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Protocol publication

[…] Time-lapse microscopy was performed using a Leica DMIRE2 inverted microscope system. Autofocus and image acquisition were done by using custom macros in μManager (an open source software program) to control the microscope, stage, shutters, and camera. The microscope was placed in a large incubator box (Life Imaging Systems) that controls the temperature to an accuracy of 0.1°C. GFP- or YFP-expressing bacteria were imaged using Yellow GFP filter (Ex-500nm, Em-535nm, Chroma USA); mCherry signal was measured by HcRed1 filter (Ex-575nm, Em-640nm, Chroma USA). Excitation was performed with LEDs (Coolled, United Kingdom) and images were acquired with a cooled CCD camera (−75°C) (Orca II, Back-illuminated, Hamamatsu) and processed with ImageJ ( X100 NA 1.40 oil objective was used for individual bacterial observation on agar pads; X63 NA 0.70 long-distance air objective NA for imaging Hela and bacteria in 24-well plates; X20 was used for imaging growing colonies on a very thin LB + 1.5% agar layer.To observe the growth of individual bacteria, a LB + 1.5% agarose pad was prepared in a polydimethylsiloxane (PDMS) square mold and dried for 10 min at 37°C. Bacterial samples of 1 μl (~105 cells) were placed between the microscopic slide and the pad inserted into the same PDMS mould and covered with a coverslip. For microscopic observation of self-aggregation, agarose pads were not dried and bacteria (5 µl, ~105 cells) originated from suspended colony were placed between the microscopic slide and the pad. […]

Pipeline specifications

Software tools μManager, ImageJ
Application Microscopic phenotype analysis
Organisms Bacteria, Escherichia coli, Homo sapiens
Diseases Infection