The light microscope is perhaps the most well-known and well-used tool in the laboratory. Yet, some laboratorians may be unaware of the full range of features available in today’s light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments should be fully examined and understood from a practical use standpoint in order to gain the most advantageous cost/benefit ratio.

Most laboratorians know that the practice of viewing and analyzing small objects does not simply involve a high degree of magnification. Rather, when it comes to viewing and analyzing specimens, the biggest concerns tend to involve:

• Obtaining sufficient contrast
• Finding the focal plane
• Obtaining good resolution
• Recognizing the subject when one sees it 

The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification, but bacteria tend to be invisible when using bright field microscopes. In order to make the best use of modern clinical microscopes, it is necessary to understand the types of optics used to obtain contrast, the best ways to view and focus on specimens, and how to properly use measurement devices with a light microscope.

Types of Light Microscopes
While the bright field microscope is quite common in biologic research settings, clinical laboratories are likely to be equipped with 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. Furthermore, fluorescence and confocal microscopes are specialized instruments used for research and clinical applications. Fluorescence (or epifluorescence) microscopy using fluorescent-labeled antibodies is useful for medical diagnosis such as detection of bacteria, viruses, fungi, or parasitic organisms. It also can pick up the presence of toxins and can be used to diagnose autoimmune diseases by identifying antibodies that attack the patient’s own tissues.¹ Confocal microscopy uses a laser to optically section tissues, allowing very high resolution of layers within three dimensional specimens. This process has been used for cancer detection in some cases.² 

Other than compound microscopes, a simpler instrument for low magnification use also may be found in the laboratory—the stereo microscope, or dissecting microscope, which usually has a binocular eyepiece tube, a long working distance, and a typical magnification range of 5x to 35x or 40x. Some instruments supply lenses for higher magnifications, but there is little to no improvement in resolution.

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 called the ocular or eyepiece. The user is able to 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 single Plasmodium falciparum, one of five known species of protozoan parasites that cause malaria, should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria will not show up at all unless the viewer hits the focal plane and distorts the image by using maximum contrast. 

A good quality microscope should have a built-in illuminator, condenser with aperture diaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. Many clinical microscopes have phase contrast, in which case an aperture diaphragm is not used. Phase contrast microscopes are almost always used for bright field, which is suitable for viewing stained specimens. The magnification of the image is simply the product of the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

Laboratorians certainly should be aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. However, some may be unaware of adjustments to the condenser that can affect resolution and contrast. Fully adjustable condensers allow a user to optimize the contrast and resolution to obtain what is called Köhler illumination—near perfect illumination that affords the truest obtainable picture of a specimen. Some condensers are fixed in position, whereas others are focusable so 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. Are you looking for formed elements in a blood sample or for evidence of bacteria? Are you examining a stained tissue sample or searching a stool sample for intestinal parasites? Some of these applications require low magnification and no special optics, while some, such as spotting bacteria, require very high magnification and either staining or specialized optics to detect colorless specimens. Basic search criteria include the size of the target, whether it is pigmented, and whether it will show up in bright field. Thus, it is important to determine the maximum magnification and contrast/resolution required, as well as whether it is a stained specimen, before setting up the device for viewing via the following steps:

Mount the specimen on the stage
The cover slip must be up if there is one. High magnification objective lenses cannot 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 in order to provide high intensity illumination at high magnifications, as well as low intensity illumination for comfortable viewing at low magnifications. Higher-end microscopes have a built-in illuminator, and the best microscopes have controls over the light intensity and shape of the light beam. 

Adjust the condenser
Proper adjustment and alignment of the microscope should be detailed in the instrument’s manual, but short of that, these are basic guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as possible. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). The light that comes up through the specimen should change brightness as the aperture diaphragm lever is manipulated.

Consider what you are looking for
It is a lot harder to find something when you have no expectations as to its appearance. 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, it can be tricky to find stained bacteria. 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 concentrate on 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 available, 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

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 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. 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 using the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye), and adjust the eyepiece, not the microscope. 

Select an objective lens for viewing
The lowest power lens (sometimes called a scanning lens) is usually 3.5x or 4x, and is used primarily for initially locating a specimen. 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, refocus and recenter the specimen. Higher magnification lenses must be physically very close to the specimen itself, so there is a risk of jamming the objective into the specimen; therefore, be very cautious when focusing. Of note, good quality sets of lenses are parfocal, that is, when there is a switch in magnifications, the specimen remains in or close to focus. 

Keep in mind, bigger is not always better. All specimens have three dimensions and unless a specimen is extremely thin, it will be difficult 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 the magnification is raised, the area of illuminated, visible specimen is smaller. By looking at a smaller area, less light reaches the eye, and the image darkens. Thus, with a low power objective, it may be necessary to cut down on illumination intensity. With a high power objective, the light source should be maximized, especially with less sophisticated 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; it is of much less utility for living specimens of bacteria or unstained cell suspensions. Below are examples of specimens that might be observed using bright-field microscopy and their appropriate magnifications (preferred final magnifications are included):

• Prepared slides, stained: bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x)
• Smears, stained: blood (400x, 1000x), negative stained bacteria (400x, 1000x)

Proper Microscope Care
As a general rule, every part of a good quality microscope is expensive, so exercising caution and care when using and maintaining a microscope is imperative. When handling the microscope, grasp it firmly by the stand only; never grab it by the eyepiece holder. Likewise, pull the plug (not the cable) when unplugging the illuminator. Since bulbs are expensive, and have a limited life, turn the illuminator off when the microscope is not in use.

Never use a paper towel, an article of clothing, or any abrasive material other than good quality lens tissues or a cotton swabs to clean an optical surface. Be gentle and use an appropriate lens cleaner or distilled water to help remove dried material as organic solvents may separate or damage the lens elements or coatings. Lastly, always make sure the stage and lenses are clean.

In the clinical laboratory setting, no one should be satisfied with an inferior image. Performing microscopy is much easier with a bright image, good contrast, good resolution, and a choice of optics that is best suited for the specimen to be observed. With a little experience, a clinical investigator can obtain a satisfactory image from just about any moderately priced, clean, and well adjusted microscope.

References

1. Sanborn WR, Heuck CC, Al Aouad R, Storch WB. Fluorescence microscopy for disease diagnosis and environmental monitoring. World Health Organization, Regional Office for the Eastern Mediterranean, Cairo, Egypt. 2005. Available at: http://applications.emro.who.int/dsaf/dsa281.pdf.
2. Yale Medical Group. Confocal microscope is a giant step in esophageal cancer. Yale School of Medicine News. November 2010. Available at: http://www.yalemedicalgroup.org/confocal2010

David Caprette, PhD, joined the biology department at Rice University as a laboratory coordinator in 1987 and currently teaches laboratory courses as a professor in practice with the department of biochemistry and cell biology. He earned his BS in biology from Case-Western Reserve University and his PhD from Cleveland State University.