Fluorodishes

Cells were concentrated by centrifugation (500×g for 5 min) and resuspended either in 0.5 mL of spring water in coverglass bottomed FluoroDishes (World Precision Instruments FD35-100) or in 0.2 mL spring water on a coverslip (FisherScientific, 12-545-D) for imaging. No more than three cells were kept in 0.5 mL imaging samples and only one cell was ever kept in 0.2 mL imaging samples in order to minimize cell-cell interactions. Cells were observed to exhibit spontaneous walking activity on coverglass. Walking cells in FluoroDishes were imaged under brightfield illumination using a Zeiss Z.1 Observer and Hamamatsu Orca Flash 4.0 V2 CMOS camera (C11440-22CU) with a 20x, 0.8 NA Plan-Apochromat (Zeiss) objective. Cells on coverslips were imaged under brightfield illumination with coverslips inverted over a well containing a small amount of distilled water to reduce evaporation using a Zeiss Axio Zoom.V16 and a PCO pco.dimax S1 camera. Importantly, in both imaging systems, the focal plane was set at the interface between cirri of walking cells and the glass surface upon which they were walking. Images were acquired at 0.033 seconds per frame with a 0.005 second exposure in order to capture all cirral dynamics during walking with minimal blur.