Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-phot on microscopic imaging and image correlation analysis may be combined to me asure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coeffic ients as large as 10(-8) cm(2) s(-1) that matched theoretical predictions f or samples of fluorescent microspheres suspended in aqueous sucrose solutio ns. We also show the sensitivity of the method for measuring microscopic fl ow using analogous test samples. We demonstrate explicitly the advantages o f the image correlation approach for measurement of correlation functions w ith high signal-to-noise in relatively short time periods and discuss situa tions when these methods represent improvements over non-imaging fluorescen ce correlation spectroscopy. We present the first demonstration of two-phot on image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed las er. We also demonstrate cellular application of two-photon ICS for measurem ents of slow diffusion of green fluorescent protein/adhesion receptor const ructs within the basal membrane of live CHO fibroblast cells.