When it comes to “depth of field” in a scanning electron microscope (SEM), photography offers a good comparison. With both microscopes and cameras, the goal is to obtain high-quality pictures. And both instruments involve making decisions about what areas of a given image to bring into focus.
When taking a picture, the object of interest should always be in focus and appear as sharp as possible. Artistically speaking, this communicates to the observer where the photographer’s attention is. And on a practical level, it reveals the greatest amount of detail in the area that’s properly in focus.
But what percentage of the subject is really in focus and how can the size of the area be manipulated? The portion of an image that’s 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’s “close enough” to the focus plane. This portion is referred to as the depth of field.
There are several factors that influence the depth of field, and manipulating these parameters can create an image that contains either more or fewer relevant details. The aperture diameter and the design of the focusing device (the lens in photography and the electron column in SEM) play important roles.
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. When the subject is closer, the depth of field decreases, but the sharpness or resolution increases, revealing details that are invisible when the sample is too far away.
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, and then the electromagnetic lenses within the electron column and the aperture at the end of it shape the beam and define its maximum possible size.
As the beam diameter approaches the minimum possible value, 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.
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 increasingly blurred, until it is no longer possible to recognize them.
How Working Distance Affects the Depth of Field
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.
There are some cases in which resolution becomes less important and a greater depth of field is desirable, for example, when dealing with tall samples. When imaging an insect, it’s 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 a complete overview of the sample requires that the entire wire and the bone pad be in focus in the same image. In such cases, a longer working distance helps to obtain a greater depth of field and to have more details clearly visible in the image.
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.
On the other hand, when the sample is positioned farther 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.
In general, the depth of field on an SEM can be increased from a few microns to several millimeters. By changing the depth of field, scientists can hone in on the exact features the seek to research, obtaining the high-quality images they need for successful analyses.
To learn more about the depth of field in scanning electron microscopes, fill out this form to speak with an expert.
Erik Luyk is a marketing communications specialist at Thermo Fisher Scientific.
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