SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy
Anton Arkhipov 1, Jana Hueve 2, Martin Kahms 2, Reiner Peters 2 and Klaus Schulten 3*
1 University of Illinois at Urbana-Champaign
2 University of Muenster, Germany
3 University of Illinois 3143 Beckman Institute
* To whom correspondence should be addressed. E-mail: kschulte{at}ks.uiuc.edu.
Submitted on February 28, 2007
Revised on April 5, 2007
Accepted on 26 July 2007
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Abstract |
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Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy which features an axial resolution of approximately 100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal-to-noise ratio was small due to the small number of fluorophores involved.
Key Words:
4Pi microscope, correlation spectroscopy, diffusion, fluorescence microphotolysis, high-resolution light microscopy, numerical methods
