Microscope Alignment Using Real-Time Imaging FCS

AO_SCPLOWBSTRACTC_SCPLOWModern EMCCD and sCMOS cameras read out fluorescence data with single-molecule sensitivity at a rate of thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real-time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for Total Internal Reflection and Light Sheet Fluorescence Microscopy (TIRFM, LSFM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRMF and LSFM data acquisition. SignificanceWe developed a direct camera readout (DCR) software tool that allows access to camera data during acquisition and provides real-time Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) analysis. DCR displays live feedback and due to the sensitivity of correlation analysis provides a sensitive tool for microscope alignment using simple solutions of fluorescent particles. DCR is adaptable to different cameras and evaluation strategies, reduces alignment time, accelerates experiments, and can be used for automation.