Computational protocol: Imaging of Cellular Oxidoreductase Activity Suggests Mixotrophic Metabolisms in Thiomargarita spp.

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Protocol publication

[…] Cells were imaged with an Olympus IX-81 inverted microscope equipped with a long working distance 40× objective (numerical aperture, 0.6; working distance, 2.7 to 4.0 mm) and a 17.28-megapixel DP73 color camera. Images were collected with CellSens dimension (Olympus, Japan) software under constant (manually set) exposure and white balance settings. ImageJ was used to subtract the background and convert the image to an XYZ color profile. A 40- by 40-pixel region of interest representing an area of cytoplasm with a low density of sulfur globules was chosen for quantification of dye change in each cell. The average pixel intensity of this region was then measured in the luminance channel (Y). Reciprocal intensity was calculated as described in reference for quantification of chromogen intensity. The change in intensity relative to that at time zero was reported, and a Student's t test was used to determine the likelihood that the imaged intensities were distinct from the mean of the controls by chance alone (). Copying of the selection region in ImageJ from image to image in the time series for an individual cell ensured that the same area of the cell was measured at each time point. Uniform adjustment of color levels was used for presentation of images in , but this adjustment was performed after image analysis. […]

Pipeline specifications

Software tools cellSens, ImageJ
Application Microscopic phenotype analysis
Organisms Escherichia coli, Homo sapiens
Chemicals Carbon, Succinic Acid