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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 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.

Image Stacking: Top Layer

Image Stacking: Middle Layer

Image Stacking: Bottom Layer

EDF (Extended Depth of Field) Processing

Extended Depth of Field (EDF) is an image processing extension that involves analysis of each image layer of the image stack to identify the best focused parts. These are re-composited into a new image called the EDF Layer.

EDF Image (Everything in Focus)

While the image viewer for the image stack lets you select the focal point as you navigate around the scanned region, the image viewer for the EDF processed image offers only a single layer where everything is in focus.

You can see in the image to the right that nearly every feature is in focus.

There are a few things to note about scanning multiple layers and creating an EDF Layer:

• Increasing the number of layers in the image stack achieves better focus for features at the extremes of the stack.
• Reducing the distance between each layer increases the quality of focus for each layer.
• Both of the above increase the time required to scan the selected region.

EDF processing is a processor-intensive operation that requires several seconds for each field in the scan (depending on the number of layers in the image stack). Once the EDF processing is complete, the re-composited image fields must be stitched and combined into a whole slide image.

Deep Zoom Image Viewer

The uScope Navigator application from Microscopes International performs image stacking and EDF processing. Whole slide images are created in the Deep Zoom image format which can be viewed by a number of viewers including the one built into uScope Navigator.

The following deep zoom images illustrate the multi-layer viewer and the composite EDF image composited from those layers:

Availability

Image Stacking (multi-layer) scanning is being introduced in uScope Navigator v4.5. Early adopters can contact our sales department to request a beta software release.

EDF (Extended Depth of Field) support is offered as an additional software license at an additional cost. Contact our sales department for a quote and availability details.