Original Paper

European Biophysics Journal

, Volume 38, Issue 6, pp 813-828

Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy

  • Gerrit HeuvelmanAffiliated withResearch Group Genome Organization and Function, Deutsches Krebsforschungszentrum and BioQuant
  • , Fabian ErdelAffiliated withResearch Group Genome Organization and Function, Deutsches Krebsforschungszentrum and BioQuant
  • , Malte WachsmuthAffiliated withCell Biology and Biophysics Unit, European Molecular Biology Laboratory Email author 
  • , Karsten RippeAffiliated withResearch Group Genome Organization and Function, Deutsches Krebsforschungszentrum and BioQuant Email author 

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Abstract

The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 μs time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.

Keywords

Fluorescence correlation spectroscopy Single-particle tracking Promyelocytic leukemia nuclear body Green fluorescent protein Chromatin-remodeling complex Protein dynamics in living cells