Microscopes

Microscopes help you see things that are small. This makes them a key tool for studying structure and function in all kinds of organisms. You'll need to get comfortable with two kinds of microscopes for Bio 6A: dissecting and compound scopes.

Dissecting microscopes

Dissecting microscopes, also called stereo microscopes, have some key characteristics that make them useful for looking at large specimens and doing dissections:

• Low magnification. For large specimens, you don't need much magnification to be able to see the important details. Many dissecting microscopes have a zoom feature, allowing you to change the magnification.
• Large field of view. In other words, you can see a relatively large area of your sample; field of view is described in more detail below.
• Large working distance: you'll have plenty of room between your specimen and the microscope's lens, so you can reach in with forceps or other tools to move things around.

With the dissecting scopes, you can use either reflected light or transmitted light. Reflected light is the way you normally see things: light comes from a light source, bounces off something, and you see it. If you use the upper light source on the dissecting scope, you're using reflected light. Transmitted light is light that passes through your specimen, instead of bouncing off. This only works with specimens that are somewhat transparent. If you're using the light source that comes from below your specimen, you're using transmitted light. When using the dissecting scope, try both kinds of lighting to see which works best. Usually it's one or the other; if you have light from above and from below, your image will be washed out and you'll see less. Properly controlling the light will make invisible things become visible.

Overall, dissecting microscopes are simple to understand and use. Compound microscopes are a little more complex.

Compound microscopes

Compound scopes provide higher magnification than dissecting scopes. The high magnification makes compound scopes powerful tools, but it also creates some diffuculties. Important characteristics of compound scopes:

• High magnification.
• Small field of view: you can only see a small part of your sample.
• Must use transmitted light (light that shines through your sample); therefore...
• Samples must be thin & transparent, and mounted on glass microscope slides.

In this lab, you'll mostly use the compound scope with commercially prepared microscope slides. These slides generally hold ultra-thin slices of specimens. The specimens are so thin that they are more or less transparent, so the slides are stained to allow you to see specific structures. In general, the colors you see on commercially prepared slides are the colors of the stains, not the colors of the living organism. This is a good thing, because the people who make the slides can choose particular stains to highlight particular features on the sample.

See "How to use the compound microscope," further down this page, for more detail.

Definitions for microscopy

Magnification means how much larger an object appears in the microscope as compared to without the microscope. If you look at a leaf without a scope, you're seeing it at 1x magnification -- in other words, life size. If you put the leaf under the scope, you might see it at 100x, or one hundred times life size. The total magnification of a compound microscope depends on two sets of lenses:

Ocular (also called eyepiece). This is the lens you look into; it typically has a magnification of 10x.

Objective. This is the lens that's closest to the object you're looking at. Compound scopes usually have several objectives, such as 4x, 10x, 40x, and 100x.

The total magnification is the ocular magnification times the objective magnification. With a 10x ocular and 40x objective, the total magnification is 400x.

Field of view is the diameter of the area you can see when you look through the microscope. When you use higher magnification, the field of view becomes smaller. It is possible to measure the field of view by placing a tiny ruler, called a stage micrometer, on the scope instead of a normal microscope slide.

It's useful to know what your field of view is so you can estimate the size of things you're looking at. If the field of view at a given magnification was 200 microns, and you were looking at a cell that was half as long as the field of view, the cell would be about 100 microns.

Scale bars. Most photomicrographs (pictures taken with a microscope) should have a scale bar -- a small line on the picture, with text indicating the length of the line.

For example, the picture at right (a mushroom cross-section) shows a scale bar labeled 200 microns. Of course, this scale bar appears much bigger than 200 microns on your computer screen, because it's magnified. How much is it magnified? That depends not only on the magnification of the microscope, but also on your computer screen. You could also print this picture larger or smaller. However you enlarge the picture, you'll always be able to see that the reddish-brown spores are much smaller than 200 microns. That's why scale bars are better than writing something like "100x" on the picture.

Depth of field describes how much of your image is in focus. A typical small digital camera has vary large depth of field: everything will be in focus, from close to the camera to far away. With a compound microscope, the depth of field may be only a few microns. You may find that you can't get your whole sample in focus at once when you're using the higher-magnification objectives.

How to use the compound microscope

Taking a few minutes to learn how to use compound microscopes will speed you through the labs and greatly enhance your viewing pleasure. Remember that you should only use the compound scope for thin samples on microscope slides.

Use a cover slip! Cover slips flatten the sample so you can see through it better, and they keep samples from messing up the objective lenses. Commercially prepared slides already have cover slips glued on, but if you make a slide yourself, you need to put one on. In microbiology class, you might skip the cover slip; that's because the samples are strongly stuck to the slide. For Bio 6A, always use a cover slip.

Go through the following steps, in order:

• Turn on the light. There is a toggle switch for on/off (1 is on; 0 is off). There is also a rheostat (dimmer) to adjust the light intensity. Turn the rheostat all the way down before you turn on the scope, then gently turn up the light until you can see it. (The expensive lightbulbs will last longer this way.) Don't turn the light all the way up; it's too bright.
• Lower the stage all the way using the coarse focus knob. This gives you room to work.
• Start with the 4x objective. Rotate the nosepiece of the microscope until the 4x is pointing toward your sample. This low-power objective makes it easy to find the sample and get it in focus. If you start with a high-power objective, you'll probably have trouble finding the focus point. Because the depth of field is so limited, you may see nothing at all until you have the focus right.
• Put the slide on the stage, held in place by the little spring clip. Make sure both the slide and the stage are dry.
• Move your sample until you can see that the light is shining through it. Do this before you even put your eyes to the oculars.
• Look through the scope and focus. Use the coarse focus knob at first, until the image is more or less in focus; then switch to the fine focus. Since you're starting with the stage all the way down, you know you'll be focusing by moving the stage up.

• Look at the whole slide on the lowest power, so you get an overview before you switch to higher magnification.
• Adjust the light. Not too bright, not too dim.
• Adjust the oculars. First, move them further apart or closer together so they match the distance between your eyes. That way, you can look through both eyes at once and see a single image. Many people only look through one eye, but this gets tiring fast. Also, there may be an ocular micrometer in one eyepiece or the other; you won't know unless you look through both. This could be important on a lab exam! The second eyepiece adjustment is for focus. One eyepiece has an independent diopter adjustment so you can get it just right for your eyes.

• Switch to the 10x objective. If the slide is in focus with the 4x, it should be more or less in focus when you switch to the 10x. A slight adjustment with the fine focus knob should get it just right. If you lose the focus and can't see your specimen at all, go back to the 4x and start again.
• Switch to the 40x objective if you want to see more detail. It's up to you to decide what magnification is best for what you want to see.
• When you want to look at a new slide, switch back to the 4x before changing slides. This will make it easier to get the new specimen in focus.
• When you're done with the scope, switch to the 4x, turn the light all the way down before turning it off, and bring the stage all the way down. Don't put away the scope with a slide still on the stage!

Can't see anything?

If you look through the oculars and it's all dark, check to make sure that:

• The light is shining.
• The nosepiece is clicked in place so that one of the objectives is pointing straight down.

If you see light but can't find your specimen, check to make sure that:

• There is something on the slide. If you can't see anything without the microscope, perhaps there's nothing there.
• The focus is right. Go back to the 4x and try to focus. If you have trouble finding the specimen, try focusing on the edge of the cover slip, then scanning across the slide. If you can't find anything with the 4x objective, you probably won't find it on high power.