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Single-particle tracking of filaments was performed manually in Volocity.
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The aim of this work was to develop a protocol for automated tracking of actin filaments in electron tomograms of lamellipodia embedded in negative stain.
The images in Figure 2A, J and visual inspection of the movies provided suggests tracking of individual filament ends may not be possible using the current microscopy methods.
In detail, myosins can transport cargoes inside the cell on the tracks of actin filaments [ 165] and walk like motor proteins to generate force and displacement along actin filaments.
We agree with the reviewer that difference mapping is a powerful tool that can often eliminate some of the perils of tracking individual filaments, but the limitations of our host/phage system have complicated its use in this case.
Notably, these calculations were very recently confirmed by experimental data showing short tracks of processive filament elongation by single VASP tetramers in solution using TIRF microscopy (Hansen and Mullins, 2010).
The sample number (N) was conservatively taken as the number of independent experiments, not the number of filaments tracked.
When hundreds of filaments are tracked, a total distance moved by all the filaments can be determined from the tabulated data list.
A network of filaments called microtubules forms tracks along which so-called motor proteins carry these items.
To quantitatively assess movement of NFs in general, the leading edges of filaments moving out of the photoactivated neuritic segment were tracked by using the Manual Tracking plugin of ImageJ and by using the cell body as a reference point.
DOI: http://dx.doi.org/10.7554/eLife.06088.001 Cells contain a network of filaments known as microtubules that serve as tracks along which proteins and other materials can be moved from one location to another.
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