[Frontiers in Bioscience E3, 476-488, January 1, 2011]

Multi-confocal fluorescence correlation spectroscopy

Remi Galland1, Jie Gao1, Meike Kloster1, Gaetan Herbomel2, Olivier Destaing3, Martial Balland1, Catherine Souchier2, Yves Usson4, Jacques Derouard1, Irene Wang1, Antoine Delon1

1Universite de Grenoble 1, CNRS, Laboratoire de Spectrometrie Physique UMR 5588, BP 87, 38402 Saint Martin d'Heres, France, 2Universite de Grenoble 1/ INSERM, Institut Albert Bonniot, U823, equipe 10, Stress et DyOGen, La Tronche, BP 170, 38042 Grenoble Cedex 9, France, 3Universite de Grenoble 1/ INSERM, Institut Albert Bonniot, U823, equipe DySAD ERL CNRS 3148, La Tronche, BP 170, 38042 Grenoble Cedex 09, France, 4Universite de Grenoble 1, Institut d'Ingenierie et de l'Information de Sante, Laboratoire TIMC, La Tronche, 38706 La Tronche cedex, France

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
3.1. Optical set-up
3.2. Phase map calculation
3.3. Data acquisition and treatment
3.3.1. Data acquisition
3.3.2. Data treatment
3.4. Sample preparation
3.4.1. Dye solutions
3.4.2. Cell culture and reagents
3.5. Heat shock treatment
4. Experimental results
4.1. Measurements in solution
4.1.1. Single spot measurements
4.1.2. Multiple spot measurements
4.2. Measurements in living cells
4.2.1. Assessing G actin diffusion
4.2.2. Response to heat shock
5. Discussion and conclusion
6. Acknowledgments
7. References

1. ABSTRACT

We report a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique that combines a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM, calculated by using the spherical wave approximation, makes it possible to produce several diffraction limited laser spots. The fastest acquisition mode leads to a time resolution of 100 Ás. By using solutions of sulforhodamine G we demonstrated that the observation volumes are similar to that of a standard confocal set-up. mFCS experiments have also been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock.