Techniques for Scanning Impossibly Thick Specimens

In slide preparation, typical tissue sections are really thin—4 to 8 microns thin. This makes focusing fairly straightforward for computer software. But, what happens when the specimen or section is really thick (100-400 microns) or has wide variations in thickness across the region of interest? That's a lot more difficult.

Thick sections have important details that require focus changes of tens or even hundreds of microns. When some features are perfectly focused, other features (in the same field of view) can become completely invisible (due to the extreme difference in focus).  

If the specimen was only a little thick (10-20 microns), we could use an objective that has a very high depth of field. A 10x 0.3NA objective might work well. But, the depth of field of that objective is nowhere near 400 microns. And, to get the extra depth of field you must give up magnification and resolution.

Fortunately, we have two suggestions for scanning those really thick sections: Image Stacking and EDF (Extended Depth of Field) Processing. These methods are complementary and are best when used together.

Image Stacking

A technique known as image stacking or multi-layer scanning is the first part of a solution to this problem. It involves scanning each field of a specimen at multiple focus layers. A whole slide image is created for each "layer" and an image viewer provides a way to navigate the image and the various layers. Image stacking comes with some requirements and caveats:

§ Each layer is located at a fixed distance from the prior layer. This distance must be less than the depth of field of the objective in order for everything to be in focus on one layer or another.

§ The time required to scan the specimen increases dramatically when there are a large number of scanned layers (which may be the case for very thick specimens).

§ Lower magnification (greater depth of field) is required to keep the scan time reasonable.

§ Image sizes increase as well. Rather than one image that is 500MB, you could end up with 20 images that are 500MB each for a total of more than 10GB.

The following images show different layers at various objective positions. Red arrows point to the different features that are in focus in each layer.

Scanning Thick Sections

Techniques for Scanning Impossibly Thick Specimens

In slide preparation, typical tissue sections are really thin—4 to 8 microns thin. This makes focusing fairly straightforward for computer software. But, what happens when the specimen or section is really thick (100-400 microns) or has wide variations in thickness across the region of interest? That's a lot more difficult.

Thick sections have important details that require focus changes of tens or even hundreds of microns. When some features are perfectly focused, other features (in the same field of view) can become completely invisible (due to the extreme difference in focus).  

If the specimen was only a little thick (10-20 microns), we could use an objective that has a very high depth of field. A 10x 0.3NA objective might work well. But, the depth of field of that objective is nowhere near 400 microns. And, to get the extra depth of field you must give up magnification and resolution.

Fortunately, we have two suggestions for scanning those really thick sections: Image Stacking and EDF (Extended Depth of Field) Processing. These methods are complementary and are best when used together.

Image Stacking

A technique known as image stacking or multi-layer scanning is the first part of a solution to this problem. It involves scanning each field of a specimen at multiple provides a way to navigate the image and the various layers. Image stacking comes with some requirements and caveats:

§ Each layer is located at a fixed distance from the prior layer. This distance must be less than the depth of field of the objective in order for everything to be in focus on one layer or another.

§ The time required to scan the specimen increases dramatically when there are a large number of scanned layers (which may be the case for very thick specimens).

§ Lower magnification (greater depth of field) is required to keep the scan time reasonable.

§ Image sizes increase as well. Rather than one image that is 500MB, you could end up with 20 images that are 500MB each for a total of more than 10GB.

The following images show different layers at various objective positions. Red arrows point to the different features that are in focus in each layer.