1 Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection Clemens Hesch 1 , Jan Hesse 2 , and Gerhard J. Schütz 1 1 Biophysics Institute, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria 2 Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstrasse 6-8, 4020 Linz, Austria Correspondence to: Gerhard J. Schütz, Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, 4040 Linz, Austria. gerhard.schuetz@jku.at. Phone: +43/732/2468-9284, fax: +43/732/2468-29284 Published in and © by “Biosensors and Bioelectronics”, February 2008. ABSTRACT. We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects – e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer – the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera read-out yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to ~10nm was shown by scanning immobilized 100nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two- color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application. KEYWORDS: Single molecule microscopy, microarray, biochip, fluorescence, colocalization 1. Introduction In recent years chip technology has become the prevalent method for large-scale characterization of biological samples (Mir, 2006, Heller, 2002, Sauter et al., 2003, Teruel and Meyer, 2002, Zhu et al., 2003). More and more researchers have been using this tool in combination with live cell imaging to obtain mechanistic insights into biomolecular function under native conditions (Huh et al., 2003, Teruel and Meyer, 2002). In particular, knowledge about expression and subcellular localization of fluorescent proteins allows for a comprehensive description of cellular signaling and sorting mechanisms, down to the level of single molecules (Huh et al., 2003, Heise et al., 2005, Cai et al., 2006, Yu et al., 2006). It turned out that cell biology imposes multiple constraints on the detection schemes. For unambiguous discrimination of the fluorescent species from cellular autofluorescence or even more so for multicomponent analysis a multi-color