Effects of donor and acceptor's fluorescence lifetimes on the method of applying F örster resonance energy transfer in STED microscopy

Summary Förster resonance energy transfer (FRET) probes being used to improve the resolution of stimulated emission depletion (STED) microscopy are numerically discussed. Besides the FRET efficiency and the excitation intensity, the fluorescence lifetimes of donor and acceptor are found to be another key parameter for the resolution enhancement. Using samples of FRET pairs with shorter donor lifetime and longer acceptor lifetime enhances the nonlinearity of the donor fluorescence, which leads to an increased resolution. The numerical simulation shows that a double resolution improvement of STED microscopy can be achieved by using Cy3–Atto647N samples when compared with that of using standard Cy3‐only samples. Lay description STED microscopy, with its capability to discern details far beyond the diffraction limit, has become a most popular imaging modality in life science. The achievable resolution in STED microscopy is theoretically unlimited by indefinitely elevating the STED laser power. However, in practice, the resolution is limited by some factors. For example, under the high STED laser illumination, a high risk of irreversible destruction to the samples exists due to photobleaching or other detrimental effects, especially when the samples are biological specimen. Therefore, without lifting the depletion intensity, the methods able to increase the resolution and the availability of the STED microscopy are highly desirable. In this paper, the phenomenon of Förster ...
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research