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In addition, our Leica SP8 confocal microscope was set at the 8 kHz fast scanning mode.
The in-plane FOV of the microscope was set to acquire the whole slide in multiple stages at a spatial resolution of 1 μm/pixel, and individual images were stitched together with 5%% overlapping such that the edges matched and became invisible.
The pinhole of the microscope was set to obtain an optical slice of 0.5 μm and samples were scanned two times at 512 × 512 pixel resolution with a time interval of 15 s.
Cells were maintained at 34° to 36° using a custom-built Precision Weather Station and imaged every 10 min for brightfield and DAPI with PI imaging (TRITC filter) every 30 min. The microscope was set to autofocus every 30 min. Slidebook software was used to program the microscope and to process the images.
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In this Part II we discuss details of microscope operation, how the microscope is set up in a systematic fashion, and we present typical results.
First, the focal plane of the microscope is set on the substrate, followed by recording and turning the magnetic field on.
This is because of the lack of conductivity in the wet sample and because the focused electron beam appears to destroy the three-dimensional biofilm structure when the microscope is set to magnifications of ×10,000 and greater (Alhede et al. 2012).
Microscopes were set to 200 kV and a magnification of 59,000× (pixel size of 1.017 Å prebinning).
The microscope was set-up to mimic in vivo excitation and emission spectra.
The microscope incubator was set at 37°C and 5% CO2.
The climate chamber covering the microscope stage was set at 28°C.
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minute was set
use was set
ass was set
light was set
transmission was set
illumination was set
lens was set
focus was set
magnification was set
lumen was set
microscope was used
microscope was constructed
microscope was employed
microscope was coupled
microscope was equipped
microscope was performed
microscope was driven
microscope was fitted
microscope was focused
microscope was controlled
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