THE LIGHT MICROSCOPE
The function of the light microscope in the study of human
anatomy is to magnify structures that are too small to be
seen with the unaided eye. Thus, most structures at the
cellular and tissue level of organization require the use of a
light microscope. For study of sub-cellular structures, the
transmission electron microscope (TEM) is used. Orders of
magnitude greater magnification is possible using the TEM.
The microscope is one of the most important tools used by
biologists.
OBJECTIVES
•
Identification and function of light microscope
components; proper techniques for use of individual
components.
•
Use of the light microscope for study: how to focus on
an object; proper procedures for carrying and storage;
maintenance and care.
•
Properties of a light microscope: image inversion;
parfocal; total magnification and how the following
relate to this concept - diameter of field of vision,
working distance.
MICROSCOPE COMPONENTS
•
OCULAR LENS: also called the eyepiece; lens you look
through; magnifies object.
•
BODY/TUBE: mirrors or prisms inside; reflect/refract
light to form image at ocular lens.
•
ARM : connects body/tube to base; coarse and fine
adjustment knobs located near base.
•
NOSEPIECE: objective lenses attached; revolves for
specific objective lens use.
•
OBJECTIVE LENSES: attached to nosepiece; each lens
has its own magnification.
•
STAGE: flat surface under nosepiece/objective lenses
where object (slide) to be viewed is placed; center hole
with condenser and diaphragm below it.
•
MECHANICAL STAGE: a stage (see above) with a
spring-loaded caliper slide-holder and two knobs to
move slide-holder left/right, front/back.
•
CONDENSOR: underneath stage; often combined with
a diaphragm; concentrates light going to the stage
from the illuminator. There is a knob to adjust height
to the stage.
•
DIAPHRAGM: (DEYE-uh-fram) regulates the size of
opening that light passes through to condenser/hole
in stage; increases or decreases light to stage. There is
a small lever for adjustment.
•
COARSE ADJUSTMENT KNOB: larger knob located at
bottom of arm near base; turning the knob moves the
stage (or nosepiece on some microscopes) a large
distance up/down compared to fine adjustment knob.
•
FINE ADJUSTMENT KNOB: smaller knob in center of
coarse adjustment knob; moves the stage a small
distance up/down.
•
ADJUSTMENT KNOB LOCK: lever located on outside of
coarse adjustment knob; when engaged, inhibits
adjustment knobs from turning; locks stage in place.
•
ILLUMINATOR: light source located in base.
•
ILLUMINATOR POWER SWITCH: located on front, lower
part of base; turns light on and off.
•
RHEOSTAT: (REE-oh-stat) adjusts the current going to
the light; thus, brightness of the light source.
•
BASE: platform on which all other components rest;
connects to lower arm; illuminator, on/off switch,
rheostat, and power cord associated with base.
•
POWER CORD: located at back of base; plugs into
electrical outlet; transfers electricity to microscope.
MICROSCOPE TRANSPORT: CABINET - TABLE
•
Form an orderly line at the cabinets. If everyone
crowds together, there is a greater likelihood that the
microscope or person holding it will be bumped. YOU
DO NOT WANT TO DROP THE MICROSCOPE. They are
expensive!!
• Grab the microscope arm with one hand; carefully lift it
off the shelf, making sure you do not hit the ocular lens
on the shelf above. Immediately place your other hand
under the base, cradling the microscope with two
hands.
•
Carry the microscope in an upright position with both
your hands. The ocular lens has no set screw, so it can
easily slide out if the microscope is not carried in this
position.
•
Carry the microscope so it is oriented with the front-toback of the base cradled against your body. Carry it as
close to your body as possible (it won’t bite you!).
•
When you get to your lab table GENTLY set the
microscope down so the ocular lens is facing you. If
you need to adjust its position, do not try to slide it. The
rubber feet grip the table, and will provide a “bumpy
ride” for the microscope. Ultimately, this results in
body prism, condenser and other component
misalignment.
• Treat the microscope with respect; it is a delicate and
expensive piece of equipment.
TURNING ON THE MICROSCOPE
•
If lenses are dirty, use only lens paper to clean them.
Kimwipes® is not lens paper, nor are paper towels, or your
shirt.
•
Before unwrapping the power cord from the lower arm
and plugging it into the electrical receptacle, make sure
the microscope had been stored properly.
o The lowest power objective lens should be in place
(or the cap with no objective lens).
o
The stage should be at its lowest position with
condenser and diaphragm at their highest position
as close to the stage as possible. Always leave the
condenser and diaphragm in that position.
o
The power switch is in the off position. Most
importantly check that the rheostat is at its lowest,
either 0 or 1; if not, turn it to that position.
o
Plug the cord into the receptacle located at the front
or back of the table. Turn the power switch on.
Slowly (reasonable rate) turn the rheostat to 7. You
should need more current than that.
o
NOTE: If your illuminator does not produce light after
doing the previous steps, check the floor to see if
the main power cord to the table is properly plugged
in. Because of their location on the floor, they often
get kicked and dislodged from the receptacle.
MAGNIFICATION
•
The magnification at which you view an object on the
stage is determined by the magnifying power of the
ocular lens and the magnifying power of the objective
lens you have in place:
TOTAL MAGNIFICATION = OCULAR MAG. X OBJECTIVE MAG.
•
The magnifying power of the ocular lens is always 10X
with our microscopes (or: 10 times the object’s normal
size if you viewed it through that lens only); it is usually
inscribed on the top of the lens.
•
The magnifying power of the objective lenses, if you were
to view the object through that lens only, is engraved on
the lens casing. There is also a colored stripe on the
casing; usually standardized for newer microscopes: red
= 4X; yellow = 10X; blue = 40X. Notice the length of each
objective lens casing. Which lens is shortest? Which lens
is longest?
