Microscopes Intl. LLC
April 20, 2018
Power vs Pixels!
Nowhere is the magnification versus resolution question more prevalent than in digital microscopy. In many cases, these two terms are used interchangeably. However, they are distinct and should not be confused.
Each objective lens has a magnification printed on its side. It's easy to understand. A 40x objective makes things appear 40 times larger than they actually are. Comparing objective magnification is relative—a 40x objective makes things twice as big as a 20x objective while a 60x objective makes them six times larger than a 10x objective.
The eyepiece in a typical desktop microscope is 10x. The product of the objective magnification and the eyepiece magnification gives the final magnification of the microscope. So, a 60x objective and a 10x eyepiece gives a total magnification of 600x.
So, what happens when you couple an objective to a 2 or 5 or 8-megapixel camera with no eyepiece? Then, what is the magnification? If you use a 20x objective, is the final image 20 times larger? 200 times larger?
In digital microscopy, we use a term called pixel mapping to answer the question of digital image magnification. In general, a 20x objective maps 0.5 microns (of the specimen on the slide) to a single pixel on the camera. The final magnification is obtained by dividing the display pixel size (in microns) by the pixel mapping.
For a 70" HD TV (1920x1080), the pixel size is about 0.8mm (800 microns). And 800 divided by 0.5 gives a final magnification of 1,600x. For more magnification, you need a larger monitor!
Instead of displaying the image data pixel for pixel, we could display one image pixel on 4 (2x2), 9 (3x3) or 16 (4x4) display pixels and achieve double, triple, or quadruple the apparent magnification. But, that only gives us bigger pixels—not better resolution.
Resolution is the objective's ability to resolve really small stuff. In objective terminology, this is the NA (numerical aperture) specification, which, like the magnification, is also printed on the side of the objective. The higher the NA, the more (smaller) stuff can be resolved. In general, higher magnification objectives have higher NA.
In general, for digital scanners, the maximum magnification of an objective is approximately 1000x the objective's NA. So, an objective with a .65NA can achieve approximately 650x in the digital domain. You might assume that we would always want the highest NA objective we could get, but that's not always the case.
Increased NA comes at a price: reduced depth of field and increased cost.
Numerical Aperture and Depth of Field are two sides of the same coin, and they are (more or less) inversely proportional. As NA increases, depth of field decreases, and vice-versa. Matching resolution and depth of field to the subject material is a key factor in choosing the right objective for the job at hand.
Depth of Field is the distance between the nearest and farthest objects that are in focus without moving the objective. Anyone who regularly uses a desktop microscope knows that a 4x or 10x objective is much easier to focus than a 60x or 100x objective. The reason is because the depth of field of the lower NA objectives is very large (so, more stuff is in focus at the same time). And that means that getting the objective "close" to ideal focus is good enough because a deeper volume of the specimen is in good focus.
High NA objectives have a lower depth of field which means setting the focus is more critical and difficult to achieve. Getting the objective close-enough just doesn't work. Changing the focus only slightly can reveal different features in the specimen. Using an objective with a lower depth of field, objects may look different depending on how "deep" into the specimen you focus.
After all of the above detail, you might think it would be best to find a microscope objective with great magnification, high NA, and large depth of field. Such an objective does not exist—at any cost. So, we need to find a happy medium that satisfies as many requirements as possible. We make these kinds of trade-offs with whole slide scanners because of their general purpose nature.
The best way to answer this question is to present a list of whole slide scanner truths to keep in mind.
So, just what does all of this information and detail mean and what can we take away from it?
Choose the objective that matches the depth of field and resolution requirements of the job.
In any case, we are here to help. Just drop us an email or phone call to discuss your whole slide scanner requirements.