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.
"In principle, this can be done at enough positions to derive a mobility map with the caveat that during the data acquisition period cell dynamics usually become significant. Depending on mobility and brightness of the particle the acquisition time for a correlation analysis can be reduced to some seconds or even tens of milliseconds to increase the number of cellular regions that are sampled (Roth et al. 2007). By extending the FCS setup to a two-color system and by labeling potentially interacting proteins with two spectrally distinct fluorophores, fluorescence cross-correlation spectroscopy provides a highly sensitive readout for bimolecular interactions (Ricka and Binkert 1989; Rippe 2000; Schwille et al. 1997). "
[Show abstract][Hide abstract] ABSTRACT: The genome of eukaryotes is organized into a dynamic nucleoprotein complex referred to as chromatin, which can adopt different functional states. Both the DNA and the protein component of chromatin are subject to various post-translational modifications that define the cell's gene expression program. Their readout and establishment occurs in a spatio-temporally coordinated manner that is controlled by numerous chromatin-interacting proteins. Binding to chromatin in living cells can be measured by a spatially resolved analysis of protein mobility using fluorescence microscopy based approaches. Recent advancements in the acquisition of protein mobility data using fluorescence bleaching and correlation methods provide data sets on diffusion coefficients, binding kinetics, and cellular concentrations on different time and length scales. The combination of different techniques is needed to dissect the complex interplay of diffusive translocations, binding events, and mobility constraints of the chromatin environment. While bleaching techniques have their strength in the characterization of particles that are immobile on the second/minute time scale, a correlation analysis is advantageous to characterize transient binding events with millisecond residence time. The application and synergy effects of the different approaches to obtain protein mobility and interaction maps in the nucleus are illustrated for the analysis of heterochromatin protein 1.
Chromosome Research 01/2011; 19(1):99-115. DOI:10.1007/s10577-010-9155-6 · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aus der Einzelmolekülfluoreszenzspektroskopie sind eine Reihe unterschiedlicher Techniken hervorgegangen, mit denen molekulare Prozesse im thermodynamischen Gleichgewicht gemessen und Subpopulationen heterogenerer Molekülverteilungen aufgedeckt werden können, die sonst im Ensemble verborgen bleiben. Die Anwendungen von Einzelmolekülexperimenten reichen von den Lebenswissenschaften über die Materialwissenschaften bis in die Photophysik und Photochemie. Einige dieser Forschungsfelder, z.B. die chemische Katalyse, wurden erst vor Kurzem erschlossen. Dieser Aufsatz will die wichtigsten Prinzipien der Einzelmolekülfluoreszenzspektroskopie zusammenfassen und einen Überblick über wichtige Anwendungen geben, die bis zur Entwicklung neuer mikroskopischer Verfahren mit Nanometer-Auflösung reichen.
Single-molecule fluorescence spectroscopy evolved to a variety of tools to investigate molecular dynamics in thermodynamic equilibrium and to reveal subpopulations in heterogeneous molecular distributions which usually remain hidden in bulk experiments. Applications of single-molecule experiments range from life sciences and material sciences to photo-physics and photo-chemistry. Some of these research fields, like chemical catalysis, have just recently been entered. This article summarizes major principles of single-molecule fluorescence spectroscopy and gives an overview on some important applications up to the development of novel microscopic techniques with nanometer resolution.
Chemie in unserer Zeit 06/2008; 42(3):192 - 199. DOI:10.1002/ciuz.200800448 · 0.26 Impact Factor
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