This presentation will take us through the steps required to view a specimen in a bright field light microscope. We will cover proper mounting of a specimen, adjusting the condenser and oculars, and strategies for finding a target and for working up in magnification. We will discuss how the thickness of a specimen can limit the range of useful magnifications, and how to use oil immersion objective lenses to obtain the best resolution possible in a bright field microscope.

Prior to mounting a specimen, one should put the lowest available magnification in the light path. An objective of low magnification is shorter than one of high magnification, giving one more room for placing the slide. More importantly, when we look for an object on a slide, we search for it in three dimensions, namely the x-y plane and the vertical dimension (i.e., the focal plane). At low magnification, we see a much greater area of specimen and have a much deeper focal plane than at high magnification.
    
A mechanical stage makes it convenient to search an area systematically for objects of interest and to collect replicate data. Using the translational controls, one can manually “chase” a fairly fast moving living organism around a microscope slide without losing it from view.
    
Whether you have a prepared slide, wet mount, or a smear with no coverslip, it is critical to mount the slide with the specimen toward the objective lens. Usually, that means the specimen will be facing up, although some microscopes (inverted microscopes) have the stage above the objectives.
    
If the slide is upside-down, you may be able to focus at low magnifications without compromising the view. You will not be able to focus at a high magnification, though. High resolution requires that the half angle at which the cone of light enters the objective (alpha in the equation for resolution) be as large as is practical. Proximity to a specimen is necessary to obtain a large enough half angle when the light comes from a very small area. It follows, then, that to obtain the necessary resolution, a high magnification objective lens must be brought very close to the specimen.
    
Coverslips are made of very thin glass or plastic for two reasons. One is to allow an objective to approach within a very short distance of a specimen. The other to prevent the thickness of the glass, which is not optically perfect, from significantly compromising contrast or resolution.

You may not need your eyeglasses when using a microscope, unless they correct for astigmatism. Using a single ocular, the focus control alone can bring an image into sharp focus. If you have a binocular microscope, the eyepieces should be adjusted to compensate for eye differences.

Anyone who has used binoculars should find it easy to adjust the oculars on a binocular microscope. Before even focusing on a specimen, you should be able to adjust for eye separation so you will see a single field of view. When the oculars are separated to match your eyes, you should be able to look into them with both eyes relaxed, just as if you are looking across a room. If you have trouble with binocular vision, you could be among the minority of users with eyes set close together, making such viewing difficult. It is  more likely, though, that the individual oculars are simply out of adjustment, which prevents you from bringing the image into focus for both eyes at the same time.

Your microscope may be equipped with one fixed and one adjustable eyepiece, or with both eyepieces adjustable. Either way, the first step is to place each adjustable eyepiece in the center of its range of travel, giving you the most latitude for adjustment either way. The next step is to obtain an image at high enough magnification so that you can see fine details. Step three is to observe with the fixed eyepiece only (or one of the two adjustable eyepieces) with the appropriate eye, and focus the microscope on the image. Recalling one or two specific details from the image, observe with the other eye only, and this time, adjust only the eyepiece until the details come into focus. From this point on, when you focus the microscope, you should be able to look comfortably using both eyes.
    
If you had trouble seeing a single image when adjusting for eye separation, it may be worth trying again once the oculars are adjusted to match your eyes.

To obtain a sufficiently bright image at high magnification, we need a very intense light beam. The condenser lens gathers light from a wide cross-sectional area and concentrates it in the area of the specimen, giving us a far more intense light beam than we could obtain directly from the source. With a good quality illumination system, one usually has to reduce the intensity of the light source to view a specimen at low magnification.

As important as the condenser is for intensifying the light beam, its primary function is to condition the beam before it enters the specimen and objective lens. To obtain full resolution from a given objective, the condenser should be adjusted so that the back lens of the objective lens is filled with light. Such adjustment is accomplished using an aperture diaphragm control that is built into the condenser. The aperture diaphragm is similar to the aperture of a camera lens. The size of the opening is adjustable so that the diameter of the light beam as it passes through a particular plane can be varied.
 
