Computational protocol: Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

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Protocol publication

[…] Bacteria strains were observed in brightfield and excitated in DUV with a Zeiss Axioobserver Z−1. The selected objective was a 100× Zeiss ultrafluar objective needing glycerine immersion. The Fle fluorescence was recorded by excitation at 290 nm, using the dichroic mirror of 300 nm (OMEGA Optical, Inc., USA) and emission bandpass filter for 420–480 nm wavelengths (OMEGA Optical, Inc., USA). To detect fluorescence images, we used CCD camera from Hamamatsu C9100-13 (HAMAMATSU PHOTONICS France SARL, France). The integration time of camera was 2 min for applied excitation and filter combination used to visualize the Fle fluorescence. In addition, to increase the signal to noise ratio, images were recorded with binning of the pixel (2×2) in Micro-Manager program , used to manipulate the CCD camera.The image analyses were performed with Image J (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,, 1997–2011). Illumination being in-homogeneous, it was corrected before background subtraction. First, threshold was automatically adjusted using a triangle algorithm; thereafter, bacteria were analysed as the remaining particles. The mean intensity coming from each bacterium was automatically calculated considering its pixel area. Finally, all bacteria signal taken from one image were averaged. For each condition, two different localisations with minimum 30 bacteria per plane were recorded and averaged. Resulting fluorescences were analysed using Microcal Origin 8 (Microcal Software Inc, Northampton, MA).In order to present images of bacteria with fluorescence signal from Fle, the contribution of autofluorescence to the instinct fluorescence of bacteria was measured from Fle-untreated bacteria with follow subtraction from Fle-treated bacteria (e.g. bacteria incubated with Fle in CCCP absence and/or presence). The same contrast was chosen for all images for better visual comparison. […]

Pipeline specifications

Software tools μManager, ImageJ
Application Microscopic phenotype analysis
Chemicals Quinolones