Epifluorescent video microscopy (EVM) for platelet-biomaterial interactions: elimination of photoactivation and dye effects.

Abstract

The use of two intracellular dyes for epifluorescent video microscopy (EVM) in observations of cell-surface interactions is evaluated and discussed. This methodology permits determinations of cell adhesion, detachment and movement at the surfaces of biomaterials in the presence of flow and physiological haematocrit. Two tests, one which examines for the effect of incident light on platelet adhesion and one which checks for sufficient light for accurate observation of cells, have been designed. Evaluations were made of the adhesion of platelets labelled with the fluorescent dyes mepacrine and acridine orange, used singly and in combination. The use of a number of light level-dye level combinations with glass and several polymers and the addition of a plasma level of fibrinogen did not show any photoactivation effects. This methodology paves the way for longer than previous exposures to light with our system, from 1 min up to 30 min now. Washed platelet suspensions are preferred; these allow for the selective labelling of specific cells and the removal of dye from the surface of the cell.