Ultrafast fluorescence microscopy

Fluorescence microscopy is an indispensable tool in biophysics for imaging samples either by their autofluorescence or by fluorescent labels such as dyes or fluorescent proteins. However, fluorescence detection inherently provides information only about the final, emission state of molecules after photoexcitation. In contrast, ultrafast spectroscopy methods such as transient absorption allow monitoring of the initial state of the molecules after absorption and the subsequent excitation dynamics. Typically, ultrafast nonlinear spectroscopy requires bulk samples and is detected coherently. Recently, we have developed a new way to measure transient absorption by detecting sample fluorescence. Within the FluoTRAM (Fluorescence-detected transient absorption microscopy) project, we have implemented our technique in a scanning confocal fluorescence microscope. This PhD project is a combined experimental and theoretical work. The experiment starts with the improvement and robust implementation of the FluoTRAM technique, which will allow the monitoring of excitation dynamics from absorption to emission, with the spatial resolution and sensitivity of a fluorescence microscope. The theoretical part involves the description of the acquired signals and the manifestation of excitation dynamics. The main focus of the work is on the application to biological and other samples of interest, for example, fluorescent proteins, photosynthetic complexes, or organic molecular crystals. The work is carried out in collaboration with the group of prof. Juergen Hauer from the Technical University of Munich.

References:

Malý et al., Fluorescence-detected Pump–Probe Spectroscopy, Angew. Chem. Int. Ed. 60, 18867 (2021)

Fersch et al., “Single-Molecule Ultrafast Fluorescence-Detected Pump–Probe Microscopy”, J. Phys. Chem. Lett. 14, 4923 (2023)

Malý et al., “ixFLIM: Interferometric Excitation Fluorescence Lifetime Imaging Microscopy”, arXiv:2310.17627v1 (2023), doi: 10.48550/arXiv.2310.17627