SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Spatially-resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope
Daniel R. Sisan 1, Richard Arevalo 1, Catherine Graves 1, Ryan McAllister 1 and Jeffrey S. Urbach 1*
1 Georgetown University
* To whom correspondence should be addressed. E-mail: urbach{at}physics.georgetown.edu.
Submitted on February 28, 2006
Revised on April 5, 2006
Accepted on 9 August 2006
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Abstract |
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We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to ~105 independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10-7 cm2/s or smaller. This speed is adequate to measure small microspheres (dia. 200 nm) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially-varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.
Key Words:
ICS, anomalous diffusion, collagen, hindered diffusion, hydrogels, spatially-varying diffusion
