Dfc9000 gtc cmos camera

Nostoc cultures were grown on agar plates and resuspended in BG11 medium. OD₇₅₀ was adjusted to ~0.8 and 10 ml of the cultures were transferred into a petri dish. Cultures were treated with two Sankyo Denki Germicidal 68 T5 UV-C lamps for 5 mins in a repurposed Herolab UV DNA Crosslinker CL-1. Ten μl of Nostoc cultures were spotted on a BG11 agar plate and left until the liquid was fully absorbed. An area was cut out and mounted on a cell culture imaging dish. Time-lapse movies were recorded using a 40x objective on a Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera (2048 × 2048 pixels, pixel size 6.5 μm). Images were recorded every 60 s over a time course of 5 h. To analyze the survival rate after UV-treatment on culture level, 3 ml of Nostoc cultures were treated 1 min with UV light and a dilution series was spotted on a pre-warmed BG11 plate. The plate was incubated in the dark for 24 h at 28 °C. Afterwards, incubation was continued for 10 days with continuous light.

Prior to harvest, cells were treated with 150 ng/ml colcemid for 1 hour and then collected by centrifugation. Cell pellets were shortly resuspended in 0.075 M KCl hypotonic buffer and then fixed in fixation buffer overnight (3:1 methanol:acetic acid). Fixed cells were extensively washed with hypotonic buffer and then spotted on microscope slides with Vectashield antifade mounting medium with DAPI (4',6-diamidino-2-phenylindole, Vector Laboratories). Metaphase spreads were imaged using a Leica DFC9000 GTC cMOS camera with a 100X objective. Each condition was repeated in three independent biological experiments and ∼50 metaphases were analyzed per condition. The two-tailed Student’s t-test was used for statistical analysis.

For sample preparation, 1.5% agar pads were freshly poured into a 35-mm-high glass-bottom µ-dish (ibidi). After 30 min, the agar pad was inverted and five 1.5 µl drops of respective bacterial culture were equally spotted onto the pad. The bacterial cultures were adjusted to an OD₆₀₀ of 0.05 for fLM experiments and to an OD₆₀₀ of 0.01 for timelapse imaging. After drying, the agar pad was mounted on a 35-mm-high glass-bottom µ-dish (ibidi) supplemented with a wet Kim wipe and closed with parafilm and vacuum grease.
Images were recorded using a ×100 phase-contrast objective on a Leica Thunder Imager 3D Cell Culture equipped with a Leica DFC9000 GTC CMOS camera (2,048 × 2,048 pixels, pixel size 6.5 mm) at a stage temperature of 25 °C. The Leica Application Suite X (LAS X) software platform was used for acquisition and the resulting images were analysed using Fiji^{65 (link)}, GraphPad Prism and Microsoft Excel. Different cell types were quantified using the cell counter function in Fiji. For timelapse imaging of Y. entomophaga chi2-sfGFP, single images were recorded every 10, 15 or 20 min over several hours using a highspeed software autofocus with a local range of 25 µm using the brightfield channel.