General Biology 1 | Madelyn De La Rosa

LESSON 1: MICROSCOPY

Microscopy - The technical field of using microscopes to view objects and areas of objects that
cannot be seen with the naked eye.

MICROSCOPE PARAMETERS:
• MAGNIFICATION
• RESOLUTION OR REVOLVING POWER --- Numerical Aperture (NA) -- ability to
distinguish detail depends on the wavelength of light used and, on a value, called the
numerical aperture (NA) a characteristic of microscopes that determines how much light
enters the lens.

PROPER USE OF MICROSCOPES:

• To avoid breaking a cover slip and/or microscope slide while focusing (more importantly
scratching a lens), first locate the specimen using the low-power objective, and then switch
to the higher power objective.
• Never focus the high-power objective with the coarse adjustment knob, and never use
these lenses when examining thick specimens or whole mounts of specimens.
• To avoid dropping the microscope or banging it against a laboratory bench, carry the
microscope in an upright position using both hands.
• When carrying the microscope, place one hand on the base and the other hand around
the arm.
• DO NOT PLACE THE MICROSCOPE IN AN UPSIDE-DOWN POSITION. PIECES WILL
FALL OUT.
• Keep microscope away from the edge of the bench, particularly when not in use.
• Never force the microscope parts to work.
• Use lens cleaners and paper both before and after use.
• Always store the microscope in low power objective.
• Place the cover over the microscope after each use.
• Bring all defective microscopes to your teachers’ attention.

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PARTS OF A COMPOUND MICROSCOPE:

• Eyepiece/Ocular Lens: The eyepieces are the lenses you look through. The eyepiece of
most binocular microscopes can be adjusted to match the distance between the eyes of
different observers (interpupillary adjustment). Other microscopes may have different
magnifications which are stamped on the side of the eyepiece. Most are 10X. You may
have to remove the eyepiece from its holder to determine its magnification.

• Body Tube: Light travels from the objectives through a series of magnifying lenses in the
body tube to the ocular. In some microscopes, the body tube is straight; in others, the
oculars are held at an angle. The body tube contains a prism that bends the light rays so
that they will pass through the oculars.

• Objective Lens: Attached to a rotating nose piece, or turret, at the base of the body tube
are a group of 3 or 4 objectives. Locate the turret and notice a click as each objective
snaps into position.

o The objective lenses focus the light that comes through the specimen, up the body
tube, and through the oculars. Each objective has numbers stamped on it. One of
these numbers identifies the magnification of the objective (e.g., 43X). Objective
lenses are usually named according to their magnifying power, as follows:
o scanning power 4X; low power 10X; high power 43X; oil immersion 93X or 100X
o The total magnification for each objective is calculated by multiplying the
magnification of the ocular and objective lens on your microscope. On your
worksheet below calculate the total magnification for each ocular/objective
combination on your microscope.
• 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. On some microscopes, the stage
is stationary and has clips to hold the slide in place. On other microscopes, the stage is
movable and is called a mechanical stage. Movement is controlled by 2 knobs located on
the top, side, or bottom of the stage. Note the horizontal and vertical scales on the
mechanical stage.
o Substage: The area under the stage, called the substage, may contain a
diaphragm, a condenser, or both.
o Diaphragm: The diaphragm regulates the amount of light passing from the light
source through the specimen and through the lens system of the microscope. By
properly adjusting the diaphragm, you can provide better contrast between the
surrounding medium and your specimen, thus greatly improving your image of the
specimen. The diaphragm may be either annular or iris. An iris diaphragm consists
of a circle of overlapping thin metal plates. The lever that projects from the side of
the diaphragm opens and closes these plates, thereby regulating the amount of
light that enters the microscope. Most of you have a microscope with an iris
diaphragm. An annular diaphragm consists of a circular plate with holes of different
diameters. You can rotate this plate to place the various holes in the light path,
thereby regulating the amount of light that passes from the light source through the
specimen.
o Condenser: 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.
• The Light Source: Your microscope has an illuminator 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. You can also control the light intensity by
adjusting the voltage of a transformer attached to the illuminator. Use low or medium
voltage settings for most microscopic observations. You will need a higher setting when
using the oil-immersion lens.
o Some compound microscopes have an attached mirror instead of a built-in
illuminator. The mirror is usually concave on 1 side and flat on the other. The flat
side of the mirror is usually used with the scanning and low-power objectives. The
concave mirror is used with the higher power objectives. The light source for the
mirror is usually a lamp; natural light can be used but it is not preferred because
its intensity is too variable.

