Fluorescence shading effects can often cause difficulty, due to the fact there is little or no light to measure the shading pattern. Nevertheless there are strategies to consider:
-
Improve the optics. In some cases the solution is simply to use a better fluorescence illuminator that gives more even illumination. For instance, with the Leica DM6000 microscope and its external fluoro source, there is the potential for very little shading.
-
Use the brightfield pattern. In fluorescence the issue is still the same, i.e. the optics transmit light more efficiently through the centre of the lens, so you can try to set a shading pattern in brightfield illumination and then switch to fluorescent mode. There's no guarantee of success due to the differences in bulb alignment and the condenser itself - but definitely try it.
- Use an evenly fluorescing sample slide to acquire the shading correction. Potentially you can obtain slides useful for this from places like these:
https://www.chroma.com/products/diagnostic-slides
http://www.tedpella.com/histo_html/fluor.htm.aspx
- Use a “sample averaging” approach. Surveyor supports this using the “Map tools” which is the little hammer icon in Surveyor’s Scan window. The trick here is to first scan a fluorescent channel to get the mosaic. Then the Map tools can be used and it will go through the set of tiles trying to figure out foreground vs background, using a low pass filter that you can specify. This will then build what it thinks is the shading image by comparing all of the tiles, and attempting to build a correction. This can help, but in fluoro it still suffers from the issues related to there being little signal in the blank areas.
- Use a region-of-interest of the camera frame. By extracting a sub-region of the frame you can avoid the outer edges which exhibit the worst of the shading effects. Depending on your camera model you may have an option to set the width and height of the camera frame in the camera properties dialog (where you adjust the exposure/gain etc, and it's usually in the Process or Advanced tab). Alternatively right-click on the live image window and you'll see a popup menu with options related to showing and setting the ROI. You can show the ROI box overlay, drag the edges of the box to a reduced size, and then right-click again to set this ROI. Try scanning with this new ROI and you may see the shading effects reduced.