) has been built and evaluated using two-layer tissue phantoms, fabricated to mimic the optical properties of melanin in different epidermal thickness and hemoglobin in dermal layers. Multiple tissue-mimicking phantoms with varying top layer thicknesses were measured for optical system calibration and performance testing. Phantom properties were characterized and validated using SkinSpect. The resulting analysis shows that the proposed system is capable of distinguishing and differentiating the layer-dependent absorption spectra and the depths at which this absorption occurs.

We present a polarization-sensitive hyperspectral imaging system (SkinSpect) that employs a spectrally-programmable light source in the visible and NIR domains. Multiple tissue-mimicking phantoms were fabricated to mimic the optical properties of normal skin as well as pigmented light and dark moles. The phantoms consist of titanium dioxide and a mixture of coffee, red food dye, and naphthol green as the scattering and the three absorptive agents in a polydimethylsiloxane (PDMS) base. Phantoms were produced with both smooth and rough textured surfaces and tested using Spatial Frequency Domain Imaging (SFDI) and Spatially Modulated Quantitative Spectroscopy (SMoQS) for homogeneity as well as determining absorption and scattering variance, respectively. The reflectance spectral images were also recorded using the SkinSpect research prototype; the spectral signatures of the phantoms were calculated using a two-flux single-layer Kubelka-Munk model and non-negative least square fitting routine was applied to extract the relative concentrations of the individual phantom components. © 2013 Copyright SPIE.

This study presents a new multimodal imaging approach that includes high-frequency ultrasound, fluorescence intensity, confocal, and spectral imaging to improve the preclinical evaluation of new therapeutics in vivo. Here we use this approach to assess in vivo the therapeutic efficacy of the novel chemotherapy construct, HerDox during and after treatment. HerDox is comprised of doxorubicin non-covalently assembled in a viral-like particle targeted to HER2+ tumor cells, causing tumor cell death at over 10-fold lower dose compared to the untargeted drug, while sparing the heart. Whereas our initial proof-of-principle studies on HerDox used tumor growth/shrinkage rates as a measure of therapeutic efficacy, here we show that multimodal imaging deployed during and after treatment can supplement traditional modes of tumor monitoring to further characterize the particle in tissues of treated mice. Specifically, we show here that tumor cell apoptosis elicited by HerDox can be monitored in vivo during treatment using high frequency ultrasound imaging, while in situ confocal imaging of excised tumors shows that HerDox indeed penetrated tumor tissue and can be detected at the subcellular level, including in the nucleus, via Dox fluorescence. In addition, ratiometric spectral imaging of the same tumor tissue enables quantitative discrimination of HerDox fluorescence from autofluorescence in situ. In contrast to standard approaches of preclinical assessment, this new method provides multiple/complementary information that may shorten the time required for initial evaluation of in vivo efficacy, thus potentially reducing the time and cost for translating new drug molecules into the clinic.

Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications.

Image blur in acousto-optic tunable filters (AOTF's) has been a persistent problem. Here we describe the connection between transducer structure and image blur and experimentally measure it by using a 5-cm 12 [degrees]-cut Te[O.sub.2] crystal of our design. With these quantitative results, we develop an image-processing method that minimizes AOTF-related image degradation. The combination of long crystal design and image processing results in substantially improved image contrast and spatial resolution relative to conventional AOTF imaging devices. We present high-magnification images of fluorescent actin fibers in cells in which we obtain a resolution of approximately 0.35 [[micro]meter], representing the first successful use of an AOTF for ultra-high-resolution microscopy. Further improvements are also predicted. Key words: Acousto-optic tunable filter, imaging, fluorescence, microscopy.

A fluorescence microscope, based on standing wave excitation of fluorescence, effects an axial resolution greater than 0.5 micrometer. Standing wave excitation technique involves the generation of standing waves due to interference in laser rays which, in turn, gives rise to an excitation field. Compactly packed nodes and antinodes characterize such an excitation field and facilitate optical scanning of the specimen at an enhanced resolution. This technique is used in three-dimensional imaging of actin fibers and filaments in static cells, actin filaments and myosin II filaments in viable cells. Mathematical representation of standing wave excitation includes an axial shift of optical transfer function in reciprocal vector space, which facilitates retraction of reduced signals of information.