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Imaging diffusion in living cells using time-correlated single-photon counting.

Analytical chemistry (2007-09-07)
Christian M Roth, Pia I Heinlein, Mike Heilemann, Dirk-Peter Herten
ABSTRACT

Current efforts to monitor the diffusion of proteins in living cells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, these methods cannot generate a map of diffusion times. Here, we introduce a new method termed diffusion imaging microscopy that combines scanning confocal microscopy, time-correlated single-photon counting, and FCS and thus allows us to measure spatially resolved diffusion times. In our approach, we record scan images with time-resolved photon streams within each individual pixel. By extending the pixel dwell time to 25-100 ms, a software correlation of individual photons within each pixel yields the average diffusion time. Additionally, information on fluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sort fluorescence 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 method to living cells and recorded a diffusion map of a living 3T3 mouse fibroblast incubated with dT20-ATTO488.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 488 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 488, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 488-Biotin, BioReagent, suitable for fluorescence, ~75% (HPCE)
Sigma-Aldrich
Atto 488 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 488 amine
Sigma-Aldrich
Atto 488 azide
Sigma-Aldrich
Atto 488 iodoacetamide

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