19 June 2003 Detectability of contrast agents for video-rate confocal reflectance microscopy of skin and microcirculation in vivo
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The lack of structure-specific contrast limits the usefulness of confocal reflectance microscopy to morphologic investigations at the cellular- and nuclear-level in human and animal skin in vivo. Morphologic and functional imaging at specific organelle- and ultrastructure-levels will require contrast agents that may be used and detected in vivo. High-resolution confocal reflectance imaging is based on the detection of singly back-scattered photons, where contrast is provided by variations in the refractive indices of microstructures. We carried out a quantitative Mie back-scatter analysis and imaging experiments to understand signal detectability of reflectance contrast agents for visualizing human skin and animal microcirculation. When imaging at video-rate with illumination of 10 milliwatts at 1064 nm, we detect 100-104 photons/pixel from the epidermis to dermis, relative to a background of 100 photons; this provides a signal-to-noise ratio of 3-40 and signal-to-background of 1-100. Organelles of size (d) 0.1-1.0 μm with refractive indices (n) of 1.34-1.45 (relative to n=1.34 for epidermis, n=1.38 for dermis) back-scatter 10-104 photons/pixel. Exogenous contrast agents such as liposomes (n=1.41, d=0.7 μm) and polystyrene microspheres (d=0.2-1.0 μm, n=1.57; 100-105 photons/pixel) are detectable and they strongly enhance the contrast of microcirculation in the dermis of Sprague-Dawley rats. Topically applied 5% acetic acid causes the intra-nuclear 30-100 nm-thin chromatin filaments to condense into 1-5 μm-thick strands, increasing back-scattered signal from 100 to 104 photons/pixels, making the nuclei appear bright and easily detectable in basal cell cancers. Such analyses provide a basis for optimizing confocal microscope design for detectability of contrast agents in vivo.
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Milind M. Rajadhyaksha, Milind M. Rajadhyaksha, Salvador Gonzalez, Salvador Gonzalez, } "Detectability of contrast agents for video-rate confocal reflectance microscopy of skin and microcirculation in vivo", Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); doi: 10.1117/12.477894; https://doi.org/10.1117/12.477894

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