uScope HXII uScopeHXII Objective Depth of Field
uScope HXII

Information in this article applies to:

  • uScopeHXII (All Models)

Article ID: HXB2351 — Created: 15 Nov 2020 — Reviewed: 13 Dec 2020


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.


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:

λ × n
n × p
M × NA


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, uScopeHXII objectives have the following depth of field:

Depth of FieldRough Focus
Focus Steps
Focus Steps

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.

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 10x objective can take a long time to find focus. Moving too many steps with a 40x 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 between ½ and ¾ of the rough focus for 20x and 40x objectives and between ½ and ⅓ of the rough focus for 10x objectives.
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