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Depth of Field in Microscopy 

Last Updated: 30.11.20

 

Good microscopes can help you learn a lot of interesting things about everything surrounding us. When used properly, these devices can help children and youngsters fall in love with science and even allow them to dream about careers in fields like medicine, biology, and research. 

However, understanding how microscopes work is a little difficult, as each device contains numerous parts that should be explained separately. If you’re looking to find out more about microscopes in general and their depth of field, continue reading this article. 

 

Magnification rate

Before getting into technical details about the importance of the depth of field, we should first talk about the magnification rate. Most magnifying items on the market (including magnifying glasses, binoculars, telescopes, cameras, and microscopes) use the same optical principle and glass lenses to magnify an image and allow you to see far or big objects in more detail.

Depending on the type of device used, magnifying rates range from 1x to 100,000x for the professional microscopes and, sometimes, even beyond that number. 

Optic microscopes usually help you magnify an object 40x to 400x times but this doesn’t mean that the image will be very clear. To make the most out of your microscope’s technical performance, you need to take a closer look at other aspects such as resolution and depth of field. 

By comparison, beam microscopes use an alternative technology that allows higher resolutions and clarity. 

 

Understanding the field of view and depth of field

The field of view (FOV) allows you to understand how much of a specimen is visible at any given moment in the lateral plane or perpendicular to the optical axis. 

You can also understand it as the diameter of the circle of light visible when looking through the lens of a microscope. The field of view is inversely proportional to the magnification rate which means that a higher magnification rate will result in a narrower field of view. 

On the other hand, the depth of field usually refers to the resolution in the longitudinal plane or parallel to the optical axis. You can measure it as the distance from the farthest object plane to the nearest object plane in focus and it is usually expressed in microns. Similar to the field of view, the depth of field also decreases once you enhance the magnification rate. 

To better understand the concept, you can think of two hairs set in a crisscrossed position on a microscope slide. With a lower magnification rate, you can easily get to focus on both hairs at the same time but, once you increase the magnification rate, the lens will mainly focus on one hair, as the other one will be blurry. 

 

Depth of field, depth of focus, and image depth

These three concepts are all important when trying to understand how a microscope or camera work. Any lens can transform a 3D object into a 2D image, and a person with good eye accommodation can later view this 3D image because the eyes see objects at a different distance if they are located within the accommodation range of the eye. 

When you’re using a microscope, the object you observe with your eyes through the provided eyepieces is transferred into the intermediate plane of the microscope. 

A simple definition of the depth of field would be that it represents the longitudinal or axial distance between the farthest and nearest parts of an object that are in sharp focus in the image of the object. In other words, it shows you exactly how big of a section you can see on the microscope from a certain object. 

This depth of field is determined by the distance from the nearest sharp object plane to the farthest object plane. 

The depth of focus represents the tolerance of placing the sensor in relation to the lens. To put it simply, it is determined in the image of the object which is located in the area behind the objective lens, as opposed to the depth of field that is determined in the area in front of the objective lens. 

The depth of field depends on a series of factors, including lens aberrations, geometrical optics, the degree of eye accommodation, microscope magnification, and others. For instance, older people will experience a shallower or smaller depth of field than people in their 20s without eye problems. 

Calculating the depth of field is done after various methods proposed by researchers, each using rather complicated mathematical formulas. 

 

How to read depth of view

By applying one of the formulas for determining a microscope’s depth of field you can also use a simple technique to increase the depth of field. If you want to decrease the collection angle and, therefore, to reduce the numerical aperture, all you have to do is to reduce the condenser aperture by reducing its diaphragm. 

The procedure will help you decrease your collection angle and the lighting beam will become parallel. 

In the photography field, the depth of view is a common concept that allows professionals to play with different lenses and objectives to create amazing results. For example, wide-angle lenses and slow lenses with small effective apertures have a deep depth of field, allowing for amazingly clear and detailed pictures. 

The depth of field is rather large in photography and usually measured several centimeters for fast long-focus lenses but can reach up to hundreds of meters for short-focus objectives. 

However, microscope objective lenses are different and don’t usually feature the diaphragm mechanisms required to do so. There are only a few variable numerical aperture objectives that come equipped with internal iris diaphragms that allow you to increase the image contrast or axial resolution. 

One easy way to do so is to make a DIY diaphragm using a thin piece of metal or a black piece of plastic. Alternatively, you can find a small washer to place in front of the microscope objective lens. The result is a reduction of the numerical aperture and an increase in the depth of field. 

However, the amount of light you will receive on the image sensor or through the eyepiece will also be reduced. Overall, the results provided by the DIY diaphragm are visible with the naked eye, so you can enjoy an improved depth of field that will allow you to observe more details on the specimen. 

To sum up, obtaining a high imaging resolution requires a high numerical aperture objective but one of the consequences is a fairly small depth of field. This emphasizes the quality and the performance of the focusing stage but will force you to constantly slide the specimen to notice other details from the fragment you’re researching. 

 

Conclusion

Understanding how the field of view, depth of field, and magnification work is mandatory if you want to get the best results using a microscope. Also, even though they are based on the same principles, camera lenses and microscope lenses work differently so you cannot expect similar results using the same techniques for both devices. 

Generally speaking, the bigger the magnification rate, the narrower the field of view, which means when looking through the lenses of a microscope, you won’t be able to see too much of your specimen. The depth of field is also altered by the magnification so, as the magnification increases, it is harder to focus on the specimen. 

Although these struggles can be fixed with the help of DIY diaphragms, you will never obtain a clear image with the help of a regular optic microscope. Electron microscopes use different technology and can provide high-resolution pictures even when the magnification rate exceeds 100,000x. 

However, most of these products cost a small fortune and are only available for specialists and scientists. They also need highly-skilled professionals to handle them and preparing the specimens might take days. 

Therefore, you need to understand the limits of an optic microscope and work around its flaws when using the device for your research. With practice and the help of the right lenses, you can still use both alive and dead specimens and see every detail. By comparison, beam or electron microscopes can only use dead or dried specimens, and, as we previously mentioned, preparing them will take hours or even days. 

 

 

Ioana Moldovan

Ioana’s professional experience in the optics field has helped her understand the value of passing her knowledge forward. Her curious personality helps her gather useful information for her readers and her goal is to make technical information fun and accessible to everyone.

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