Fast rasterscanning enables FLIM in macroscopic samples up to several centimeters

Abstract

Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) is nowadays a well established technique that is very often realised as an add-on for confocal laser scanning microscopes. However, the standard laser scanning technique limits the maximum scan range in these setups to a few millimetre, making it therefore unsuited for e.g. fluorescence multiplexing in multi well plate based assays or for macroscopic material science studies on solar cells, wafers and similar material. In order to also realize larger scanning ranges, we have developed a sample scanning approach based on a xy-cross stage equipped with piezo linear motors. Using online position monitoring, this approach permits fast acceleration and scanning as well as precise positioning and features scan ranges from 100×100 microns up to 80×80 mm with submicron positioning accuracy. Standard upright and inverse microscope bodies can easily be equipped with this scanning device. Along with the necessary excitation and detection components "largearea" FLIM thus becomes possible. We will show new results obtained with a modified MicroTime 100 (PicoQuant GmbH) illustrating the system capabilities for lifetime based imaging in macroscopic samples such as the improvement of the fluorescence sensitivity in 2D gel electrophoresis or the possibility to perform lifetime based fluorescence multiplexing in μ-well plate based assays. Even Two Photon Excitation (TPE) imaging is possible with this widerange sample scanning approach and first FLIM results on cockroach salivary glands, loaded with a chloride sensitive dye (MQAE) will be presented.