Single-molecule tracking

Figure 1 Modes of diffusion of individual membrane proteins as revealed by single-molecule tracking techniques. The hypothetical trajectory of an individual plasma membrane protein as traced by single-particle tracking techniques is shown. An individual protein can switch between several different modes of over time, which include confined diffusion (region 1), free diffusion (region 2), and immobilization (region 3).

An obvious advantage of single molecule tracking is that it allows for highly detailed analyses of the movements of individual molecules that are obscured in population-based measurements such as FRAP and photoactivation experiments. However, SPT... 

Kusumi A, et al. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid high-speed single-molecule tracking of membrane molecules. Ann. Rev. Biophys. Biomolec. Struct. 2005 34 351-378. 

Quest dynamics through single-molecule tracking... 

Fig. 27.1. Comparison between transmission electron microscopy (TEM) and single-molecule tracking (SMT) by optical wide-field microscopy, (a) High-resolution TEM gives the landscape of a channelar structure of a hexagonal mesoporous system and (b) SMT gives the trajectories of the movement of single molecules as guests in the nanoporous host...

Murase, K. and Fujiwara, T. (2005) Single-molecule tracking of membrane molecules plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules. Sem. Immunol., 17, 3-21. 

Fig. 13 Single molecule tracking data of dye-labeled PMOx lipopolymers as a function of area per molecule. The plots of the lateral diffusion coefficient, D, vs area per molecule for DiCisPMOx3o and DiCisPMOxso show two different diffusion regions (labeled I and II). Unlike in Region II, D follows Rouse scaling in Region I [31] (reproduced with permission from the American Chemical Society)...

K. Iwasawa, T. Fujiwara, A. Kusumi, Detection of non-brownian diffusion in the cell membrane in single molecule tracking, Biophys.J. 2005, 88, 2266-2277. 

The analysis of single-molecule data is a field of ongoing development. We therefore only provide a relatively simplified view on a basic approach to obtain the diffusion coefficient from the analysis of single-molecule tracks. In principle, each track can be analyzed individually, which yields a diffusion coefficient for each tracked molecule. However, due to the stochastic nature of the... 

For highly concentrated polymer solutions, FCS measurements revealed subdiffusive motion as an additional mode on an intermediate timescale between the fast collective diffusion and the slow self-diffusion [24]. In such slow systems, however, FCS reaches its limits when probe motion becomes so slow that the number of molecules moving into or out of the confocal volume within the measurement time is too small to allow for reliable statistics. Increasing the measurement time is often not straightforward since all fluorescence dyes have only a limited photostability. If a dye bleaches within the confocal volume, it will fake a faster diffusional motion than its real value. Therefore, for the study of such concentrated systems, wide-field fluorescence microscopy and subsequent single molecule tracking is a much better method [120] and has been utilized to study the glass transition [87, 121]. 

W611 D, Kolbl C, Stempfle B, Karrenbauer A (2013) Novel method for automatic single molecule tracking of blinking molecules at low intensities. Phys Chem Chem Phys 15 6196-6205... 

Siebrasse, J. R Kubitscheck, U. Single molecule tracking for studying nucleocytoplasmic transport and intranuclear dynamics. Methods Mol. Biol. 2008, 464, 343-361. 

Frost NA, Lu HE, Blanpied TA (2012) Optimization of cell morphology measme-ment via single-molecule tracking PALM. PLoS One 7 e36751... 

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