For any given objective lens, there is an ideal position for the aperture diaphragm. "Stopping down" the aperture (making it smaller in diameter) beyond the ideal diameter dims and distorts the image. "Opening up" the aperture increases glare and washes out the image. Usually, to reduce glare, we sacrifice some resolution and stop down the aperture so that about 75% of the back lens of the objective is illuminated.

Inexperienced users may employ the aperture diaphragm control in the condenser to regulate the amount of light reaching the eye. However, misusing a condenser that way will sacrifice the resolving power of the microscope.

The lowest power objective lens is often called the scanning lens. Scanning lenses are seldom of the highest quality and are not of much use in collecting information. Their purpose is primarily to find a specimen readily and to bring it to the center of the light path and roughly in focus.

In a typical microscope field at 40x (calculated by multiplying the power of the ocular lens by the power of the lens), the field diameter is 5 mm. The advantage of the scanning lens is depth of focus and large viewing area. Although you cannot see much detail, you should be able to find what you are looking for, provided (1) the image is visible in bright field and (2) you know what to look for.

The only concern with finding an object at a very low magnification is that a specimen may not be recognizable. Therefore, it is essential that you know something about your specimen before setting up to view it. Think about the size of the target, how much (or little) contrast it should have in bright field, and how the material is likely to be distributed on a slide. Here are a few suggestions for finding hard-to-locate objects.

Try stopping down the aperture diaphragm (in the condenser) to increase the contrast of the image. Objects will not be well resolved, but the goal at this point is to find them, not to take data. Try focusing on an artifact, such as an air bubble, the edge of a coverslip, or a piece of visible debris. Among the most difficult specimens that are suitable for bright field microscopy are very small Gram negative bacteria. Stained bacteria at low magnification resemble dust on the slide surface. You might use a glass marking tool to make a shallow scratch on the slide surface. (Obviously, you mustn't scratch a prepared slide that is meant to be re-used.) Just as the scratch begins to come into focus, you should be at the level of the specimen, although it still may be hard to find. 

Unless you are so familiar with a type of specimen that you can go straight to an appropriate magnification and find your target immediately, it is best to take the same approach to finding specimens each time you observe. The most consistently effective strategy is to start at low magnification, find the target, adjust illumination, resolution and contrast, focus and center the object, and then raise magnification. Most sets of objective lenses are parfocal, meaning that the objectives are matched, so that if a specimen is in focus using one objective, it will be very nearly in focus when you raise the magnification using the next objective lens. Thus, if you re-focus, using only the fine focus control, and center the target each time you change magnification, you should have no trouble obtaining the image you seek at the desired final magnification.

After reaching 100x magnification, it is a good to re-adjust the microscope for binocular viewing, if you have a binocular eyepiece tube. You can see more detail now, and the better the oculars are adjusted to match your eyes, the more satisfactory the viewing.

At low magnifications (up to 100x or so total magnification), you should use the coarse focus control. Not only does it take too long to move a distance with the fine control, but the limit of travel with the fine focus may be less than with the coarse. Trying to focus past the limit of travel can damage a focusing mechanism.

When you bring in a high dry objective (a high power lens which is used without oil, usually a 35x or 40x lens) with the specimen in focus, the end of the objective will approach the specimen closely. It is unwise to use the coarse objective with such a lens, because it is too easy to ram the lens into the slide. In this case, use the fine control only.

Suppose you mount your slide upside-down. You will be able to focus at 40x total magnification, and again when you go to 100x magnification by swinging in the 10x objective. However, the thickness of the slide may exceed the depth of focus with the high dry objective (35x or 40x). If so, you won't be able to focus at all. If you don't pay attention, you probably will bump the slide with the end of the objective. Good high power lenses will telescope so as to buffer such shocks, but if you reach the limit, further movement will damage the slide and also may scratch the objective, and even the exit lens of the condenser. Such damage cannot be repaired.

