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BP: Fachverband Biologische Physik

BP 20: Poster VIII

BP 20.25: Poster

Dienstag, 17. März 2020, 14:00–16:00, P2/4OG

Development of a flat-top laser excitation for TIRF microscopy-based single molecule FRET experiments — •Wubulikasimu Yibulayin and David Dulin — Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Total internal reflection fluorescence microscopy (TIRFM) is a widely used technique for single molecule fluorescence spectroscopy studies, in particular in combination with single molecule fluorescence resonance energy transfer (smFRET). The advent of large detector scientific CMOS camera has enabled high throughput observation of single molecule using TIRFM. However, the Gaussian distribution of intensity in the excitation laser renders the illumination inherently inhomogeneous, whereas a flat-top intensity distribution would be ideal. Several approaches have been developed to convert the Gaussian laser excitation beam into a flat-top beam, such as injecting the excitation laser(s) into a mechanically vibrated multimode fiber (MMF). Here, we have assembled a custom two-colors TIRF microscope with the excitation lasers (532 nm and 640 nm) are injected into a MMF. To remove the speckle added by the MMF to the laser intensity profile, we couple to the MMF a piezo element that vibrates at high frequency, enabling a uniform flat-top illumination, while maintaining the TIR conditions. This all-custom microscope will provide a platform for hybrid force-fluorescence spectroscopy measurements, e.g. TIRFM-magnetic tweezers.