Quantitative Determination of Cellular-and Neurite Motility Speed in Dense Cell Cultures

Abstract

Mobility quantification of single cells and cellular processes in dense cultures is a challenge, because single cell tracking is impossible. We developed a software for cell structure segmentation and implemented 2 algorithms to measure motility speed. Complex algorithms were tested to separate cells and cellular components, an important prerequisite for the acquisition of meaningful motility data. Plasma membrane segmentation was performed to measure membrane contraction dynamics and organelle trafficking. The discriminative performance and sensitivity of the algorithms were tested on different cell types and calibrated on computer-simulated cells to obtain absolute values for cellular velocity. Both motility algorithms had advantages in different experimental setups, depending on the complexity of the cellular movement. The correlation algorithm (COPRAMove) performed best under most tested conditions and appeared less sensitive to variable cell densities, brightness and focus changes than the differentiation algorithm (DiffMove). In summary, our software can be used successfully to analyze and quantify cellular and subcellular movements in dense cell cultures.

Noninvasive measurement of cell/colony motion using image analysis methods to evaluate the proliferative capacity of oral keratinocytes as a tool for quality control in regenerative medicine

“…Nonetheless, as shown in Figure 4(c), the identical order of the two indices obtained from three colonies and the similar outcomes obtained from the two algorithms indicate that each compensates for the limitations of the other, suggesting that the quantitative measurements acquired using two different algorithms collaborate efficiently. 36…”

“…However, as cells proliferate, sampling of distinct colonies will be impossible, due to an increase in cell confluency. 36 Thus, the current protocol is not appropriate to measure the status of the entire cell population. In a clinical setting in which a larger number of cells are present in culture, an alternative protocol is needed to produce a culture vessel-based measurement, instead of a single colony base evaluation, for the later phase of cell culture, in order to eliminate a substandard cell population as a QC.…”

Viability and Contractility of Rat Brain Pericytes in Conditions That Mimic Stroke; an in vitro Study

“…For Alexa Fluor 555 staining analysis, images were further processed with self-written SynoQuant Ver. 10.69 software (for details see Henkel et al, 2019). The images were deblurred with a non-linear algorithm (“Level background”) that removed specifically homogenous stain corresponding to background staining, while enhancing structured any objects.…”

“…Contraction and dynamics of large processes are quantified by the area/perimeter ratio (A/PR), while small membrane movements could be measured by the fractal dimension (D f ) of the cellular perimeter (red border around cells in Supplemental Figure 3). The mobility speed was quantified by spatial intensity difference changes between successive image frames; this protocol was described in detail previously (Henkel et al, 2019).…”

“…Details of all procedures have been described previously (Henkel et al, 2019). The following parameters were measured: single cell membrane mobility (SCMM), Area/Perimeter ratio (A/PR), which corresponds to cell surface–cell volume ratio and is well-suited to monitor cell process dynamics.…”