VISION — an open-source software for automated multi-dimensional image analysis of cellular biophysics

Environment-sensitive probes are frequently used in spectral/multi-channel microscopy to study alterations in cell homeostasis. However, the few open-source packages available for processing of spectral images are limited in scope. Here, we present VISION, a stand-alone software based on Phyton for spectral analysis with improved applicability. In addition to classical intensity-based analysis, our software can batch-process multidimensional images with an advanced single-cell segmentation capability and apply user-defined mathematical operations on spectra to calculate biophysical and metabolic parameters of single cells. VISION allows for 3D and temporal mapping of properties such as membrane fluidity and mitochondrial potential. We demonstrate the broad applicability of VISION by applying it to study the effect of various drugs on cellular biophysical properties; the correlation between membrane fluidity and mitochondrial potential; protein distribution in cell-cell contacts; and properties of nanodomains in cell-derived vesicles. Together with the code, we provide a graphical user interface for facile adoption. We anticipate that VISION will find a broad range of applications in different fields of biology, spanning from molecular and tissue biology to immunology and biophysics. Summary statement VISION, an open-source software, enables high throughput and correlative analysis of cellular biophysical properties. ### Competing Interest Statement The authors have declared no competing interest.