Measurement of concentration distribution in endothelial surface layer using super resolution LIF technique

Abstract

In order to investigate an effect of glycocalyx layer covering vascular endothelial cell surface on molecular transport, a super resolution LIF (Laser Induced Fluorescence) technique for concentration distributions measurement with high spatial resolution based on super resolution microscope, stimulated emission depletion (STED) microscope, was developed. Culturing of endothelial cells on side wall of a microchannel with the width of 500 μm and the depth of 88 μm made from PDMS (Polydimethylsiloxane) was achieved by optimizing surface modification. Time series of concentration distributions of dextran stained with Alexa Fluor 488, excited at 495 nm and emitted at 519 nm, near surface area of an endothelial cell including glycocalyx layer attached on side wall was obtained. The visualized images were 2048×2048 pixels, 16 bit grayscale and pixel size of 37.9 nm corresponded with 77.5×77.5 μm, and time interval of 165 seconds. Furthermore, a dimension of glycocalyx by stained with wheat germ agglutinin Alexa Fluor 488 conjugate was measured using super resolution microscopy. Although concentration of dextran within glycocalyx layer was constant as time proceeds, that around outside of the cell surface increased gradually. The measurement results suggested that glycocalyx layer with 0.43 μm thickness affected molecular transport out of the layer with a distance of around 10 μm from the layer.