Fluorescence lifetime imaging microscopy for quantitative biological imaging.
Fluorescence lifetime imaging microscopy (FLIM) is a method for measuring fluorophore lifetimes with microscopic spatial resolution, providing a useful tool for cell biologists to detect, visualize, and investigate structure and function of biological systems. In this chapter, we begin by introducing the basic theory of fluorescence lifetime, including the characteristics of fluorophore decay, followed by a discussion of factors affecting fluorescence lifetimes and the potential advantages of fluorescence lifetime as a source of image contrast. Experimental methods for creating lifetime maps, including both time- and frequency-domain experimental approaches, are then introduced. Then, FLIM data analysis methods are discussed, including rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis. After, data analysis methods are introduced that improve lifetime precision of FLIM maps based upon optimal virtual gating and total variation denoising. The chapter concludes by highlighting several recent FLIM applications for quantitative biological imaging, including Förster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.
Leng-Chun
LC
Chen
William R
WR
Lloyd
Ching-Wei
CW
Chang
Dhruv
D
Sud
Mary-Ann
MA
Mycek
D000465:Algorithms; D003627:Data Interpretation, Statistical; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016018:Least-Squares Analysis; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis; D054046:Plant Stomata / Q000378:metabolism; D021381:Protein Transport; D059010:Single-Cell Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
Fluorescence correlation spectroscopy FLIM; Fluorescence lifetime imaging microscopy; Förster resonance energy transfer FLIM; Laguerre polynomial fitting; Multiexponential fitting; Multiphoton FLIM; Multispectral FLIM; Optimal gating; Phasor analysis; Total variation denoising