Because high magnification lenses come so close to the specimen, to reduce the risk of a disaster, you might want to take your eyes from the eyepieces and instead watch the lens as you rotate it carefully into place. Until you are used to your microscope, you should check the position of the lens frequently while focusing, or (better) have someone else watch the objective and warn you if it contacts the slide.

Even with parfocal objectives, each time you change lenses, you will have to make some adjustment to focus. When you go to high dry magnification, and especially to an oil immersion lens, the depth of focus is so narrow that the specimen may be faint or invisible. If you lose track of a specimen completely, it usually is fruitless to remain at high power and search. You are searching in three dimensions, and your quarry occupies only a small volume in the three dimensional search area. It is best to enlarge your field of view in all three dimensions by going back to a lower magnification, re-focusing and re-centering a specimen before returning to high magnification.

A trick for spotting your target when it is out of focus is to jiggle the slide with the mechanical control and focus on any object that appears to be moving. You should be aware, though, that your slide consists of multiple surfaces, and that you naturally must focus on the surface that bears your specimen.

With the objective lens well away from the specimen so that no part of the slide can be in focus, suppose you begin moving the stage toward the objective. The first surface that comes into focus is the top of the coverslip (A), although in bright field you may not see anything without stopping down the aperture diaphragm in the condenser. You may see scratches, dust, and fibers on the surface. The next surface is where the coverslip contacts the specimen. In a wet mount, the bottom of the coverslip (B) might be above the target material. To focus on a specimen at the surface of the slide (C) you would need to raise the stage. The next surface is the bottom of the slide (D). If you raise the stage too far, you actually may focus on the surface of the condenser lens (E). You can tell if you are focused on the condenser by jiggling the slide. Anything on the slide will be seen to move, but the condenser image will remain still.

The thickness of a specimen limits the maximum functional magnification with which we can expect to obtain useful information. Objective lenses, as you may recall, have limited depth of focus, so while one part of a thick specimen is in focus, the other parts that are out of focus make it difficult to see detail. But the structures of some specimens are arranged in such a way that one can focus on one level at a time and obtain much more information than by using low magnification to see everything at once.

For example, a strand of the filamentous alga Spirogyra is, for the most part, in focus at 40 power. At 100 power, some part of the cell, either toward or away from the objective lens, is out of focus at any given distance. At 400 power, so little of a cell is in focus at any given time that one can begin with the specimen below the focal plane and optically section the filament by slowly raising the stage. First, the chloroplasts just beneath the cell wall appear in focus. As the stage is raised, chloroplasts come into and go out of focus in a spiral pattern. A middle view of a cell shows chloroplasts in focus only at the edges, revealing that the chloroplasts of Spirogyra are arranged near the cell walls and are not found in the centers of the cells. Raising the stage further brings the bottommost chloroplasts into focus. Unless you are aware of the direction in which you are moving the stage, you cannot tell the difference between top and bottom.

When a stream of photons travels through a medium, such as air, and strikes or exits a surface of a different medium, such as glass, its velocity and its direction change. The extent of deviation from the original path depends on the angle of the light ray from the perpendicular, and also varies with wavelength. White light consists of a spectrum of wavelengths, so on its way through a specimen, through glass, and into air, light passing through any one point is fairly well scattered. Scattering may not be noticeable at low magnification, but at magnifications above 200x, it will make what should be a discrete point look like a fuzzy ball. Similarly, what should be separate cells, such as in a chain of bacterial rods, will look like a melted thread.

Immersion oil typically has a refractive index* of 1.5, nearly the same as that of glass. That means that as light passes between glass and oil, it does not change velocity significantly, and does not bend as much. Using oil, we can exercise much greater control in concentrating light on the back lens of an objective lens, matching its numerical aperture and improving resolution tremendously.

By the way, light passing at 90 degrees to a surface doesn't bend at all. We obtain very good resolution with parallel light rays (i.e., with light coming in at right angles to the surface of a lens). At the center of a lens, light rays are parallel. This is the basis for the pinhole camera, and is the reason why near-sighted people can see more clearly when they squint.

* refractive index = n = (velocity of light in a vacuum)/(velocity of light in a transmitting medium)