Summary Our increasing ability to resolve fine details using light microscopy is matched by an increasing need to quantify images in order to detect and measure phenotypes. Despite their central role in cell biology, many image analysis tools require a financial investment, are released as proprietary software, or are implemented in languages not friendly for beginners, and thus are used as black boxes. To overcome these limitations, we have developed PyJAMAS, an open-source tool for image processing and analysis written in Python. PyJAMAS provides a variety of segmentation tools, including watershed and machine learning-based methods; takes advantage of Jupyter notebooks for the display and reproducibility of data analyses; and can be used through a cross-platform graphical user interface or as part of Python scripts via a comprehensive application programming interface. Availability PyJAMAS is open-source and available at https://bitbucket.org/rfg_lab/pyjamas. Supplementary information Supplementary data are available at Bioinformatics online.

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Integrins a5b1 and avb3 both mediate cell focal adhesion: a5b1 supports firm adhesion and spreading, whereas aVb3 mediates early-stage mechano-signal transduction and rigidity sensing. Integrin functions are correlated with its global conformation, which is regulated by mechanical force-force pulling facilitates bent integrins to unbend. However, (un)bending conformational changes were never experimentally observed on purified integrins, nor was it clear whether they have distinct mechano-sensitivity among different integrins. Using two single-molecule force spectroscopies, we visualized in real-time that purified a5b1 and avb3 can both undergo unbending and bending conformational changes while binding to fibronectin, but manifest distinctive mechano-sensitivity. The conformational changes of a5b1 were ''digitally'' triggered by a force threshold of 7.4 pN, so that forces above/below this threshold respectively stabilize the integrin in the extended/bent conformation. In contrast, the conformation of avb3 was ''analogously'' regulated by force over a wide range (0 to >40 pN), where an increase in force accelerated the unbending but decelerated the bending. The distinctive mechano-sensitivity of a5b1 and avb3 potentially comply with their respective roles in focal adhesion: the ''digital'' unbending of a5b1 allows quick activation by extracellular stretch to mediate strong adhesion, whereas the ''analogous'' force-regulation of avb3 (un)bending enables them to collectively act as a 'ruler' to 'measure' the extracellular stretching and matrix rigidity. The fact that avb3 can bend against large tensile force is intriguing. Based on the experimentally measured (un)bending kinetics and our molecular dynamics simulation results, we constructed energy landscapes for both integrins. Unlike a5b1 wherein bent and extended states are separated by a single energy barrier, avb3 also possesses several semi-stable intermediate states, which realize a ''protein machinery'' that can accumulate energy from small-scale random thermodynamics to allow large-scale directional motion against force.