Single Molecule Microscopy in Living Cells: Subtraction of Autofluorescence Based on Two Color Recording Manuel Mörtelmaier A) , Eva J. Kögler B) , Jan Hesse A) , Max Sonnleitner C) , Lukas A. Huber B) and Gerhard J. Schütz A) A) Biophysics Institute Johannes Kepler University Linz Altenbergerstr.69 A-4040 Linz, Austria B) IMP, Research Institute of Molecular Pathology A-1030 Vienna, Austria C) Center for Biomedical Nanotechnology Upper Austrian Research GmbH Scharitzerstr.6-8 A-4020 Linz, Austria Correspondence to Gerhard J. Schütz Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, A-4040 Linz, Austria phone +43-732-2468-9265 fax +43-732-2468-9280 email gerhard.schuetz@jku.at submitted 02 May 2002 acepted 17 Jul 2002 published 02 Aug 2002 keywords: single fluorophore detection, image processing, CD44, spectroscopy, singular value decomposition 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. Introduction In recent years, single fluorophore detection has become a standard technique for the investigation of a wide range of molecular properties (for reviews see [1,2]). In synthetic environments, the identification of single molecule signals can be achieved routinely due to full control over environmental parameters. The study of biomolecules, however, requires experiments under physiological conditions; the ultimate goal would be the investigation of biological processes in the living cell. The frequent occurrence of many species of endogenous fluorescent molecules inside cells makes such studies difficult. Up to now, successful detection of single molecules in vivo has been achieved through the deliberate choice of cell type, metabolic state, and excitation wavelength [3-11]. Such RESEARCH PAPER Molecules Single Single Mol. 3 (2002) 4, 225-231 WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2002 1438-5163/01/0408-0225 $17.50+.50/0 225