Fundamentals of Light Microscopy

OBJECTIVES:

Upon completion of this exercise the student should be able to:

1. name the parts of a compound light microscope and describe their function

2. know how to properly:

a. carry a microscope, clean the lenses, and prepare the microscope for use and storage;

b. set and control illumination;

c. focus on a specimen;

d. determine the total magnification, the size of the field of view at any magnification, and estimate the size of a specimen;

e. make reasonably accurate drawings of specimens observed through the microscope.

INTRODUCTION TO THE MICROSCOPE

IMPORTANT TERMS

compound microscope – magnifies objects using a series of lenses

resolution – detail; clarity of an image; the ability to recognize two separate points as in fact being separate

magnification – increase in the size of an object’s image

depth of field – amount of thickness of the specimen that is in focus at any one time

field of view – total area visible through the microscope

permanent slide – prepared so that the object and coverslip are permanently attached to the slide (object is not alive)

wet mount – freshly prepared slide; living object is dropped on slide in water/media and covered with a coverslip

contrast – use of a dye or adjusting the amount of light to distinguish the specimen from the background

Front of microscope

Rear of microscope

Functions of the parts of a compound microscope

The stage of the microscope, is a rectangular plate on which the slides are placed. There will be a hole in the stage through which the light will pass.

The stage finger assembly, are two metal strips laying flat on the upper surface of the stage, which will be used to hold slides. You should never place your slide under these strips, they are spring loaded to pinch/hold your slide in place gently so that the mechanical stage knobs can easily move the slide around the stage.

The mechanical stage adjustment knobs, these knobs are used to move the slide left and right or up and down on the stage to allow you to view the whole slide. Generally, the knobs are located below the stage, on the opposite side of the microscope from the condenser adjustment knob.

The condenser is just under the stage. It will gather the light from the illuminator and focus it on the specimen laying on the stage. The condenser is mounted on a track and can be moved up and down beneath the stage.

The condenser adjustment knob controls the height of the condenser and is located just beneath the stage on the side opposite the mechanical stage adjustment knobs.

The iris diaphragm control ring or lever, is a rotating device attached to the condenser, or a metal tab or arm sticking out horizontally from the condenser. The iris diaphragm allows the amount of light passing through the condenser to be regulated. This device is very important, and you will need to adjust it most every time you change slides and change magnifications. The amount of light passing through the iris diaphragm is critical for proper viewing of the image.

The objective lenses are on a turret or nosepiece just above the stage. They magnify the image of the specimen to varying degrees. Each lens will have thin stripes of a different color. They will also have their magnification level etched on them (4x, 10x, etc.). The objective lenses ranging from 4x to 40x are “dry” lenses. The objective lens of 100x is an “oil immersion” lens, and before it can be used, a special oil must be added to the slide.

The nosepiece holds 3 to 4 objective lens and allows these lenses to be rotated into use. Usually you will feel the lenses “click” into place when they are properly aligned.

The ocular lens in the eyepiece. It further magnifies the image that has already been magnified by the objective lenses. It commonly magnifies 10x. It should contain a pointer (often in only 1 of the 2 lenses), which appears as a dark line when viewed through the microscope. The pointer can be used to specifically indicate features within the specimen.

The coarse focusing adjustment knobs and the fine focusing adjustment knobs on either side of the arm just above the base. There is one of each on each side. The coarse adjustment knobs are usually the larger of the two. These knobs are used to focus the specimen. Note that it doesn’t matter whether you use the right- or left-hand coarse adjustment knob. They are interlocked, thus they move in unison. When you turn the coarse focus knobs the stage is moved rapidly with poor precision. This focus knob is only used with the lowest power objective lens.

The fine focusing adjustment knobs move the stage is moved very slowly with great precision and are used to bring your specimen into precise focus. Usually they are located as the inner knob with the coarse focusing adjustment knob. Use this knob with the higher magnification objective lenses.

The arm is the main part of the microscope that holds the ocular lenses above the stage. Some microscopes have a handle in the back part of the arm. To carry a microscope properly you need to hold the arm directly (or the handle if available) and the base of the microscope. NEVER hold the microscope by the stage when moving a microscope.

The base of the microscope is the bottom part containing the plug and the light source.

The light source is located in the base of the microscope and shines the light up through the condenser, iris diaphragm to the stage. It is controlled by both an on/off switch and a variable, rheostat knob.

Rheostat control knob (dimmer switch) is located on the base of the microscope and control the amount of light that reaches the condenser. At low power you will need to turn this down to decrease the amount of light on your slide but as you move to higher magnification objective lenses you will need to increase the amount of light.

CARE OF THE MICROSCOPE

Microscopes are very expensive and delicate pieces of equipment. When using a microscope, you must:

1. Carry it upright with two hands with one hand holding the arm and the other hand supporting the base.

2. Use only the lens paper/Kimwipes and the solution provided to clean the lenses. Paper towels or cloths could scratch or leave lint on the lenses.

3. Begin with the low power (4X) objective when bringing an object into focus, and return the microscope to the cabinet with the low power (4X) objective in place. Also return the microscope with the stage in the lowest possible position.

4. Never use the coarse focus adjustment above low power.

5. When cleaning start cleaning lower the stage to the lowest position, then start with the 4x, then move to the 10X and finish with the 40X objective with one piece of lens paper soaked with lens cleaning solution. Use a separate piece of lens paper soaked with lens cleaning solution to clean the 100X oil immersion lens to make sure you don’t move oil from the 100X to the other lens.

How to Use the Microscope

Watch the following two videos. When you get into a lab the instructor will show you their preferred way to handle, focus and clean a microscope.

Properties of the Microscope

A. Magnification

The magnification of an image occurs by bending light through a series of lenses. Note that it is the image of the specimen that is magnified, not the specimen itself. The total magnification of the image is the product of the magnifications of the objective lens and of the ocular lens, as shown in this equation:

total magnification = objective lens magnification x ocular lens magnification