Pump–probe fluorescence lifetime imaging microscopy

Abstract

Pump–probe spectroscopy is the most popular technique to resolve ultrafast photo-induced dynamics. Its fluorescence-detected variant connects it to fluorescence microscopy, for a combined spatial and temporal resolution in sensitive samples. In this work, we introduce fluorescence-detected pump–probe spectroscopy (F-PP) with fluorescence lifetime imaging microscopy (FLIM) detection, which we call pump–probe FLIM (PP-FLIM). Like FLIM, PP-FLIM images the time-resolved fluorescence decay in three spatial dimensions. At each voxel, furthermore, the full pump–probe spectrum is obtained, measuring spectrally resolved transient dynamics. We demonstrate the PP-FLIM principle on a microliter–volume mixture of two fluorescent dyes, oxonol VI and cresyl violet, whose transient spectra PP-FLIM disentangles by their fluorescence lifetime. We then showcase the high-resolution non-invasive ultrafast imaging by measuring individual chloroplasts within intact spinach leaves.