Transient State Imaging of live cells by Single Plane Illumination Microscopy

Long-lived, photoinduced transient dark states of fluorophores have a great potential for environmental sensing. Transient State (TRAST) Imaging based on time-modulated excitation has recently been shown to provide imaging of the kinetics and populations of these states in biological samples and has been realized for confocal, wide-field and total internal reflection microscopy settings. In this study, we demonstrate the applicability of Single Plane Illumination Microscopy to TRAST imaging, offering optical sectioning and reduced overall excitation light exposure of the specimen. This concept was first verified by showing that transition rates of free dye in solution can be determined accurately. Thereafter, experiments on MCF-7 cells were performed, showing that fluorophore triplet state decay and oxidation rates can be resolved pixel-wise. Furthermore, a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains has been derived. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.