title: Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy.
abstract: Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.
authors.affiliation: University of Pittsburgh School of Medicine, Renal-Electrolyte Division, Pittsburgh, Pennsylvania 15261, United StatesbWestern Carolina University, Department of Biology, Cullowhee, North Carolina 28723, United States.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Youker
authors.affiliation: Carnegie Mellon University, Molecular Biosensor and Imaging Center (MBIC), Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Haibing
authors.initials: H
authors.lastname: Teng
mesh_terms: D002138:Calibration; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D042541:Intracellular Space / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D016011:Normal Distribution; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D011506:Proteins