We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.
Spatial Fluorescence Cross Correlation Spectroscopy is a rarely investigated version of Fluorescence Correlation Spectroscopy, in which the fluorescence signals from different observation volumes are cross-correlated. In the reported experiments, two observation volumes, typically shifted by a few µm, are produced, with a Spatial Light Modulator and two adjustable pinholes. We illustrated the feasibility and potentiality of this technique by: i) measuring molecular flows, in the range 0.2 -1.5 µm/ms, of solutions seeded with fluorescent nanobeads or rhodamine molecules (simulating active transport phenomenons); ii) investigating the permeability of the phospholipidic membrane of Giant Unilamellar Vesicles versus hydrophilic or hydrophobic molecules (in that case the laser spots were set on both sides of the membrane). Theoretical descriptions are proposed together with a discussion about Fluorescence Correlation Spectroscopy based, alternative methods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.