Slide scanning: Troubleshooting for fluorescence
Acquiring a fluorescent background subtraction image is problematic and should only be done as a last resort to troubleshoot tiling.
Do not use this method if you intend on using the virtual tissue for pixel counting and thresholding.
Since there is no area on the slide with a uniform spread of fluorophore to reveal the variations in the fluorescent light path, one option is to obtain a slide with a uniform fluorophore coating. But it is still difficult to determine how bright the background-subtraction image should be.
Here are some tips that may help you to achieve proper illumination of your field with fluorescent imaging without performing a background subtraction image.
- Verify that your fluorescent light source is centered and provides uniform illumination.
- If the problem persists, try Scan settings: pixel trimming to prevent your virtual tissue montages from looking like mosaics.
- If the problem persists, try to darken the background using the Camera Histogram.
- To adjust the dark point, drag the black triangle towards the graph data but not too far from this (BEFORE)
to this (AFTER) 
. - As a result, your background will be darker and lines in your virtual tissue will be less likely.
Adjusting the dark point reduces the dynamic range. It may lead to data loss if you push the dark point so far right into the histogram that you start to remove signal. Pre-acquisition adjustments lead to less flexibility in performing image adjustments after acquisition (due to a reduced dynamic range). Keep in mind that images can be adjusted after acquisition to preserve the full dynamic range.