Single Molecule Microscopy in Living Cells: Subtraction of Autofluorescence Based on Two Color Recording

Abstract

A significant limitation of ultra-sensitive microscopy on living cells is set by background signal arising from cellular autofluorescence. Up to now, most strategies to circumvent this limitation were based on choosing long-wavelength dyes and selecting cell lines with reduced metabolism. In this article, we present a new strategy to identify and eliminate signal arising from autofluorescence. Two images are recorded simultaneously in distinct spectral channels. An algorithm, based on singular value decomposition, separates the contributions by the fluorophore of interest and autofluorescence. A first application of the method for imaging CD44-YFP in living cells is given.