Orca flash v2 digital cmos camera

Exponentially growing cells were transferred to slides containing a thin pad of 1.5% agarose (Cambrex) with TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KH₂PO₄ pH 7.6, 8 mM MgSO₄, 0.2% CTT, covered with a coverslip and imaged with a temperature-controlled Leica DMi8 inverted microscope. Phase contrast and fluorescence images were acquired using a Hamamatsu ORCA-flash V2 Digital CMOS camera. Cells in phase contrast images were automatically detected using Oufti software^{72 (link)}. Fluorescence signals were identified and analyzed using a custom-made Matlab v2016b (MathWorks) script. E. coli cells were induced with 0.05 mM isopropyl-β-D-thiogalactopyranosid (IPTG) for 2 h and treated with 30 μg ml⁻¹ chloramphenicol for 30 min before DAPI staining. For DAPI staining, cells were incubated with 1 mg ml⁻¹ DAPI for 10 min at 32 °C prior to start of microscopy. Image processing was performed using Metamorph_ v 7.5 (Molecular Devices).

Exponentially growing M. xanthus cultures were harvested (3 min, 6,000 × g at RT) and resuspended in MC7 buffer (10 mM morpholinepropanesulfonic acid [pH 7.0], 1 mM CaCl₂) to a calculated density of 7 × 10⁹ cells ml⁻¹. Ten-microliter aliquots of cells were placed on TPM agar (10 mM Tris-HCl [pH 7.6], 1 mM K₂HPO₄-KH₂PO₄ [pH 7.6], 8 mM MgSO₄), and 50-μl aliquots were mixed with 350 μl of MC7 buffer and placed in a 24-well polystyrene plate (Falcon) for development in submerged culture. Cells were visualized at the indicated time points using an M205FA stereomicroscope (Leica) and imaged using a Hamamatsu ORCA-flash V2 digital CMOS camera (Hamamatsu Photonics), DMi8 inverted microscope, and DFC9000 GT camera (Leica). Images were analyzed as previously described. After 120 h, cells were collected and incubated at 50°C for 2 h and then sonicated as described above. Sporulation levels were determined as the number of sonication- and heat-resistant spores relative to the WT.