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o You can focus your microscope by using the coarse and fine adjustment knobs;
these raise or lower either the body tube or the stage, depending on the type of
microscope you are using. Try this with the low-power objective in position, about
1/4 inch above the stage. Rotate the coarse adjustment knob 1/4 turn clockwise
while watching the low-power objective. Do the same with the fine adjustment
knob.

USING COMPOUND MICROSCOPE:

Before using your microscope, thoroughly clean the oculars and objectives with lens
paper. Use a circular cleaning motion to avoid scratching the lens. When using the microscope,
keep your eyelashes from touching the ocular: because oil from your lashes will adhere to the
oculars and smear them. (Students should avoid wearing mascara in microscopy labs.)
Thoroughly clean the stage and microscope slides to prevent damage to the microscope.

Focusing

1. Clean the oculars and objectives using lens paper.

2. Cut out a letter "e" from a newspaper or other printed page. Clean a microscope slide and
prepare a wet mount of the letter, using the procedure described below.

3. Put the scanning (4x) objective in position, and then place the slide on the stage in its normal
viewing position.
4. Turn on the illuminator and open the diaphragm fully. If there is a condenser, position it as
high as it will go, so that the top lens of the condenser unit is level with the stage aperture.
Center the specimen over the stage aperture.
5. Position the scanning objective (4x) as close to the slide as possible; then, while looking
through the oculars, use the coarse adjustment knob to back off slowly until the specimen
comes into focus.
6. Use the diaphragm (and/or the transformer voltage regulator) to adjust the light intensity as
necessary, and again center the specimen by moving the slide.
7. Switch from the scanning lens (4x) to the low-power objective (10x). Make certain the objective
clicks into position. If the specimen stays in focus, your microscope is parfocal. Parfocal
means that the microscope has been designed in such a way that once the specimen is clearly
focused and centered it remains in focus while changing objectives, You can sharpen the
focus by small adjustments of the fine adjustment knob.

If your microscope is not in focus after changing objectives, you may have to use the
coarse adjustment knob and then the fine adjustment knob. But remember, do not do this
with the high-power or oil-immersion objectives in position. Ask your instructor for help if
you have difficulty focusing your microscope.

Recenter the specimen, adjust the diaphragm, and adjust the position of the condenser to
increase the contrast of the specimen.
8. Switch to the high-power objective (43x) and adjust the focus using the fine adjustment knob.

These procedures are usually used when examining a wet mount or a commercially
prepared microscope slide. Always use clean microscope slides. Always proceed from the
lowest power to the highest power objectives, making minor focus and light corrections
as necessary. Learn to fine-tune your microscope.

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MICROSCOPIC IMAGE

The image that you see in the microscope is affected by several factors: the orientation of
the image, the total magnification, the size and brightness of the field of vision, the plane of focus,
the depth of focus and the contrast of the specimen.

1. Orientation of the Image

Hold your slide of the letter "e" so that the letter is in a normal reading position. Then place
it on the stage in that position and examine it with the low-power objective. Move the image to the
right, left, upward, and downward, and notice the direction in which you have to move the slide.

