Massively-parallel fluorescence correlation spectroscopy using a spinning disk confocal microscope

We describe 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 $\sim $10$^{5}$ independent locations simultaneously. Complex media---e.g., a tumor, cell nucleus, or extracellular matrix---are spatially-heterogeneous, making this spatially-resolved technique an ideal tool to understand hindered diffusion. There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, can be much faster at high resolution---potentially up to 1000 Hz at full resolution for the fastest available cameras---and without temporal delays between pixels. We show how to correct for a pixel size effect not encountered with standard or scanning FCS, and we introduce a method to correct for photobleaching. Finally, we apply this technique to microspheres diffusing in Type I collagen, which show non-trivial spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.