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

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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.

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Fig. 1
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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

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Acknowledgments

We are indebted to Christoph Cremer for his continuous support of the development of the STFM instrument at the Kirchhoff-Institut für Physik, and thank Felix Bestvater, Zahir Seghiri, Moon Sik Kang, Katharina Müller, and Michael Tewes for help and discussions as well as Werner Knebel from Leica Microsystems and Peter Vogt from Coherent for technical support. The detailed comments and suggestions of one of the reviewers for the revision of the paper are gratefully acknowledged. The work was funded by the Volkswagenstiftung and by the Deutsche Forschungsgemeinschaft within the Research Training Group “Molecular imaging methods for the analysis of gene and protein expression” (GRK 886/1) and by grants Ri 1283/5-3 (SPP1128 priority program “Optical analysis of structure and dynamics of supramolecular biological complexes”) and Ri 1283/8-1.

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Authors and Affiliations

  1. Research Group Genome Organization and Function, Deutsches Krebsforschungszentrum and BioQuant, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Gerrit Heuvelman, Fabian Erdel & Karsten Rippe

  2. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117, Heidelberg, Germany

    Malte Wachsmuth

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  1. Gerrit Heuvelman
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  2. Fabian Erdel
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  3. Malte Wachsmuth
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  4. Karsten Rippe
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Correspondence to Malte Wachsmuth or Karsten Rippe.

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This article has been submitted as a contribution to the festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.

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Heuvelman, G., Erdel, F., Wachsmuth, M. et al. Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy. Eur Biophys J 38, 813–828 (2009). https://doi.org/10.1007/s00249-009-0499-9

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  • DOI: https://doi.org/10.1007/s00249-009-0499-9

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