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The Light Microscope

The-light-microscope

ABOVE: FIGURE 1.4 The eyepiece is positioned close to the real image formed by the objective. It cannot therefore form a real image and instead the rays appear to originate from a magnified virtual image.

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The simplest microscope is a single lens, and as we saw in the Introduction this was for many years the best microscope. Simple microscopes, as they are called, can give high-resolution images, and once we understand how one lens works, it is equally easy to see how they are combined to form both ancient and modern compound microscopes.

There is one fundamental property of a lens from which many of its other characteristics follow on automatically. A lens, by definition, makes parallel rays of light meet at a point, called the focus of the lens (Figure 1.1). The distance from the lens to the focus is called the focal length.

For a simple understanding of the microscope the focal length is the only thing we need to know about a lens. This ignores any imperfections of real lenses, assuming that all lenses are perfect, but it is a great starting point.

We can use this property to draw some very simple ray diagrams, which will show us how and where a lens will form an image and from there how different lenses work together in the complete microscope.

Since all parallel rays are deflected through the focus, while rays that pass through the exact center of the lens will go straight through, undeflected, it is easy to plot where the image of a specimen will be.

Provided that the object is farther away from the lens than its focal length, we can draw these two rays, as shown in Figure 1.2, from any point on the object. Where they meet is where the image of that point will be. (If the sample is closer to the lens than its focal length, the two rays will not meet.)

This shows us that the objective lens forms an inverted, real image—real in this context meaning that it is an actual image that can be projected on a screen. In fact you can easily do just that if you take out an eyepiece from a microscope in a darkened room.

Put a piece of tracing paper over the hole and you will see this image, since it is formed at a point near the top of the microscope tube. A slight adjustment of the fine focus should bring it into sharp focus.

How then can we get a greater magnification? As Figure 1.3 shows, this is done by using a lens with a shorter focal length. A "higher power' lens really means one that has a shorter focal length.

how a light microscope works

ABOVE: FIGURE 1.3 A high power objective has a shorter focal length. It is positioned closer to the specimen and will then form a more highly magnified image but at the same place.

If we are going to still form the image at the same place we must bring the specimen closer to the lens, which is why high power objectives are always longer than low power ones.

They are designed to be parfocal—as you rotate the turret of the microscope each lens should come into approximate focus once one has been focused.

In theory (and indeed in practice) we could make the image larger with any given focal length lens by forming the image farther away (i.e., bringing the specimen closer to the lens). But lenses are corrected for one image distance, and the image quality will suffer if we do that. The distance from the back of the objective to the image plane is called the tube length and is marked on the lens.

Having a fixed tube length brings us the added benefit that we know what magnification an objective of a given focal length will give, and we can mark this on the lens. If you look at an old microscope objective, from the days when tube lengths were not standardized, you will find that it gives the focal length rather than the magnification.

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microscope focus lens function

ABOVE TOP: FIGURE 1.1 A lens makes parallel rays meet at a point, called the focus of the lens.

ABOVE BOTTOM: FIGURE 1.2 Plotting the paths of the parallel and central rays shows where a lens of given focal length will form an image and what the magnification will be.

e do not usually view the image on a sheet of tracing paper but through another lens, the eyepiece. The eyepiece is positioned so that the real image projected by the objective is closer to the lens than its focal length.

This means, as we have seen, that it cannot form a real image. However, if we plot the rays in the same way (Figure 1.4) we see that they appear to come from a point behind the lens, which is thus a virtual image.

The virtual image is the same way up as the original real image but much larger, so the final image remains inverted relative to the specimen but is magnified further by the eyepiece.

We normally focus a microscope so that the virtual image is located somewhere around the near point of our eyes, but this is not essential. Where it forms is under our control as we adjust the focus.

Figure 1.5 shows these same ray paths in their correct position relative to an actual microscope. For the sake of simplicity an old single-eyepiece microscope is used since its optical path is in a straight line. This also emphasizes the point that microscope resolution has not improved since the end of the 19th century; the modern microscope is not really any better optically.

Each operator may focus the microscope slightly differently, forming the virtual image at a different plane. This does not cause any problems in visual microscopy, but once we start to take photographs it can make life difficult. The camera needs an image at the plane of the film, not the plane at which an individual operator finds comfortable for viewing.

A microscope fitted with a camera will also have crosshairs visible through the eyepiece, and the eyepiece will have a focusing adjustment to bring these into focus. Then the microscope focus is also adjusted so that the image is seen in sharp focus with the crosshairs superimposed; it will then also be in focus for the camera.

parts of early microscope

ABOVE: FIGURE 1.5 An early 20th century Zeiss "jug-handle" microscope. By this stage the microscope had developed to the point where its resolution was only limited by the wavelength of light, and it has therefore not improved since. Modern improvements are fundamentally matters of convenience.

The final magnification that we see will be the objective magnification multiplied by the eyepiece magnification, provided that the tube length is correct. The image captured by a camera will have a different value, determined by the camera adaptor.

Modern microscopes rarely just have the one eyepiece, which sufficed up until the mid-20th century. Binocular eyepieces just use a beamsplitter to duplicate the same image for both eyes; they do not give a stereoscopic view (Figure 1.6).

(In this they differ from the beautiful two-eyepiece brass microscopes of the 19th century, which were stereoscopic but sacrificed some image quality by looking through the objective at an angle.)

There always needs to be an adjustment for the interocular spacing to suit different faces. This has to be done without altering the tube length. Sometimes there is a pivoting arrangement so that the actual length does not change.

On other microscopes the eyepieces move out as they come closer together and in as they move farther apart.

On some older microscopes it must be manually adjusted; the focusing ring on each eyepiece must be adjusted to match the scale reading on the interocular adjustment.

microscope lens operation

ABOVE: FIGURE 1.6 Typical arrangement of prisms in a binocular microscope. Both eyes see the same image.