1 4 trimethylammoniumphenyl 6 phenyl 1 3 5 hexatriene p toluenesulfonate tma dph

Vegetative and sporulating cells and dormant spores were collected by centrifugation at 6,500×g for 1 min and immobilized on 2% agarose pads. Phase-contrast and fluorescence microscopy were performed using an Olympus BX61 microscope equipped with an UplanF1 100× phase contrast objective lens and a monochrome CoolSnapHQ digital camera (Photometrics). Membranes were stained with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH; Molecular Probes) at a final concentration of 50μM. Live-dead staining was performed with propidium iodide (PI; Invitrogen) at a final concentration of 5μM. Exposure times were typically 100, 200 and 400 ms for TMA-DPH, GFP, and PI, respectively. Image analysis and processing were performed using MetaMorph software (Molecular Devices; version 7.7).

Fluorescence microscopy was performed with an Olympus BX61 microscope as previously described [58 (link)]. Cells were mounted on a 2% agarose pad containing resuspension medium using a gene frame (BioRad). Fluorescent signals were visualized with a phase contrast objective UplanF1 100x and captured with a monochrome CoolSnapHQ digital camera (Photometrics) using MetaMorph software version 7.7 (Molecular devices). The membrane dye 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH, Molecular Probes) was used at a final concentration of 50 μM and exposure times were typically 500 ms. The DNA dye 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes) was used at 2 μg/mL and exposure times were typically 200 ms. At least two biological replicates were performed for all microscopy experiment. Images were analyzed, adjusted and cropped using MetaMorph software.