Exact(7)
Images of single, in focus cells were selected for analysis.
After release of the block the proportion of single ori1 focus cells had reduced to 13%.
These single cells were now gated for in focus cells based on bright field gradient root mean square (GRMS) feature (> 300 GRMS).
Four categories of cells according to the number of foci were distinguished, namely, cells without DNA DSB, cells with only one focus, cells with two to five foci and cells with more than five foci.
Single focus cells (20%25%%) had the focus positioned in the midcell region (>85% in the midcell third), while those with two foci generally had the foci in different cell halves, with the mean position close to the nucleoid quarter.
Potential improvements that could be made to reduce the impact of imperfections in the samples are focus stacking, to reduce the impact of out of focus cells, and taking into account image texture, from the phase contrast image, to differentiate debris and thresholding artifacts from cells.
Similar(53)
First dimensional electrophoresis was performed using a Protean iso-electronic focus cell unit (BioRad).
We used appropriate size thresholds to exclude small objects not representing entirely a cell, such as ramification portions from out-of-focus cells, and we distributed acquisition frames equally by an operator non-dependent sampling of the region of interest, thus limiting the impact of non-stereological acquisition.
This would prevent partially out-of-focus cells and some mutant morphologies from being analyzed.
Color deconvolution was able to separate signal from kinetoplasts and nuclei for all cells, including cells with approximately wild-type morphology, zoids, cells with disrupted nuclear structure and out-of-focus cells.
Other issues with accurate analysis of cells primarily arose from technical issues with sample preparation (debris and multiple cells lying in contact with each other) and image capture (out-of-focus cells).
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