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.
We introduce a multimode dermoscope (SkinSpectTM) we developed for early detection of
melanoma by combining fluorescence, polarization and hyperspectral imaging. Acquired reflection
image datacubes were input to a wavelength-dependent linear model to extract the relative
contributions of skin chromophores at every pixel. The oxy-hemoglobin, deoxy hemoglobin,
melanin concentrations, and hemoglobin oxygen saturation by the single step linear least square
fitting and Kubelka-Munk tissue model using cross polarization data cubes were presented. The
comprehensive data obtained by SkinSpect can be utilized to improve the accuracy of skin
chromophore decomposition algorithm with less computation cost. As an example in this work, the
deoxy-hemoglobin over-estimation error in highly pigmented lesion due to melanin and deoxy
hemoglobin spectral cross talk were analyzed and corrected using two-step linear least square fitting
procedure at different wavelength ranges. The proposed method also tested in skin with underlying
vein area for validating the proof of concept.
We investigated the use of a near-infrared (NIR) fluorescent dye, Rhodamine 800 (Rhod800, λexc = 693 nm, λem > 720 nm) as a flow-dependent molecular tracer for NIR spectroscopy and high-resolution cardiac imaging. Rhod800 accumulates in isolated mitochondria in proportion to the mitochondrial membrane potential (ΔΨ). However, in the intact myocardium, Rhod800 binding is ΔΨ-independent. Rat hearts were perfused in a Langendorff mode with Krebs-Henseleit buffer containing 45-nM Rhod800 at normal (100%), increased (150%), or reduced (50%) baseline coronary flow (CF) per gram, for 30 to 60 min. In a different group of hearts, the left anterior descending artery (LAD) was occluded prior to Rhod800 infusion to create a flow deficit area. Rhod800 deposition was analyzed by: 1. absorbance spectroscopy kinetics in the Rhod800-perfused hearts, 2. Rhod800 absorbance and fluorescence imaging in the short-axis heart slices, and 3. dynamic epicardial/subepicardial fluorescence imaging of Rhod800 in KCl-arrested hearts, with a spatial resolution of ∼200 μm. Rhod800 deposition was proportional to the perfusate volume (CF and perfusion time) and there was no Rhod800 loss during the washout period. In the LAD-ligated hearts, Rhod800 fluorescence was missing from the no-flow, LAD-dependent endocardial and epicardial/subepicardial area. We concluded that Rhod800 can be used as a deposition flow tracer for dynamic cardiac imaging.
Diffuse reflectance was applied to the biomedical studies (muscles, cardiac tissues etc.) in a form of either a direct
pseudo-optical spectrum or its second derivative. The first derivative adopts advantages of both direct spectrum (high
signal-to-noise ratio) and its second derivative (simplifying the consideration of light scattering contribution, S). In
contrast to spectrophotometry of solutions, diffuse reflectance application to the analysis of turbid medium
chromophores leads to non-trivial problems of contribution of light scattering, the choice of reference, and light
pathlength. Under certain conditions, the first approximation of the Taylor series of S results in the known linear
dependence of S on wavelength in the 650-1050 nm wavelength range. Then the light scattering contribution to the first
derivative becomes a wavelength-independent offset. In contrast to the second derivative, the information on light
scattering inside the tissue is not lost. Effect of reference on the measured spectra becomes negligible. Application of the
first derivative allowed (i) determination of NIR light pathlength in muscle tissue, and (ii) quantification of hemoglobin
+ myoglobin absolute concentration (in mM) in cardiac tissue during open-heart surgery. The first derivative approach
may in general be applied to any chromophores in turbid (biological) media.
The existing non-invasive optical methods of the hemoglobin (Hb) and myoglobin (Mb) estimation in cardiac tissues
imply knowledge of the light pathlength (L) when various modifications of Lambert-Beer law for either
spectrophotometry or light diffuse reflectance is applied. For Hb and/or Mb quantification in tissue, a few invasive
(biochemical) approaches were applied. For L (differential pathlength factor; DPF) determination in tissue, special
optical methods were used. No approaches have been proposed to simultaneously and non-invasively determine Hb/Mb
and L in cardiac or other muscle tissues. In the present study, the first derivative of the NIR diffuse reflectance spectrum
is shown to be effective in simultaneous determination of Hb+Mb concentration (in mM) and L (in mm) in cardiac tissue
in vivo. The results showed that measured in a few minutes in a normal pig heart in vivo the total Hb+Mb concentration
was 0.9-1.2 mM of heme, tissue oxygen saturation parameter (OSP) was approximately 65%, and DPF at 700-965 nm
was of 2.7-2.8. At the experimental ischemia, total [Hb+Mb] decreased by 25%, OSP reduced to zero, while DPF did not
change. These results correlated with the previously published. The method may be applied during open-heart surgery,
heart studies ex vivo or to any muscle tissue to continuously and non-invasively monitor the [Hb+Mb] content and
oxygenation as well as L, which may reflect the changes in tissue structure.
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