Spatially Resolved Fluorescence Correlation Spectroscopy Using a Spinning Disk Confocal Microscope
| Autor: | Daniel R. Sisan, Catherine Graves, Jeffrey S. Urbach, Richard C. Arevalo, Ryan G. McAllister |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Magnetic Resonance Spectroscopy
Microscope Materials science Light Confocal Biophysics Fluorescence correlation spectroscopy 01 natural sciences Polyethylene Glycols law.invention Diffusion 010309 optics Matrix (chemical analysis) 03 medical and health sciences Optics Spectroscopy Imaging Other Techniques law Confocal microscopy 0103 physical sciences Animals Diffusion (business) 030304 developmental biology 0303 health sciences Microscopy Confocal Models Statistical Pixel business.industry Reproducibility of Results Photobleaching Microspheres Rats Spectrometry Fluorescence Spectrophotometry lipids (amino acids peptides and proteins) Collagen business Software |
| Zdroj: | Biophysical Journal. 91:4241-4252 |
| ISSN: | 0006-3495 |
| DOI: | 10.1529/biophysj.106.084251 |
| Popis: | 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 approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) 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. |
| Databáze: | OpenAIRE |
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