European Biophysics Journal

, Volume 38, Issue 6, pp 813–828

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

Authors

  • Gerrit Heuvelman
    • Research Group Genome Organization and FunctionDeutsches Krebsforschungszentrum and BioQuant
  • Fabian Erdel
    • Research Group Genome Organization and FunctionDeutsches Krebsforschungszentrum and BioQuant
    • Cell Biology and Biophysics UnitEuropean Molecular Biology Laboratory
    • Research Group Genome Organization and FunctionDeutsches Krebsforschungszentrum and BioQuant
Original Paper

DOI: 10.1007/s00249-009-0499-9

Cite this article as:
Heuvelman, G., Erdel, F., Wachsmuth, M. et al. Eur Biophys J (2009) 38: 813. doi:10.1007/s00249-009-0499-9

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 spectroscopySingle-particle trackingPromyelocytic leukemia nuclear bodyGreen fluorescent proteinChromatin-remodeling complexProtein dynamics in living cells

Abbreviations

GFP

Green fluorescent protein

MSD

Mean-squared displacement

SPT

Single-particle tracking

FRAP

Fluorescence recovery after photobleaching

FCS

Fluorescence correlation spectroscopy

FCCS

Fluorescence cross-correlation spectroscopy

STFM

Spatial and temporal fluctuation microscope/microscopy

CLSM

Fluorescence confocal laser-scanning microscope

EM-CCD

Electron-multiplying charge-coupled device

PML-NB

Promyelocytic leukemia nuclear body

PSF

Point-spread function

TIRF

Total internal reflection fluorescence

Copyright information

© European Biophysical Societies' Association 2009