What is depth of field and how can I optimize it in a scanning electron microscope?

By Luigi Raspolini - July 25, 2019

Imaging with a scanning electron microscope (SEM) consists of taking pictures of small features. So why not consider a comparison with photography? Let’s analyze how similar the behaviors of a SEM and a camera are when it comes to focusing on your subject, and what the exact definition of depth of field is.

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What is depth of field?

When taking a picture, the object portrayed should always be in focus and appear as sharp as possible. Artistically speaking, this silently communicates to the observer where the photographer’s attention is. Practically, it reveals a greater amount of detail in the area that is properly in focus.

But what percentage of the subject is really in focus and how can this portion be manipulated? The portion of an image that is in focus is always a plane, which means that we should only be able to image perfectly flat surfaces. Luckily enough, our brain can process the portion of the subject that is ‘close enough’ to the focus plane. This portion is referred to as the depth of field.

There are several factors influencing the depth of field and manipulating these parameters can create an image that contains more or less relevant details. The aperture diameter and the design of the focusing device (the lens in photography and the electron column in SEM) play an important role.

The distance between the imaged object and the imaging tool is also a crucial aspect. In general, when the object is very far away, the depth of field increases, and more objects will be sharp enough for our brain to process and distinguish them.

When the subject is closer, the depth of field decreases dramatically, but the maximum sharpness — or resolution —- value increases, revealing details that are invisible when the sample is too far away. (see Figure 1).

depth of field on a micoroscope

Figure 1: a) A close-up of a flower – when the picture is taken from a short distance, the background will be blurred; b) A landscape picture – the longer distance increases the depth of field from a few centimeters to several kilometers. Both pictures were taken at the same lens aperture and focal length.

How focus works in an electron microscope 

In an electron microscope, the focus is intended as the position where the cone of electrons from the primary beam has the smallest diameter. The electron source emits the beam, the electromagnetic lenses within the electron column, and the aperture at the end of it, then shape the beam and define its maximum possible size.

As the beam diameter approaches the minimum value possible, the resolution improves. This value is typically obtained at a specific and optimized working distance — the distance between the bottom of the column and the sample. Positioning the sample at a longer or shorter working distance will increase the theoretical minimum size of the beam diameter. Therefore, the best resolution can no longer be achieved. In these cases it is still possible to focus on the sample.

The horizontal plane described by the beam section at the minimum value is known as the focal plane. All the features positioned at this distance from the bottom of the column will be perfectly sharp when the beam is focused. Correcting the focus means changing the height of this plane. All the features above or below this plane will look gradually more and more blurred, until it is no longer possible to recognize them.

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How working distance affects the depth of field 

Summarizing what has been discussed so far: the depth of field is a portion (specifically a range of working distances) where the image is acceptably sharp, and the ideal working distance will provide the best results in terms of resolution when the beam is focused.

Nevertheless, there are some cases where resolution becomes less important and depth of field has a much bigger impact on the results – for example, when dealing with tall samples.

When imaging an insect, it is crucial that all the features included within the frame are distinguishable – for example the legs and the top of the head. The same applies to electronic connections, where it is interesting to have the entire wire and the bone pad in focus in the same image for a complete overview of the sample.

In such cases, a longer working distance — with regard to the optimal value — helps to obtain a greater depth of field and to have more details clearly visible in the image.

Figure 2 helps to clarify what the role of the working distance is. When positioning the sample closer to the column, the angle of the beam is larger. This means that a small deviation from the focal plane will result in a consistent increase in the beam diameter and therefore a noticeable increase in the image blurriness.


depth of field on a scanning electron microscope Figure 2: Representation of the electron column, electron beam and focal planes. When the working distance is longer (a) the angle α is smaller and moving away from the focal plane does not make the image significantly blurred. When the working distance is shortened, the angle β is broader, and moving away from the focal plane determines a consistent increase in the beam diameter and therefore more blurring. On the other hand, the beam diameter on the focal plane is smaller, providing better resolution.

On the other hand, when the sample is positioned further away from the column, the beam angle is smaller and the deviation from the focal plane height will result in a small fluctuation in the diameter of the beam. Therefore, all the features located at a different height will look acceptably sharp.

The depth of field can thereby be increased from a few microns to several millimeters and adapted to optimize the results of the analysis and the overall image quality.

Choosing the correct working distance for your sample is just as important as preparing the sample correctly. Sputter coating the sample with gold, for example, can increase the backscattered electrons yield for organic samples.

Find out more about the optimal way to prepare your samples for electron microscopy imaging by downloading this sample preparation e-guide. The extended guide helps you obtain good results from the most common samples through tried-and-tested tips and tricks.


About the author

Luigi Raspolini is an application engineer for the Thermo Scientific Phenom Desktop SEM product range at Thermo Fisher Scientific. Luigi is constantly looking for new approaches to materials characterization, surface roughness measurements, and composition analysis. He is passionate about improving user experiences and demonstrating the best way to image every kind of sample.

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