Fig. 1: Experimental workflow and representative cell morphologies in different extracellular osmolarities.

A Shows the division of 3D space of an artificial cell into octants around the centroid (marked in red) and the sides associated with octants along the 3 axes. B Flowchart showing the experimental methodology prior to image acquisition. Cells were cultured in a T25 flask till passage 4, followed by the seeding of approximately 50000 cells in a 35 mm Petri dish containing a 19 mm square cover slip. Cells were allowed to adhere to the surface of the coverslip and arrive in the log phase in 12 h. After 12 h, organelles were fluorescently labeled, and tonicity treatments were carried out for 30 min followed by slide preparation in the treatment buffer as mountant and image acquisition. Both DIC and fluorescent images were captured for each field. C Shows a representative asymmetrical cell, and the overlaying ROI (blue boundary). D, E Show the representative frame reading pattern along the X and Y axes, respectively. Imaging was carried out at in UPlanSApo 60x oil objective (N.A. 1.35) in Olympus FV1200 Confocal Microscope. F, G Show Three representative images of cells from the same section of single fields in the three environmental conditions, hypertonic (i), isotonic (ii) and hypotonic (iii) in asymmetric (F) and symmetric cells (G) of RAW264.7 cells. (H) show Pie chart representing the distribution of number of asymmetric (darker shades) and symmetric (lighter shades) cell subsets in RAW264.7 cell population in three different environmental conditions.