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Article ID: NSC1202 — Created: 18 Nov 2018 — Reviewed: 27 Jul 2020
uScope Navigator v4.5 introduces a new, streamlined method for changing the settings for multi-layer scanning. Multi-layer scans are also known as Z-Stacking or Image Stacking.
Multi-layer scans involve capturing images at multiple focus layers (Z-stacking) for each field in the selected region of interest. uScope Navigator allows you to control not only the number of layers but the focus distance between each layer.
To estimate the number of layers, you first need to estimate the depth of the specimen and get the depth in focus steps. There are several ways to accomplish this.
Using a digital calliper, determine the thickness of the slide (Ts) and coverslip (Tc). Then, measure the thickness of the slide at the coverslip. Subtracting Ts and Tc from the combined measurement leaves the thickness of the specimen. Convert this number into steps by dividing by 0.0001984375mm/step.
Using the uScope Navigator, browse the slide you wish to scan. Move the objective as high as possible while achieving focus on various parts of the specimen. Move around the specimen raising the objective as necessary to achieve good focus. Write down this focus step position (the upper focus limit). Repeat the whole process setting the objective as low as possible. Move around the specimen to find the lowest focus point of the specimen. Write down this number (the lower focus limit) and subtract the upper focus limit. The difference is the specimen depth in steps.
|Objective||Depth of Field||Recommended|
(Steps per Layer)
Take the specimen depth (in steps) and divide by the recommended spacing to obtain the number of layers for the multi-layer scan. Enter the spacing and number of layers in the Multi-Layer Focus Configuration box.
Select the starting position (top, middle, bottom) to correspond with the initial focus. Typically, you would select Top then set the initial scan focus to the highest point of the specimen that is in focus.
We suggest you start multi-layer scanning with a small region of interest and a small number of layers (7-13). It can take a long time to scan a region of interest with many layers.