| Abstract
| - Current efforts to monitor the diffusion of proteins in livingcells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, thesemethods cannot generate a map of diffusion times. Here,we introduce a new method termed diffusion imagingmicroscopy that combines scanning confocal microscopy,time-correlated single-photon counting, and FCS and thusallows us to measure spatially resolved diffusion times.In our approach, we record scan images with time-resolved photon streams within each individual pixel. Byextending the pixel dwell time to 25−100 ms, a softwarecorrelation of individual photons within each pixel yieldsthe average diffusion time. Additionally, information onfluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sortfluorescence photons and to discriminate from autofluorescence. We evaluated our method by measuring diffusion times of dT20-TMR in solutions of different viscosity.We further demonstrate the applicability of the methodto living cells and recorded a diffusion map of a living 3T3mouse fibroblast incubated with dT20-ATTO488.
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