Fluorescence microscopy is an indispensable tool in many areas of research. In life sciences it has been perfected for biological sample imaging either by its autofluorescence or using fluorescent markers such as dyes or fluorescent proteins. It is thus possible to localize molecules in cells, obtaining wealth of information on their dynamics and environment. Despite its power, the fluorescence detection is, by its nature, limited to the information on the final, emissive state of the molecules after photoexcitation. Meanwhile, transient absorption spectroscopy enables to track the initial state of the molecules after absorption and the following excitation dynamics. However, such ultrafast nonlinear techniques typically require volume samples and coherent detection. We have recently developed a new way to measure transient absorption by detecting the sample fluorescence. In project FluoTRAM we will implement our technique in the fluorescence microscope, where it truly reveals its potential. Using the established imaging techniques and markers, FluoTRAM brings the additional information on the excitation event and the dynamics towards the emissive state. We will implement FluoTRAM in two parallel stages, the time resolution and the spectrally varying excitation. The time resolution will be achieved using chopped laser pulses, varying their delay by a delay stage and recording a difference fluorescence in a pump-probe fashion. The excitation spectrum scanning will be realized interferometrically, creating a phase-stable replica of the excitation pulse and scanning the delay between the two. The comprehensive additional information on the excitation dynamics from absorption to emission will be of great use in life sciences and beyond. Examples include correlation of the excitation and emission spectra (increased Stokes shift vs red shift) for dye probes, intramolecular charge transfer in fluorescent proteins, or charge transfer and recombination in organic materials.
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