11 - Real-Time Fluorescence Lifetime Imaging and FRET Using Fast-Gated Image Intensifiers

Abstract

This chapter discusses practical features and guidelines for fluorescence lifetime imaging (FLI) and Förster resonance energy transfer (FRET) for interpreting FLI and FRET measurements of complex biological systems. New technology in image intensifiers has produced fast-gated intensifiers that can modulate the gain on the cathode with GHz frequencies or tens of picoseconds gate width. This allows fluorescence lifetime measurements to be performed with a full-field setup; that is, every location of a fluorescence image falling on the cathode can be modulated simultaneously. Lifetime measurements are independent of the concentration of the fluorophores, whereas the fluorescence lifetime of a molecule is sensitive to the microenvironment surrounding the molecule. Lifetimes are attractive for biological samples because they do not require the extensive experimental controls and normalization procedures that are essential for quantifying steady-state fluorescence intensity measurements. The capability to directly measure the dynamic process is especially advantageous when determining parameters such as FRET in images in biological samples, where it is difficult to determine the control parameters necessary to interpret intensity-based FRET measurements. FLI is used to spatially distinguish areas of varying lifetime that can be resolved. There may also be multiple components to the lifetimes measured within a pixel of the fluorescence image; that is, often the different lifetime components cannot be separated spatially.