Nanosecond fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell

Abstract

Summary: Visualizing and quantifying protein–protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity‐based FRET microscopy is limited by spectral bleed‐through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed‐through is not an issue in FRET–FLIM imaging. In this paper we describe the development of a nanosecond FRET–FLIM microscopy instrumentation to acquire the time‐resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one‐ and two‐component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.