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uScope Navigator (Scan)

Parts of Scanned Region are Out of Focus
uScope Navigator (Scan)

Information in this article applies to:

  • uScope Navigator V4.0 and Later

Article ID: NSC1153 — Created: 19 Feb 2018 — Reviewed: 13 May 2018

Symptom

Half-Focused FieldWhen scanning some slides, parts of the scan are in perfect focus while some parts are out of focus (as shown in the image to the right).

Cause

There is basically only one reason that every slide is not well focused: the specimen being scanned is not perfectly flat and infinitely thin. Under ideal circumstances, every slide would be perfectly prepared and the specimen would be flat and thin. However, this is never the case. Even with the best preparation and thinnest sections, the specimen will anywhere from 4 to 8 microns thick. And, achieving correct focus for each field becomes critical.

Fortunately, focus issues are easy to overcome by selecting a different focus method or changing focus settings to give the uScope a better chance to achieve good focus all the time.

Resolution

You can change the focus method used to scan the region and you can change the focus settings. Both options will have an effect on the way the focus of your scans.

Changing Focus Method

Following are a few suggested changes to make depending on the focus method currently selected.

When Initial Only is the selected focus method...

The Initial Only focus method scans the entire region at a single focus level. It performs no focus adjustment while scanning. So, any of the other focus methods will likely yield better focus results. In this case, you should consider using the Fast Stack or Predictive focus methods.

  • The Fast Stack focus method captures a short (fast) stack of images for each field—typically, one image above and one image below the initial focus selected when starting the scan. You can adjust how many images and how far the objective is moved for each. Refer to When to use the Fast Stack Focus Method for more information.
  • The Predictive focus method captures an array of well-focused images (the prediction points) across the selected region. It builds a focus prediction map by interpolating the prediction points in an attempt to mathematically predict where the focus will be best for each field. Refer to When to use the Predictive Focus Method for more information.

When Fast Stack is the selected focus method...

The Fast Stack focus method captures a very short stack of images (typically three in total) and chooses the best one. It provides no dynamic adjustment to the focus level if it (the focus level) changes over the course of the scan. In such a case, you should consider using the Predictive or Exhaustive Stack focus methods.

  • The Predictive focus method captures an array of well-focused images (the prediction points) across the selected region. It builds a focus prediction map by interpolating the prediction points in an attempt to mathematically predict where the focus will be best for each field. Refer to When to use the Predictive Focus Method for more information.
  • The Exhaustive Stack focus method captures a deep stack of images for each field in the scan. It is the slowest focus method but provides the best focus results in almost all cases. Refer to When to use the Exhaustive Stack Focus Method for more information.

When Predictive is the selected focus method...

The Predictive focus method captures a grid of focus points and creates a focus map of the region to scan. Sometimes, the topology of the tissue being scanned defies mathematically predicted focus levels. In this case, you should consider using the Exhaustive Stack or Predictive (Fast) Stack focus methods.

  • The Exhaustive Stack focus method captures a deep stack of images for each field in the scan. It is the slowest focus method but provides the best focus results in almost all cases. Refer to When to use the Exhaustive Stack Focus Method for more information.
  • The Predictive Stack focus method is a combination of the Predictive and Fast Stack focus methods. If the unfocused fields in a Predictive focus scan are only slightly unfocused, this may be the best focus method to choose. Refer to Predictive Fast Stack Focus Method for more information.

Adjusting Focus Settings

There are settings you can change to affect the operation of most focus methods. Refer to the following articles for more information about the settings available:

Typically, there are both a focus step size and a focus range associated with many aspects of the various focus algorithms. In general...

  • Increasing the focus range allows the focus algorithm to search for the best focus in a wider range of focus (objective) levels.
  • Decreasing the focus step size provides more images in the range from which the focus algorithm can choose.

Notes
Increasing the focus range too much can cause the focus algorithm to find the best focus to be on the cover slip. You can recognize this situation if the specimen is completely unfocused but there are several sharply focused black blotches on the image.

Make certain that the range is an integer multiple of the step size. For example, if the step size is 10, the range should be 10, 20, 30, 40, 50, ...

There is a balance between large focus range values and scan speed. Increasing the range or decreasing the step size will cause the focus algorithm to capture more images for each field or prediction point. And, this increases the time to scan.

When increasing the focus range, it's better to start conservatively. If the step size is 3 and the range is 18, try increasing the range to 21 or 24 and observe the results of a scan.

One step is about 0.2 microns (0.198375 microns to be precise). So, 6 steps are about 1.2 microns and a range of 61 steps (30 above and below) is about 12.2 microns.

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