What can you see at 40x magnification

Magnification:

Figuring Total Magnification

Magnifying Objects/ Focusing Image:

When viewing a slide through the microscope make sure that the stage is all the way down and the 4X scanning objective is locked into place. Place the slide that you want to view over the aperture and gently move the stage clips over top of the slide to hold it into place. Beginning with the 4X objective, looking through the eyepiece making sure to keep both eyes open (if you have trouble cover one eye with your hand) slowly move the stage upward using the coarse adjustment knob until the image becomes clear. This is the only time in the process that you will need to use the coarse adjustment knob. The microscopes that you will be using are parfocal, meaning that the image does not need to be radically focused when changing the magnification. To magnify the image to the next level rotate the nosepiece to the 10X objective. While looking through the eyepiece focus the image into view using only the fine adjustment knob, this should only take a slight turn of the fine adjustment knob to complete this task. To magnify the image to the next level rotate the nosepiece to the 40X objective. While looking through the eyepiece focus the image into view using only the fine adjustment knob, this should only take a slight turn of the fine adjustment knob to complete this task.

Total Magnification:

To figure the total magnification of an image that you are viewing through the microscope is really quite simple. To get the total magnification take the power of the objective (4X, 10X, 40x) and multiply by the power of the eyepiece, usually 10X.

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Bacteria are tiny living microorganisms that live in enormous numbers in almost every environment on Earth. From deep within the soil to inside the digestive tract of humans. In order to see bacteria, you will need to view them under the magnification of a microscopes as bacteria are too small to be observed by the naked eye.

Most bacteria are 0.2 um in diameter and 2-8 um in length with a number of shapes, ranging from spheres to rods and spirals. Bacteria have colour only when they are present in a colony, single bacteria are transparent in appearance. At high magnification*, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick. It takes a skilled person to be able to differentiate bacteria from small dust and dirt which may be present on the slide. Some bacteria are found in bunches and therefore, makes it difficult to see the individual cells.

Magnification Strengths

Microscope objectives give you the ability to view samples at different magnification strengths. When viewing bacteria, you will notice the different objectives will obviously yield different results:

Magnification and its Result

Transparent bacteria are often difficult to see/recognize. For them, you can use phase contrast optics. This method makes the bacteria visible by making bacteria darker or lighter than the background. Alternatively you can stain bacteria for better results. But this method may introduce artifacts.

The figure below gives you an idea about the relative sizes and which resolution is used to see them.

*(i) To calculate the magnification for a compound microscope, you need to know about two sets of lenses.

• Objective lens -which is closest to the specimen slide stage, produces an enlarged, inverted image of the specimen.
• Eyepiece lens -which magnifies the image further.

The total magnification of the microscope is calculated by multiplying the magnification of the objectives, with the magnification of the eyepiece. Most educational-quality microscopes have a 10x (10-power magnification) eyepiece and three objectives of 4x, 10x & 40x to provide magnification levels of 40x, 100x and 400x.

(ii) Another thing to consider is that microscope resolution which is the  ability to see close but separate points as distinct comes from the objective lenses, not from the eyepieces. All an eyepiece can do is magnify the resolution that is already provided by the objective. To illustrate, a 40x objective and a 10x eyepiece will result in a higher resolution (more detailed) image than a 20x objective and a 20x eyepiece. Total magnification is the same, but the detail, the resolution, will be better with the 40x objective.

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.

Magnification

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?

Pixel Mapping

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

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

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.

Striking a Balance

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.

How Does This Affect Whole Slide Scanning?

The best way to answer this question is to present a list of whole slide scanner truths to keep in mind.

• Doubling the objective magnification quadruples the pixel data in the scan area. The reason for this is that the region doubles vertically and horizontally and we need to acquire four times as many images.
• Doubling the magnification quadruples the scan time (more or less). Since you're capturing four times the image data, it will naturally take four times as long to do so.
• Increasing the magnification (or NA) increases the precision (and time) required to focus. So, a scan with a 10x (low NA) objective is likely more than four (4) times faster than a 20x (high NA) objective. This is due to the greater precision required when focusing the higher NA objective. Focus gets even more critical with 40x or 60x objectives.

Conclusion

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.

• If you're looking at insects and most plant biology, a 10x or 20x objective is probably the right choice.
• If you're looking at detail inside a white blood cell, you most likely require a 60x objective.
• If you're looking at pathology, you may find that a 40x objective works well. But, a 20x objective may be just fine for many applications (the scans are certainly faster).

In any case, we are here to help. Just drop us an email or phone call to discuss your whole slide scanner requirements.