Setup of a TIRF Microscope With Attached Microfluidics for the Detection of Fluorescently Labeled Single Molecules

Abstract

At Laserinstitut Hochschule Mittweida (LHM), single molecule fluorescence microscopy is to be established for biophotonic RNA research. The aim of this work is to establish an objective-based total internal reflection fluorescence microscope (TIRFM) with alternating laser excitation (ALEX) in the millisecond range. We have designed a suitable detection beam path for color channel-selected imaging with a scientific CMOS camera. The two detection channels allow distance changes to be measured at the molecular level using Förster resonance energy transfer (FRET). In addition to the microscope setup, this work details the imaging and color channel selection of the fluorescently labeled samples and the modulated laser excitation.