Field of View and Magnification
In optical lenses, there are two incredibly important properties to understand: Field of View, and Magnification.
The field of view is the area your lens can see, and the magnification is how expanded it is, but their interaction is more complicated. Within a microscopy setting, we must understand how our total magnification works to calculate our field of view, and also be aware of how our magnification alters our working distance, which in turn affects which samples are viewed at which levels of magnification.
How Does Microscope Magnification Work?
To understand the magnification of a microscope, you need first to understand how a standard light microscope works. As your specimen is lit, light is funneled up into an objective lens which provides a large amount of magnification to ‘blow up’ the image of your subject in great detail.
These high-quality lenses have improved over centuries through research and innovation, first starting at minute levels of magnification like 3x and ballooning up to 100x in modern times. The light from our objective is then filtered up through a secondary optical lens, which has smaller magnification but is intended to make the image more visible to the human eye.
This process doesn’t increase the ‘resolution’ or level of detail on the sample but instead just makes it easier for a person to see, and most commonly provides 10x magnification, but 2x and 4x are also prevalent.
The total magnification offered by a microscope is therefore understood as the magnification of the objective lens multiplied by that of the optical lens. As an example, if we are using a 40x objective with a 10x optical, then we are producing a total of 400x magnification.
How Immersions Impact Resolution
According to Sciencing.com, the theoretical maximum for a light-based microscope is up to 1,500x; anything beyond this produces an illegible image due to the wavelengths of light.
While common light microscopes aim for 1,000x with a 100x objective and 10x optical, even these images are subject to blur and loss of quality. To help compensate, microscope researchers created a technique called ‘immersion’ in which they place a liquid substance between the objective lens and the sample.
As liquids have a higher refractive index than that of air, they are able to more directly channel the light through, resulting in cleaner images. However, lenses must be properly corrected for these techniques, and as such often feature adjustable parts, or some microscopes implement multiple lenses that can be swapped.
In addition, different fluids have different refractive indexes, so oil immersions are capable of producing the highest theoretical ‘resolution’ for a microscope lens. But, oil immersions have a low depth of field, and therefore do not work well in life-science microscopy that involves specimens submerged in water.
For these, you are better off utilizing a water immersion, which can provide a deeper depth of field, albeit with less deep clarity.
What Is a Field Number (F#)?
As MicroscopyU explains, the size of the opening a microscope sees through is known as the field-of-view number or field number for short. This is most commonly measured in millimeters, with the number representing the openings, diameter.
We know that in an eye-piece, there is an opening called the field diaphragm that is the primary contrast of the field of view; second to the limits of the objective lens’s size of course. This diaphragm is an important part of maintaining an image’s clarity, as it prevents refracted light from bouncing off the sides of the channel and refracting back into the eye lens. This causes the edges of the image to look blurry and fuzzy, as there are multiple instances of light being reflected into them.
However, it should be noted that the field number represents what the microscope can see, as defined by its own opening. To understand what amount of space the microscope sees, we need to divide our field number by the magnification of our objective lens.
If your microscope utilizes a tertiary lens between the objective lens and the optical lens, then simply multiply this lens’s magnification by the objective’s before dividing. Here’s a quick reference guide with more information on focal length and FoV.
There are two commonly used designs for eyepieces, which are the Huygens and Ramsden designs, named after their creators. The primary difference is the order in which the components occur within the eyepiece, as this somewhat alters the way in which the light is channeled through them.
An eyepiece consists of an eye lens, which is the one closest to the eye of the user, a field lens, which is nearest the image formed by the objective, and the field diaphragm that limits superfluous light.
In a Ramsden design, the field diaphragm is the first feature of the lens and limits the light before it reaches the field lens, which is the first of the two plano-convex lenses.
Conversely, in a Huygens eyepiece, we see the field lens located before the diaphragm, meaning that all light from the objective reaches this lens, but some light is blocked before arriving at the eye lens.
The difference between these two techniques is nominal but represents the ways in which microscopes continually work to offer new solutions and strive for optimal results, which we’ll touch on just ahead.
An average eyepiece has a field number between 6 and 28 millimeters, but this varies depending on the magnification of the eyepiece itself. The lower the eyepiece’s magnification, the higher the field number; for example, a 10x will have a field number between 16 and 18 millimeters on average, but a 5x will be around 20 millimeters.
However, as microscope eyepieces continue to evolve, they are able to offer corrected glass that compensates for the light within the mechanism and offers wide-field designs of 26mm or more. These eyepieces are even sometimes substituted for cameras, where the human eye is substituted for a digital sensor that captures an image for long-term analysis.
This is still a developing technology but is an extremely promising development in the world of microscopy.
What Is a Microscope’s Working Distance?
The multiple lenses of a microscope objective are all finely tuned, meaning that they will all focus to a concrete distance. As we alluded to when talking about immersions, this means that your microscope is intended to be elevated to a specific height when that lens is being used.
However, we didn’t fully elaborate on the fact that, as a microscope’s magnification increases, this working distance actually decreases. As the light becomes more tightly channeled and funneled through the objective, it is sharply directed into the objective and therefore arrives more quickly (in terms of space traveled through) at its destination.
As an example, Microbus points out that a 100x objective lens will have a miniature working distance of just 0.04 millimeters or just 40 micrometers. However, our immersion techniques also have effects on the way light reflects. Most notably, a water immersion offers greater depth than an oil immersion can.
This has immense implications for how you intend to use your microscope, as it also affects the maximum specimen height that your microscope operates. If you magnify something that is taller than 0.04 millimeters, the image would likely be out of focus or might require a water immersion to help alter the light and the field of view.
There are many considerations to make when deciding which objective is best for your project, but it is certainly possible to lessen the level of magnification to afford yourself a greater working distance and a larger field of view at the cost of magnification.
As we now know, you can calculate your microscope’s total level of magnification by multiplying the magnification of our two lenses, the objective and the optical. We also understand that our lens’s field of view is primarily determined by its objective opening and field diaphragm, which limits the light taken into the initial lens and the light projected out to the eye lens.
Finally, we learned that a microscope’s magnification shortens the working distance provided, meaning that the higher the magnification, the shorter your sample needs to be.
Of course, there is more to understand about how light is routed through a microscope, such as the intricacies of immersion techniques and other concepts like numerical aperture.
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