SCANNING 2-PHOTON FLUCTUATION CORRELATION SPECTROSCOPY - PARTICLE COUNTING MEASUREMENTS FOR DETECTION OF MOLECULAR AGGREGATION

Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by moni toring spontaneous equilibrium fluctuations in the local concentration of fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, su bmicromolar samples by directly ''counting particles.'' We introduce t he application of two-photon excitation to scanning FCS and discuss it s important advantages for this technique. We demonstrate the capabili ty of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, a nd then with B phycoerythrin. The detection of multiple species in a s ingle sample is shown, using mixtures containing both sphere sizes. Th e method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/dissociation equilibri um for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good a greement with previously published values. In addition, oligomer disso ciation induced by pH titration from pH 8 to pH 5.0 is detectable for the enzyme phosphofructokinase. The possibility of measuring dissociat ion kinetics by scanning two-photon FCS is also demonstrated using pho sphofructokinase.