Light Microscopy Tuesday, June 23, 2009

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.

A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,

• obtaining sufficient contrast
• finding the focal plane
• obtaining good resolution
• recognizing the subject when one sees it

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.

Types of light microscopes

The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.

Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. The stereo microscope, or dissecting microscope usually has a binocular eyepiece tube, a long working distance, and a range of magnifications typically from 5x to 35 or 40x. Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such "false magnification" is rarely worth the expense.

Bright Field Microscopy

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. A Paramecium should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria won't show up at all unless the viewer hits the focal plane by luck and distorts the image by using maximum contrast.

A good quality microscope has a built-in illuminator, adjustable condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

Students are usually aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. They are frequently unaware of adjustments to the condenser that can affect resolution and contrast. Some condensers are fixed in position, others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter and contrast is low.

A disadvantage of having to rely solely on an aperture diaphragm for contrast is that beyond an optimum point the more contrast you produce the more you distort the image. With a small, unstained, unpigmented specimen, you are usually past optimum contrast when you begin to see the image.

Using a bright field microscope

First, think about what you want to do with the microscope. What is the maximum magnification you will need? Are you looking at a stained specimen? How much contrast/resolution do you require? Next, start setting up for viewing.

Mount the specimen on the stage

The cover slip must be up if there is one. High magnification objective lenses can't focus through a thick glass slide; they must be brought close to the specimen, which is why coverslips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. The slide may require manual positioning, or there may be a mechanical stage (preferred) that allows precise positioning without touching the slide.

Optimize the lighting

A light source should have a wide dynamic range, to provide high intensity illumination at high magnifications, and lower intensities so that the user can view comfortably at low magnifications. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the field is bright without hurting the eyes.

Adjust the condenser

To adjust and align the microscope, start by reading the manual. If no manual is available, try using these guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). You should see the light that comes up through the specimen change brightness as you move the aperture diaphragm lever.

Think about what you are looking for

It is a lot harder to find something when you have no expectations as to its apprearance. How big is it? Will it be moving? Is it pigmented or stained, and if so what is its color? Where do you expect to find it on a slide? For example, students typically have a lot of trouble finding stained bacteria because with the unaided eye and at low magnifications the stuff looks like dirt. It helps to know that as smears dry down they usually leave rings so that the edge of a smear usually has the densest concentration of cells.

Focus, locate, and center the specimen

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is rather easy to find and focus on sections of tissues, especially if they are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens such as bacteria or unpigmented protists. A suspension of yeast cells makes a good practice specimen for finding difficult objects.

• Use dark field mode (if available) to find unstained specimens. If not, start with high contrast (aperture diaphragm closed down).
• Start with the specimen out of focus so that the stage and objective must be brought closer together. The first surface to come into focus as you bring stage and objective together is the top of the cover slip. With smears, a cover slip is frequently not used, so the first thing you see is the smear itself.
• If you are having trouble, focus on the edge of the cover slip or an air bubble, or something that you can readily recognize. The top edge of the cover slip comes into focus first, then the bottom, which should be in the same plane as your specimen.
• Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around until you have a good area for viewing.

Adjust eyepiece separation, focus

With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it.

One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.

Select an objective lens for viewing

The lowest power lens is usually 3.5 or 4x, and is used primarily for initially finding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small protists and for details in prepared slides such as cell organelles or mitotic figures, you will need a higher magnification. Typical high magnification lenses are 40x and 97x or 100x. The latter two magnifications are used exclusively with oil in order to improve resolution.

Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.

Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to "chase" a moving specimen.

Adjust illumination for the selected objective lens

The apparent field of an eyepiece is constant regardless of magnification used. So it follows that when you raise magnification the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes.

When to use bright field microscopy

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

• Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).
• Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).
• Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation.

Care of the microscope

• EVERYTHING on a good quality microscope is unbelievably expensive, so be careful.
• Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.
• Hold the plug (not the cable) when unplugging the illuminator.
• Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.
• Always make sure the stage and lenses are clean before putting away the microscope.
• NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents may separate or damage the lens elements or coatings.
• Cover the instrument with a dust jacket when not in use.
• Focus smoothly; don't try to speed through the focusing process or force anything. For example if you encounter increased resistance when focusing then you've probably reached a limit and you are going in the wrong direction.