When you want to point out something of interest in the field of vision to someone, you
can describe its approximate location by referring to the field of vision as a clock. Thus, you could
tell them to "look at 3 o'clock", or "look just off-center toward 3 o'clock", and so forth. Alternatively,
in some microscopes, a thin black line appears to cut across the field. This is a pointer that has
been added to the ocular of your microscope so that you can point out something by moving the
object under observation to the end of the pointer.

2. Brightness of the Field of Vision and Working Distance

Examine your slide, starting with the lowest power objective and progressing to the highest
power objective (do NOT use the oil-immersion objective). Note any changes in the brightness of
the field when you change objectives. When the object on your slide is in focus for each objective,
the distance between the slide and the objective lens, the working distance, decreases as the
objective magnification increases.

3. Depth of Focus
Like the human eye, the lenses of your microscope provide a limited depth of focus. This
means that only part of the object will be in sharp focus; areas above and below that plane will be
slightly out of focus or not in focus at all.

4. Magnification and Measurement

The approximate size of a specimen can be estimated by comparing it to the diameter of the
field of vision. The field of vision is what you can see when looking through the microscope.

5. Contrast
Even with sufficient magnification and resolution, you can visualize an object under a
microscope only if there is sufficient contrast between the object and its surroundings or between
the various parts of the object. You can improve image contrast by regulating the opening of the
diaphragm. This deflects the light rays from the edge of the diaphragm and causes them to enter
the specimen at an angle. Such scattering makes the specimen look darker.

Cells and sub cellular structures may contain natural pigments (e.g., chlorophyll in
chloroplasts and hemoglobin in red blood cells) that provide contrast and make these structures
visible. However, many cells and parts of cells are translucent. One way you can improve contrast
is to use dyes or stains that bind to or are taken up by various sub cellular structures, which then
absorb enough light to provide contrast.

PREPARING A SPECIMEN:

STUDY OF PROTOZOA AND ALGAE:

In your laboratory work with the microscope, many of your observations will be of living organisms
or tissues or parts of organisms that you will want to keep alive. To allow them to dry out greatly
distorts them, to say nothing of the effect death has on a study of their movements.

To observe living material, prepare a wet mount of a drop of water containing protozoa or algae
provided your instructor. (Since your specimen is already immersed in water, there is no need to
add tap water in making your wet mount. However, if your preparation begins to dry out while
under observation, add 1 drop of water at the edge of the cover slip.)

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Excess water under the cover slip can be soaked up by carefully touching a piece of tissue
(Kimwipe) to the edge of the cover slip.

Do not be too hasty in discarding a slide because you don't find any microorganisms; a systematic
survey of the preparation is often necessary to locate them. Try to identify the microorganisms
you observe, using books or by the materials in the notes section of the General Biology web
pages. To identify the smaller organisms, you may have to use the high-power objective. If the
organisms are moving too fast mix a drop of protoslow with your sample.

STUDY OF PLANT CELLS:

Prepare a wet mount of anion epidermal tissue, following the procedure demonstrated below.
Examine this tissue under low power objective.

The "lines" that form the network between the cells are non,living cell walls composed chiefly of
cellulose. The cell wall surrounds the plasma membrane, which encloses the cytoplasm. The
central part of many plant cells (which is difficult to observe in living cells) is taken up by a vacuole
that is filled with water and salts.

Next examine the cells under high power. Locate the nucleus, which appears as a dense structure
in the translucent cytoplasm. Note that in some cells, the nucleus looks circular and seems to be
lying in the central part of the cell. In other cells it seemes to be compressed and pushed against
the cell wall.

The central vacuole, nucleus, and cell wall are separated from the cytoplasm by membranes, but
the membranes are difficult to observe in the preparation that was used.

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STUDY OF CHEEK CELLS:

Follow the instructions below to examine epithelial cells obtained from the inner lining of your
cheek. First, try to determine something of their structure by adjusting the diaphragm and the
condenser. Next, add a drop of methylene blue stain to the edge of the cover slip and draw it
under as shown in figure below.