Measurement of transient transport of hyperosmotic agents across cell membranes and resulting optical clearing using differential phase contrast optical coherence microscopy
Rylander, Christopher Grady
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The response of tissue to hyper-osmotic agents is a reduction in light scattering and corresponding increase in optical clarity. “Tissue optical clearing” permits delivery of near-collimated light deeper into tissue potentially improving the capabilities of optical diagnostic and therapeutic applications. The overall objective of the proposed research is to characterize the mass transport of hyper-osmotic agents across cell membranes and the resulting optical clearing. To accomplish this task, a differential phase contrast optical coherence microscope (DPC-OCM) is configured to permit quantitative spaciotemporal optical path length (OPL) imaging of biological cell specimens. The first application of DPC-OCM is analyzing the intracellular dry mass of individual biological cells. Differences between normal and cancerous cell dry mass are investigated. Populations of normal and cancerous human dermal fibroblast cells and human prostate cells demonstrate a statistically significant difference in mean dry mass and mean en face area. Linear discriminant analysis yields a maximum of 79% accurate classification. The second application of DPC-OCM is use as a novel technique for determining cell membrane permeability parameters due to an osmotic chemical stimulus. Glycerol, a hyperosmotic agent, is perfused across an adherent layer of human keratinocytes, and the dynamic osmotic response of individual cells is imaged with DPC-OCM. A novel optical path length (OPL) mass transport model is devised relating chemical concentration to intrinsic refractive index and OPL. Hydraulic conductivity and solute permeability are determined by fitting the OPL mass transport model to transient OPL data collected with DPC-OCM. The final study investigates the mechanisms of optical clearing of cellular and collagenous tissue using hyperosmotic agents and evaporation. OCT and photographic images quantify optical scattering reduction between native and dehydrated tissue states. Air-drying optically clears tissue as effectively as the most successful hyperosmotic agent, glycerol. Tissue ultrastructural alterations due to dehydration are investigated using transmission electron microscopy. The Rayleigh-Gans model is used to simulate light scattering effects due to tissue ultrastructural alterations and measured refractive index excursion using DPC-OCM.