Microscopes Intl. LLC
July 10, 2018
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.
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:
The following images show different layers at various objective positions. Red arrows point to the different features that are in focus in each layer.
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.
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:
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.
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:
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.