It all starts with a quality scan…

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In order to get the best experience with digital pathology and take full advantage of all the applications, you must first have a high quality digital slide scan.  There are several factors that influence the quality of a slide scan, some that can be controlled while others may be outside the boundary of scanning.  Here are a few samples:

Image out of focus

There are multiple reasons an image may be out of focus, everything from the system being off calibration to a slide being placed upside down, but the three most common are: the slide sitting loose in the tray, dust on the cover slip, and focus points on objects at a different focal plane than the tissue.  The best way to proceed if an image scan looks like this is to: check the slide holding try for small glass shards or anything that may keep the slide from sitting correctly, use a lint free cloth to whip the cover slip, and manually set focus points on the tissue area.

Near-far effect

The near-far effect is when only parts of the image in focus due to a large focal differential.  You will see this when you have thick tissue sections, greater than 25 um, and not enough focus points to compensate for the focal plane differential.  This can be easily solved by adding more focal points to the areas of high differential and ensuring that both thick and thin sections are represented.  However, it is worth noting that there is a technical limit of how many points can be added so it may not be possible to get everything in focus on all sections.

Dirt and dust fragments

This may be more subtle than focus issues but can create problems for running applications, especially image analysis.  In this sample, the grayish area circled with the green box is an out of focus piece of dust on the cover slip.  This can be picked up by image analysis algorithms as positive staining and throw off the accuracy of the quantification.  The best way to avoid this is to carefully wipe cover slips with a lint free rag.

Pen marks on the cover slip

It is not uncommon for a cover slip to be marked by a pathologist or histologist with a pen to point out areas of interest.  Even with the markings, these slides can be easily scanned as long as there are no focus points on the marks.  Due to the high contrast with the surrounding glass, most automated tissue finding algorithms will select the marks for inclusion in focusing.  It is worth the step of manually overriding or deleting these focal points before scanning to ensure the tissue will be in focus.

Folds in the Tissue

Unfortunately, there is nothing that can be done to remove folds in tissue samples during the scanning process.  However, these folds can create a problem with focus in a similar manner to the pen marks.  It is also good practice during the scanning process to remove any focal points that may be placed on tissue folds.

Bubbles between the cover slip and sample

Similar to folds, there is nothing that can be done about bubbles during scanning.  Nevertheless, the bubbles create a magnification effect with the tissue inside that can lead to moving the focal plane away from the actual tissue and causing a focus problem.  The tissue inside the fold should never be used for focus because the focal plane may be moved outside of a realistic distance and make the surrounding tissue out of focus.

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