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uScope MXII uScopeMXII Objective Depth of Field
uScope MXII

Information in this article applies to:

  • uScopeMXII (All Models)

Article ID: MXB2349 — Created: 15 Nov 2020 — Reviewed: 15 Nov 2020

Summary

The depth of field of an objective determines how much of the specimen is in focus. The distance between the closest in-focus part of the specimen and the farthest in-focus part is the depth of field.

Details

Each objective has a different depth of field that depends on a number of factors including:

  • the objective's numerical aperture (NA),
  • the objective's magnification,
  • the wavelength of the illuminating light,
  • the refractive index of the medium between the specimen and objective (usually air, 1.0),
  • and the size of the camera sensor.

Various authors have proposed many different ways to calculate the depth of field. We use the following formula:

DoF
=
λ × n
NA2
+
n × p
M × NA

Where:

DoF is the depth of field,
λ is the wavelength of the illuminating light,
n is the refractive index of the medium,
NA is the objective lens numerical aperture,
M is the objective lens magnification, and
p is the pixel size of the camera's image sensor.

Based on this formula, uScopeMXII objectives have the following depth of field:

uScopeMXII
Model
Objective
Power
Objective
NA
Depth of FieldRough Focus
Focus Steps
Auto-Focus
Focus Steps
uScopeMXII-2020x0.403.81µm196
uScopeMXII-4040x0.651.42µm73
uScopeMXII-6060x0.800.82µm42

The Rough Focus column in the above table lists the number of steps to move the objective to the next depth of field range. Theoretically, moving the objective this amount moves to the next focus plane.

The Auto-Focus column in the above table lists the number of steps the uScope is configured to move when focusing a field automatically.

Note
There can be some confusion regarding the number of steps to move the objective to get (or find) good focus. Moving too few steps at a time with a 20x objective can take a long time to find focus. Moving too many steps with a 60x objective can cause the objective to skip over good focus.
  • When focusing the objective, each step of movement is approximately 0.2µm.
  • We calculate the number of steps to move the objective to achieve rough focus by dividing the objective depth of field by 0.2. The rough focus is handy for quickly getting a specimen in "rough focus".
  • For automatic focus, we overlap the depth of field when moving the objective. The overlap is about ½ of the rough focus for 40x and 60x objectives and between ⅓ and ¼ of the rough focus for 20x objectives.
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