3.1: Background Information

Types of Microscopes and Their Applications

Exploring Parts of the Microscope

The simplest optical microscope is the magnifying glass, which is good to about ten times (10 times) magnification. The compound microscope has two systems of lenses for greater magnification: the ocular, or eyepiece lens, that one looks into, and the objective lens, or the lens closest to the object.

In Figure \(\PageIndex{1}\), the bright field light microscope is displayed and the list below captures a detailed description of all the labeled parts:

1. Ocular Eyepieces: The lenses of the eye pieces (oculars) on your microscope magnify ten times. One of your oculars may have a pointer that can be moved by rotating the eye piece. The eyepiece of most binocular microscopes can be adjusted to match the distance between the eyes of different observers (inter-pupillary adjustment).
   • Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes.
2. Observation Tube: The assembly on a microscope that houses the eyepieces and directs the light path from the objective lenses to the observer's eyes or a camera.
3. Arm: In microscopy, the "arm" typically refers to the structural backbone of a standard microscope.
4. Coarse Focus: Serves the essential function of rapidly adjusting the focus of the microscope's objective lens in larger increments, to bring the specimen into a rough focus range. It helps researchers and students locate the specimen on the slide and get it roughly in focus before fine-tuning the focus using the fine adjustment knob.
5. Fine Focus: While the coarse adjustment knob facilitates initial focusing by rapidly moving the objective lens closer to or farther away from the specimen, the fine adjustment knob serves a more refined purpose. Its primary function is to fine-tune the focus of the microscope to achieve optimal clarity and detail. The fine objective knob may be used with all objective lenses.
6. Stage Control Knob: Allows precise X-Y positioning of a slide, enabling users to scan specimens horizontally and vertically. Typically located below the stage on the side, one knob manages front-to-back movement while the other manages side-to-side, centering the specimen for optimal viewing.
7. Nosepiece: This is the disc-like part that holds two or more objective lenses and can be rotated to easily change power.
8. Objective Lenses: The critical optical component closest to the specimen, responsible for primary magnification and image resolution. It gathers light from the specimen to create a magnified real image, typically ranging from 4 times to 100 times magnification, including specialized oil-immersion lens for high resolution imaging. The microscopes are parfocal, meaning that after you adjust the focus or change the objective, the image will remain in view.
   • Scanning objective: This lens magnifies four times ; it's used to center the object in the field of view. You will not be drawing at this lowest power, but it serves to orient your slide initially. This is the only objective with which you should use the coarse focus knob.
   • Low power objective: This lens magnifies ten times (total magnification = ocular X objective = 10 X 10 = 100 times total magnification). Focus with the coarse adjustment knob on scanning power first, then move the nosepiece so that the low power objective is in place.
   • High power objective: This lens magnifies 40 times (total magnification is 400 times). Use this objective only after centering an object with the low power objective. You should only use the fine focus knob when this objective is in place.
9. Stage Clip: Small metallic clip used to secure specimens to a microscope stage.
10. Stage: The surface or platform on which you place the microscope slide is the stage. Note the opening (stage aperture) in the center of the stage. A mechanical stage allows you to move the slide horizontally and vertically.
11. Iris Diaphragm: regulates the amount of light passing from the light source through the specimen and through the lens system of the microscope. The lever that projects from the side of the diaphragm opens and closes these plates, thereby regulating the amount of light.
12. Condenser Lens: The condenser consists of a series of lenses that focus light onto the specimen. It is moved up and down by a knob at its side or by a lever projecting from the condenser housing. By properly adjusting the condenser, you can greatly improve the clarity of the specimen. A filter holder may be attached to the bottom of the condenser. It usually contains a blue filter.
13. Light Source (Illuminator): An illuminator, is the light source built into the base of the microscope and controlled by an on/off switch. You can control the amount of light entering the specimen by adjusting the diaphragm.
14. Base: The bottom portion of the microscope and the only part that touches the tabletop. When carrying the microscope, the upper part of the stand (the arm) is held by one hand and the other hand should be placed under the base to support the microscope's weight.

Resolution vs. Magnification

Two parameters are especially important in microscopy: magnification and resolution.

• Magnification measures how much larger a microscope (or set of lenses within a microscope) causes an object to appear. For instance, the light microscopes typically used in high schools and colleges magnify up to about 400 times actual size. So, something that was 1 mm wide in real life would be 400 mm wide in the microscope image.
• The resolution of a microscope or lens is the smallest distance by which two points can be separated and still be distinguished as separate objects. The smaller this value, the higher the resolving power of the microscope and the better the clarity and detail of the image. If two bacterial cells were very close together on a slide, they might look like a single, blurry dot on a microscope with low resolving power, but could be told apart as separate on a microscope with high resolving power. The smaller the wavelength of light, the better the resolving power. Resolving power is also impacted by the numerical aperture of the objective lens that is being used to magnify the specimen.

Parfocal Lenses

Our microscopes are parfocal. What does this mean? Once you have focused on any of the objectives (we always start with 4 times), the other objectives will also be (mostly) in focus. You do not need to make major focus adjustments after changing objectives - usually the fine focus knob can be used to make small adjustments and obtain a crisp, clear image. In fact, you should never use the coarse focus once you've obtained an in-focus image using the 4 times objective.

Microscope Care

1. Always carry the microscope upright and use two hands. Place one hand on the arm of the scope and support the base with the other. Our individual and joint responsibility is to ensure that the microscopes remain in good working condition.
2. Be gentle. Sliding the microscope on the table could jar lenses and other parts loose. The microscope seems like a simple instrument, but each lens (eyepiece and objective) is actually made up of several other lenses; jerky movements may mis align lenses.
3. Always use clean hands when handling your microscope. Do not touch the lenses with your fingers, and do not remove any of them.
4. Use only lens paper and lens cleaner to clean all of the lenses (not Kleenex, Kimwipes, or cloth). Clean lenses regularly and oculars when they are dirty.
5. Never let an objective lens touch the glass slide. If you lose the image, go back to the scanning lens (4 times) or 10 times objective and refocus.

On completion of the laboratory exercise, return the microscope to its cabinet in its original condition. The following steps are recommended:

1. Clean all lenses with dry, clean lens paper. If necessary, use a drop of lens cleaning fluid.
2. Place the low-power objective in position and close the stage and objectives together.
3. Center the mechanical stage.
4. Coil the electric wire around the body tube and the stage.
5. Carry the microscope to its position in its cabinet as described above.