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For calculation of threshold, the intensity of signal for background was set using unstained area of tissue.
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In each image, the background value was set using the "threshold" function of the software to exclude all pixels surrounding the positive stained signals.
The background light illumination was set using a LOT Oriel LS0106 solar simulator with an AM 1.5-G filter, and light intensity was adjusted using appropriate neutral density filters; a 532-nm CryLaS (Berlin, Germany) FTSS 355 50 laser at a frequency of 18 Hz with an intensity of approximately 7 mW cm−2 was used to cause the small perturbations (1-ns pulse width) in the cells.
The background value for each image was set using the "threshold" function of the program to a level that would exclude all pixels in control tissues stained with secondary antibody alone.
Within the region of interest (ROI), the area that stained positive for vWF above a threshold that was set using background staining levels, as determined using IPLab (Scanalytics, Inc., Milwaukee, WI), was averaged between two fields as we described previously [ 35].
The background traffic in OPNET modeler is set using the "background traffic config" node, under its attributes the packet interarrival time is set as Pareto distribution.
The SSE thresholds used here are based upon the background reverberation power and may be set using prior knowledge or derived from the data.
For confocal microscopy, laser intensities/gains were set using negative control (PBS injected) heart tissue to minimize background fluorescence.
Okay, so the background is set.
Background set used was the entire set of known mouse genes.
The normalized data are background-adjusted, and probe values are summarized per probe set using a median polish function.
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