Single particle tracking

Principle of single particle tracking: The rectangles represent individual images of a time series. The tracked particles are marked in red, and in the last picture the reconstructed trajectories are shown as blue lines. ${\displaystyle {\vec {x}}_{i}(t)}$

Single particle tracking , Einzelteilchenverfolgung or conventional English single-particle tracing ( SPT ) is a measuring method of physics and especially the Biophysics , at which the trajectories of many (microscopic) particles (eg. As a liquid or in a medium cell ) can be collected individually . It allows the Brownian molecular motion to be observed and quantified directly.

description

Single particle tracking is often used in conjunction with various fluorescence microscopic techniques . The particles to be tracked are then marked with a fluorescent dye . If they are present in such a low concentration that individual particles can be differentiated and localized in the microscopic image, individual particle tracking can be used. You then take a quick series of images and locate the particles in each of these images. This gives a set of coordinates of the particles for each image in the series . Then an attempt is made to assign the particle positions from two (or more) successive images to a particle so that trajectories for the particles are obtained ( tracking ). Often one then assigns those positions and from two consecutive images that are closest to one another. ${\displaystyle t}$ ${\displaystyle {\vec {x}}_{i}(t)}$ ${\displaystyle i}$ ${\displaystyle {\vec {x}}_{i}(t)}$ ${\displaystyle {\vec {x}}_{j}(t+1)}$

This method then results in a set of trajectories that can be fed to a further statistical evaluation in order to calculate diffusion coefficients or transport speeds, for example . Different diffusion processes ( normal diffusion , anomalous diffusion , diffusion in closed pores, directed transport by motor proteins along actin filaments) can be distinguished by calculating the mean square displacement . ${\displaystyle {\vec {x}}_{i}(t)}$

As with fluorescence recovery after photobleaching (FRAP), a mobile fraction can easily be distinguished from an immobile fraction (e.g. in the case of a bound receptor to the extracellular matrix or the cytoskeleton ). In addition, fractions that diffuse at different speeds (e.g. during oligomerization ) can be quantified.

In addition to the mean square displacement, the mean angle between successive displacements can also be determined, which is a measure of the directionality of the respective trajectory.

Microscopy method

Fluorescence microscopy methods suitable for tracking individual particles are e.g. B.

• Internal total reflection fluorescence microscopy (TIRF)
• Light disk microscopy (LSFM)

In addition, scanning confocal microscopy suitable. Only a single particle can be tracked, but with a higher temporal resolution. For example, the focus of a confocal microscope can be moved continuously in circles around a particle to be tracked. If it moves, the fluorescence distribution on the circular path is no longer the same everywhere and the orbit of the focus can be shifted accordingly. The trajectory of the particle is then obtained from the successive positions of the orbit.

resolution

Camera-based processes achieve temporal resolutions in the range of 1–100 ms. Resolutions of up to 25 µs can be achieved with very fast cameras.

The spatial resolution is typically better than the resolution of the microscopy method (factor 1–5, or 20–100 nm), since the position of a single fluorophore can be determined with sub-pixel precision (see Photoactivated Localization Microscopy ).

Individual evidence

1. ^ H. Qian, MP Sheetz, EL Elson: Single particle tracking. Analysis of diffusion and flow in two-dimensional systems . In: Biophysical Journal . tape 60 , no. 4 , October 1991, ISSN  0006-3495 , pp. 910-921 , doi : 10.1016 / S0006-3495 (91) 82125-7 , PMC 1260142 (free full text).
2. ↑ Michael J. Saxton, Ken Jacobson: Single-Particle Tracking: Applications to Membrane Dynamics . In: Annual Review of Biophysics and Biomolecular Structure . tape 26 , no. 1 , June 1997, ISSN  1056-8700 , p. 373-399 , doi : 10.1146 / annurev.biophys.26.1.373 .
3. ↑ Kevin Braeckmans, Dries Vercauteren, Jo Demeester, and Stefaan De Smedt: Single particle tracking . In: Alberto Diaspro (Ed.): Nanoscopy and multidimensional optical Fluorescence microscopy . CRC Press, 2010, ISBN 978-1-4200-7886-2 , pp. 5-1-5-17 , doi : 10.1201 / 9781420078893-c5 .
4. ↑ Stas Burov, Jae-Hyung Jeon, Ralf Metzler, Eli Barkai: Single particle tracking in systems showing anomalous diffusion: the role of weak ergodicity breaking . In: Physical Chemistry Chemical Physics . tape 13 , no. 5 , 2011, ISSN  1463-9076 , p. 1800-1812 , doi : 10.1039 / C0CP01879A .
5. ↑ Thomas Bürli, Kristin Baer, ​​Helge Ewers, Corinne Sidler, Christian Fuhrer, Jean-Marc Fritschy, Huibert D. Mansvelder: Single Particle Tracking of α7 Nicotinic AChR in Hippocampal Neurons Reveals Regulated Confinement at Glutamatergic and GABAergic Perisynaptic Sites . In: PLoS ONE . tape 5 , no. 7 , July 9, 2010, ISSN  1932-6203 , p. e11507 , doi : 10.1371 / journal.pone.0011507 .
6. ^ H. Ewers, AE Smith, IF Sbalzarini, H. Lilie, P. Koumoutsakos, A. Helenius: Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes . In: Proceedings of the National Academy of Sciences . tape 102 , no. 42 , October 18, 2005, ISSN  0027-8424 , p. 15110-15115 , doi : 10.1073 / pnas.0504407102 .
7. ↑ Jörg G. Ritter, Roman Veith, Jan-Peter Siebrasse, Ulrich Kubitscheck: High-contrast single-particle tracking by selective focal plane illumination microscopy . In: Optics Express . tape 16 , no. 10 , 2008, ISSN  1094-4087 , p. 7142-7152 , doi : 10.1364 / OE.16.007142 .
8. ^ Jörg Gerhard Ritter, Roman Veith, Andreas Veenendaal, Jan Peter Siebrasse, Ulrich Kubitscheck, Jörg Langowski: Light Sheet Microscopy for Single Molecule Tracking in Living Tissue . In: PLoS ONE . tape 5 , no. 7 , July 23, 2010, ISSN  1932-6203 , p. e11639 , doi : 10.1371 / journal.pone.0011639 .
9. ↑ Valeria Levi, QiaoQiao Ruan, Enrico Gratton: 3-D Particle Tracking in a Two-Photon Microscope: Application to the Study of Molecular Dynamics in Cells . In: Biophysical Journal . tape 88 , no. 4 , April 2005, ISSN  0006-3495 , p. 2919-2928 , doi : 10.1529 / biophysj.104.044230 .
10. ↑ Akihiro Kusumi, Chieko Nakada, Ken Ritchie, Kotono Murase, Kenichi Suzuki, Hideji Murakoshi, Rinshi S. Kasai, Junko Kondo, Takahiro Fujiwara: 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 . In: Annual Review of Biophysics and Biomolecular Structure . tape 34 , no. 1 , June 2005, ISSN  1056-8700 , p. 351-378 , doi : 10.1146 / annurev.biophys.34.040204.144637 ( nanobio.frontier.kyoto-u.ac.jp [PDF]). nanobio.frontier.kyoto-u.ac.jp ( Memento of the original from March 4, 2016 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.