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Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (∼10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.

We applied single-molecule fluorescence microscopy (using organic dyes or semiconductor quantum dots) to study the lateral diffusion of glutamate receptors (AMPA and NMDA) in live synapses. We directly imaged glutamate receptors movements inside and outside synapses of live cultured hippocampal neurons. We could record exchanges of receptors through lateral diffusion between these different membrane compartments. In addition, our data suggest that this lateral diffusion might be regulated by neuronal activity. To overcome the photobleaching problem inherent to fluorescence techniques we recently developed new optical methods for the detection of individual metallic nanoparticles. We can now detect signatures of diffusing AMPA receptors labeled with 10 nm gold nanoparticles on live neurons.

An all-optical method called photothermal heterodyne imaging (PHI) is developed that allows for the detection of individual nano-objects such as gold nanoparticles with diameter down to 1.4 /spl mu/m (67 atoms) as well as nonluminescent semiconductor nanocrystals. This observation of intrinsic size effects in the optical response of gold nanoparticles is analysed within the frame of Mie theory using a size-dependent surface damping term in the dielectric constant.

Photothermal Heterodyne Imaging (PHI) is a highly sensitive optical detection method of individual absorptive nano-objects. It can be applied to absorption spectroscopy measurements Surface Plasmon Resonance spectra of individual gold nanoparticules with diameters down to 5nm were recorded. Intrinsic size effects which result in a broadening of the Resonance are unambiguously observed and analyzed within the frame of Mie theory. Preliminary results obtained with silver nanoparticles are also presented.