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Depthof focus

Lens focus

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To calculate focal distance effectively, utilize the formula 1/Focal length = 1/Image distance + 1/Object distance. This can be reorganized for simpler ...

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Focallength

Focal depthmeaning

Depth of field and depth of focus are both well-known terms in photography, filmography, and microscopy. Given how similar their names are, they have sometimes been used interchangeably. However, despite both playing a vital role in camera and microscope optics, the two are very much different. Let's look more closely at what defines the depth of field and depth of focus, their role in microscopy, and why the two should not be confused with one another.

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Focal depthcamera

Since the 4x objective lens has the least magnification, but a larger field of view, it allows for more of the specimen to be seen, as well as locating the part of the sample you wish to view. This in turn makes it easier to focus on the sample. Most microscope objective lenses are parfocal, meaning once you have one objective lens in focus, all other objectives should be in focus as well as you move from 4x to 10x, etc. Occasionally, when switching to the highest magnification lens you may need to make a slight adjustment to the fine focus, but this is not required when you start out with the 4x lens. So starting your microscopy viewing at the lowest objective is usually the most simple way to start. You can certainly use another magnification to begin, but it will likely take more time and can be a bit discouraging if you just can't seem to locate the area of your specimen you wish to view, especially if you are a microscopy novice.

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Depth of focus is the imaging complement to depth of field and is more concerned with the tilt of the sensor plane of the microscope with respect to the lens image plane. This concept relates to how the quality of focus is altered as the object stays still and the lens's sensor side changes. Therefore, the depth of focus will change significantly based on the magnification of the object and the microscope's ability to focus light, known as the numerical aperture. Depth of focus has little to do with sensor size and is more closely related to sensor perpendicularity to the optical axis and pixel count — the more pixels the sensors have, the more noticeable sensor tilt will be on the quality of the image. Given how digital camera sensors are exposed at a given focal plane, depth of focus impacts the quality of the resulting image and is an essential consideration in photomicrography.

Depthof field

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The depth of field is the distance between the closest object plane in focus and the furthest one in focus. In other words, it can be considered a lens's ability to retain the quality of an image without moving the object farther or closer to the frame. Depth of field relates to how much detail can be seen above and below the optimal focus position.  When objects are placed at a set focal distance away from a lens, detail can blur and thereby degrade the resolution. As a result, smaller objects naturally lead to reduced depth of field for the lens. It is worth noting that the depth of field will increase as the condenser numerical aperture (condenser aperture diaphragm) is closed down (approaches zero).  While depth of field increases when the condenser aperture closes, resolution decreases.

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As described above, depth of field and depth of focus relate to different perspectives. However, they are not entirely separate, as they both correspond to the axial depth of the space of a particular plane — depth of field relates to object space, whereas the depth of focus involves image space. This, in turn, influences their applications. For example, low-power objective lenses generally have lower numerical apertures and will have a higher depth of field (detail is lost quickly above or below the optimal focus plane) and higher depth of focus (sensor tilt is less impactful to image quality), while high-power objective lenses will have almost the reverse.

An important point to understand when working with compound microscopes and their objective lenses is that the field of view changes as the magnification changes. Typically a lower magnification objective lens will have a larger field of view, and a higher magnification objective lens will have a smaller field of view. For more information on how magnification affects the field of view read this article on microscope magnification and field of view.

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It is easy to think that more microscope magnification will result in seeing more detail and a better image. However, keep in mind that more magnification means you will see a much smaller area on your sample. Additionally if you get too much magnification (anything over 1000x), you will end up with empty magnification and poor resolution. So no, more microscope magnification is not necessarily better.

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