Abstract
The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. Although the skewness of the pixel intensity distribution derived from fluorescently-labeled cytoskeletons has been widely used as a metric to evaluate the degree of bundling in digital microscopy images, its versatility has not been fully validated. Here, we applied the coefficient of variation (CV) of intensity values as an alternative metric, and compared its performance with skewness. In synthetic images representing extremely bundled conditions, the CV successfully detected degrees of bundling that could not be distinguished by skewness. On actual microscopy images, CV was better than skewness, especially on variable-angle epifluorescence microscopic images or stimulated emission depletion and confocal microscopy images of very small areas of around 1 μm2. When blur or noise was added to synthetic images, CV was found to be robust to blur but deleteriously affected by noise, whereas skewness was robust to noise but deleteriously affected by blur. For confocal images, CV and skewness showed similar sensitivity to noise, possibly because optical blurring is often present in microscopy images. Therefore, in practical use with actual microscopy images, CV may be more appropriate than skewness, unless the image is extremely noisy.
Highlights
The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology
For the variable-angle epifluorescence microscopy (VAEM) images, no significant differences in skewness values were detected between the three samples (Fig. 6B, Supplemental Fig. 8B), whereas the differences in coefficient of variation (CV) values were statistically significant between the DMSO control and 5 μM and 20 μM triiodobenzoic acid (TIBA) treatments (Fig. 6C, Supplemental Fig. 8B), as was the case for the confocal laser scanning microscopy (CLSM) images (Fig. 5C, Supplemental Fig. 7B). These results suggest that CV is more suitable than skewness for evaluating cytoskeleton bundling in VAEM images
We have shown that intensity features are useful for understanding cytoskeleton organization, especially cytoskeleton bundling
Summary
The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. In cells with fluorescently labeled cytoskeletons, cytoskeleton bundles show stronger fluorescence than single filaments, suggesting that bundling level could be quantitatively evaluated by measuring the absolute values of fluorescent intensity peaks in fluorescent microscopy images[10] This method may not be accurate in some cases, such as when the abundance levels of cytoskeleton fluorescent protein markers differ according to cell status[11]. We previously reported a robust quantification method for measuring changes in the abundance levels of fluorescent protein markers[7,12], which uses the skewness of the pixel intensity distribution derived from the cytoskeleton as a metric for the quantitative evaluation of actin filament bundling in plant stomatal guard cells. The CV method allowed successful detection of cytoskeleton bundling with equal or greater sensitivity than the skewness method, and it was useful for blurred images where skewness was inappropriate
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