Here we demonstrate that SPIM-FCS/FCCS possesses the sensitivity to detect and quantify protein-protein interactions in live cells by characterizing the interaction of the subunits of heterodimeric transcription factors, c-Fos/c-Jun and IQGAP/cdc42. The protein-protein interaction clearly shows up in the cross-correlation amplitude. Analysis of the spatial distribution of diffusion coefficients of the fluorescent proteins and of their cross-correlation amplitude shows that formation of the heterodimer is correlated with regions of decreased mobility, probably related to DNA binding.
Picosecond lasers sources operating over an order of magnitude in repetition rates and pulse energies at selected emission wavelengths from the UV to IR range are presented for time-correlated single-photon counting in demanding imaging applications.
show the kinetics of antigen and antibody binding in real-time without significant contribution of signal from background fluorescence. We also show how single molecule analysis allows determination of the labeling efficiency of the antibody bound to the surface. By analyzing the bleaching steps of individual fluorophores at a given location, we can determine the number of dye molecules attached to randomly labeled antibody conjugates. Our data using this method indicates a bias towards antibody labeled with less fluorophores. Finally, we show single molecule detection of sub-picomolar concentrations of antigen using well characterized antibody reagents.
Time-domain diffuse correlation spectroscopy (TD-DCS) aims to increase cerebral blood flow (CBF) sensitivity by discriminating photon time of flight. We report on the optimization of the laser pulse shape to maximize TD-DCS performance.
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