Microscopes Intl. LLC
September 28, 2017
Choosing the Right Objective
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?
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.
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.
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.
(microns per pixel)
(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|
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.
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.
(microns per pixel)
(Limited by NA)
(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.
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.