Fluorescence Lifetime Imaging Microscopy: Quality Assessment and Standards

Abstract

The measurement of fluorophore lifetimes—the excited state duration—in the microscope provides unique quantitative information on the molecular environment and is therefore increasingly being used in cell biological questions. Perhaps the most popular use of fluorescence lifetime imaging (FLIM) is to measure Förster resonance energy transfer (FRET) to detect protein interactions, conformational changes, and activities in the context of the (living) cell. The analytical use of FLIM requires a detailed knowledge of the proper use and limitations of its different instrumental implementations, including platform standardization and calibration, and considerations regarding its optimization for increased throughput. The results obtained with FLIM are conditional on the quality of the data. Therefore, stringent data analysis assessment and analysis criteria have to be maintained in the imaging workflow. In particular, the issues of photobleaching and fluorophore saturation, and their effect and correction possibilities, are discussed. This chapter deals with the various aspects of FLIM that need to be taken into consideration when this powerful technique is to be used as an analytical tool in the life sciences.