Inverse-fluorescence correlation spectroscopy: more information and less labeling.

Inverse-Fluorescence Correlation Spectroscopy (iFCS) is a recently developed modification of standard FCS that allows analysis of particles and biomolecules without labeling. The particles generate no signal; instead the signal is generated by a surrounding medium. Particles diffusing through the FCS-detection volume displace a fraction of the surrounding medium, causing transient dips in the detected signal. These give information about the mobility and concentration of the analyzed particles. Also labeled particles can be analyzed, whereby their signal is cross-correlated with that from the surrounding medium (iFCCS). This can give information about the volume of the labeled particles, or alternatively about the size of the detection volume. Also the interaction of unlabeled particles with small, labeled ligands can be analyzed with iFCCS. This allows using cross-correlation as a sensitive indication of binding, even though only one binding-partner is labeled. This review describes the principles of iFCS and iFCCS and measurements of microspheres dissolved in a surrounding medium containing alexa 488. We also discuss practical considerations, and future possibilities for analyses of biomolecules.