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Jon Ward

Jon Ward
Microscopes Intl. LLC

September 28, 2017

Blog
Which Objective is Best for Scanning Slides?

Choosing the Right Objective

Microscope Objectives The uScopeMXII and uScopeHXII whole slide scanners include a single objective. Having only a single objective leads to real concerns over choosing the right model uScope to fit each customer's requirements. While we would like to recommend that each customer purchase two or even three uScopes with different objectives, that suggestion is not viable for most users.

The uScopeMXII is available with a 20x, 40x, or 60x objective while the uScopeHXII is available with a high-NA 10x, 20x, or 40x Plan Apo objective. But, what are the real differences among the models that are available?

Magnification

The total magnification of a traditional desktop microscope is the product of the magnification of the objective (10x-60x) and magnification of the eyepiece (usually 10x). So, a microscope with a 20x objective and a 10x eyepiece delivers 200x magnification.

An effect of excessive magnification is that at some point what is known as empty magnification occurs. This happens when the image is magnified but no additional detail is resolved. In digital microscopy, we get what are known as empty pixels. In effect, rather than collecting a green pixel, we collect a 2x2 block of green or (even worse) a 3x3 or 4x4 block of all green pixels. These empty pixels are nothing more than a waste of space because, although the image appears larger, the additional pixels add no additional image resolution. Keep this in mind as we discuss a few other aspects of digital microscopy.

Objective Power

Each objective has a power specification that is convenient to use but that doesn't provide the whole picture.

Low power objectives (10x and 20x) present a larger field of view and, therefore, allow faster scans because fewer images are required to cover a given slide area. They have a lower numerical aperture (NA) and, therefore, lower resolution than higher-power objectives. The depth-of-field of these objectives is quite high, meaning that, for specimens that are not uniformly flat, more of the specimen is in focus in each field. In a nutshell, you see better three-dimensional detail in each field, but the resolution is lower so smaller details are not as distinct.

High power objectives (40x and 60x) present a smaller field of view and a slower scan speed because they require more images to cover a given specimen area. They have a much higher numerical aperture (NA) and, therefore, much higher resolution than lower-power objectives. Conversely, the depth-of-field of these objectives is low. Focus becomes more critical for non-flat images since the range of things in focus in each field is reduced. In a nutshell, you see less three-dimensional detail in each field, but the resolution is amazing.

Scan Performance

Each doubling of magnification quadruples scan time (because magnification doubles horizontally AND vertically). The following table gives you an idea of typical scan times for each objective power.

Objective Mapping
(microns per pixel)
Scan Speed
(15mm × 15mm)
No. of Fields
10x (0.30NA) ≈ 1.0 ≈ 1.5 minutes ≈ 700
20x (0.40NA or 0.65NA) ≈ 0.5 ≈ 6.0 minutes ≈ 2,800
40x (0.65NA or 0.80NA) ≈ 0.25 ≈ 24 minutes ≈ 10,800
60x (0.85NA) ≈ 0.16 ≈ 54 minutes ≈ 24,000

Camera Resolution

The cameras used in the uScopeMXII and uScopeHXII offer two resolution modes.

  • Standard Definition (SD) Mode
    is the default image mode for the objective camera. In this mode, the camera captures images at 1920×1080 and reduces the resolution to 960×540 by halving and averaging the vertical and horizontal resolution. This has the effect of reducing the resolution and camera noise. The standard definition camera mode is up to four times faster than the high definition camera mode.
  • High Definition (HD) Mode
    is the full resolution mode of the objective camera. In this mode, the camera captures each image at its native resolution. This camera mode provides the greatest resolution but it is up to four times slower than the standard definition mode.

Typically, you would scan in SD mode. This gives the greatest balance of performance and image quality. Scanning in HD mode doubles the resolution and simulates the effects of doubling the objective magnification.

Numerical Aperture

The numerical aperture of an objective specifies the level of resolution. A rule of thumb is that the maximum digital magnification you can get is 1000 × NA of the objective. Expanding the table above, we get the following maximum magnification for each objective and camera mode.

uScope Model
Objective
Mapping
(microns per pixel)
Digital
Magnification
(Theoretical Maximum)
Digital
Magnification
(SD Mode)
Digital
Magnification
(HD Mode)
uScopeHXII
10x (0.30NA)
≈ 1.0 300× 100× 200×
uScopeMXII
20x (0.40NA)
≈ 0.5 400× 200× 400×
uScopeHXII
20x (0.65NA)
≈ 0.5 650× 200× 400×
uScopeMXII
40x (0.65NA)
≈ 0.25 650× 400× 650×
(Limited by NA)
uScopeHXII
40x (0.80NA)
≈ 0.25 800× 400× 800×
uScopeMXII
60x (0.85NA)
≈ 0.16 850× 600× 850×
(Limited by NA)

What you should glean from this table is that scanning in HD mode provides significantly more resolution for the following uScope models:

The resolution improvement for the uScopeMXII-40 and uScopeMXII-60 units is somewhat limited by the NA of the objective. Using HD mode with these models may lead to excessive magnification and, ultimately, empty pixels. So, at 60x, even though we get more pixels with an HD mode scan, we don't get significantly more image resolution.

Putting It All Together

So, what can you take away from all of this?

Most obvious should be that the 20x uScope models provide the best balance of performance, magnification, resolution, and depth-of-field for most uses except those requiring extremely high resolution.

Is the 20x the best objective for your digital microscopy tasks?

That's a question only you can answer. Hopefully, the data provided helps you to determine the objective and, therefore, the uScope model best suited to your needs.

Contact Microscopes International or speak with your local distributor.
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