MICROSCOPE USE – FOCUSING PROCEDURE
•
After turning on the microscope as described above, open
the diaphragm fully; look under the stage to find the
lever; NEVER tip the microscope to do this. Alternatively,
you can feel for it without having to look.
• Put the 4X objective in place if stored with the blank.
Make sure you feel a solid “click”. This tells you the lens is
firmly in place.
•
NOTE: NEVER GRAB THE OBJECTIVE LENS TO ROTATE IT
INTO PLACE. REVOLVE THE NOSEPIECE! The objective
lenses screw into the nosepiece; continually grabbing the
lens will begin to loosen them, resulting in incorrect
optical adjustment. Additionally, it increases the chance
you will smudge and dirty the glass lens.
• Start by using a prepared slide of the letter “e”.
•
Place a slide in the mechanical stage slide holder so that
it is secured with the caliper and centered in the stage
opening. Place the slide label to your left, with the
coverslip facing upward; do not place the coverslip so that
is on the stage
•
NOTE: Release the caliper slowly; never release it
immediately so it slams into the edge of the slide. It will
chip the slide corner. You can see evidence of slide
damage resulting from previous students releasing the
caliper all at once.
•
Take one slide at a time to your table. Do not grab a
handful, or ever stack prepared slides; this will eventually
damage them. You should have only one slide, anyway.
• Rotate the coarse adjustment knob so the stage is as
high as it can go.
•
Look through the ocular lens. Most of the microscopes
have a single ocular lens; hence, they are called
monocular microscopes (mono- =one). There are a few
microscopes with two ocular lenses; they are referred to
as binocular microscopes (bi- = two). The following
procedure will refer to monocular microscopes.
•
While looking through the ocular lens, keep both eyes
open. While it will seem awkward at first, and will take
some practice, this method reduces eye fatigue when
viewing slides for long periods of time.
TIP: Initially, try placing a hand over the non-viewing eye
while still keeping it open. You will eventually become
more comfortable with keeping both eyes open.
•
First, check that the adjustment knob lock is released.
Bring the “e” into focus using the coarse adjustment
knob. Always focus by moving the stage downward.
•
Next, use the fine adjustment knob to bring the “e” into
sharp focus.
• Adjust the amount of light going to the stage by using the
diaphragm.
TIP: The most common mistake when learning to use the
microscope is allowing too much light to reach the stage.
Decreasing the light by adjusting the diaphragm, results
in better contrast. The result: you can see many more
structures and finer details.
Rotate the mechanical stage knob so that the slide physically
moves to the right. When viewing the “e”through the ocular
lens, in which direction does it moves? Look at the orientation
of the “e” on the slide with your unaided eye. Look through the
ocular lens. How does the “e”look in comparison?
• Re-center the “e” to the middle of the stage opening.
•
Revolve the nosepiece so that the next higher power
objective (10X) is firmly in place without changing the
height of the stage.
• Now, you should only use the fine adjustment knob to
provide sharp focus. If you need to use the coarse
adjustment knob, you did not focus properly using the 4X
objective.
•
These are PARFOCAL microscopes. The term parfocal
means that once you have focused properly with one
objective lens, the next objective lens you use should
already have the object in focus (or at least good enough
so that you only need to use the fine adjustment).
• Adjust the diaphragm.
•
Slowly and carefully revolve the nosepiece so that the
40X objective lens is in place without changing the
height of the stage. It may appear as if the 40X objective
lens will hit the slide. You should be careful. If you have
properly focused at the prior steps, it will be close to the
slide, but should not hit it.
WORKING DISTANCE = THE DISTANCE BETWEEN THE SLIDE
AND THE OBJECTIVE LENS. WORKING DISTANCE
DECREASES AS OBJECTIVE LENS MAGNIFYING POWER
INCREASES.
• Only use the fine adjustment for sharp focus.
• Adjust the diaphragm.
You may have also noticed that the image “disappeared” as
you viewed it under higher total magnification. This is
because the DIAMETER OF FIELD OF VIEW (= the length
across the circular view you see while looking through the
ocular lens) decreases as the objective lens magnifying
power increases, if using the same ocular lens.
The relationship is important to remember; always center
the object you are looking at in your diameter of field of
view as you proceed to higher total magnification. With the
“e”,your diameter of field of view may have fallen within
the center area of the letter that lacked ink.
IMPORTANT - REMEMBER WHEN FOCUSING:
1.
ALWAYS BEGIN FOCUSING USING THE 4X OBJECTIVE.
EVEN IF YOU WANT TO VIEW THE OBJECT AT HIGHEST
MAGNIFICATION, YOU MUST PROCEED: 4X; 10X; 40X.
2.
THE MICROSCOPES ARE PARFOCAL; USE ONLY FINE
ADJUSTMENT TO FOCUS WHEN USING 10X; 40X
OBJECTIVE LENSES.
3. DO NOT CHANGE STAGE HEIGHT WHEN SWITCHING TO
HIGHER POWER OBJECTIVE LENSES - PARFOCAL.
4. ALWAYS CENTER IMAGE BEFORE PROCEEDING TO THE
NEXT OBJECTIVE LENS.
5.
ADJUST LIGHT TO THE STAGE USING DIAPHRAGM AT
EACH MAGNIFICATION.
STORING THE MICROSCOPE
• Remove slide from the stage. Replace it in its proper
orientation and in the proper slide folder.
•
Revolve the nosepiece so the 4X objective lens or blank is
secured in place.
• Slowly turn the rheostat to its lowest number.
• Turn off the illuminator switch.
•
Unplug power cord at receptacle. DO NOT pull the cord
itself.
• Wind cord around bottom of the arm, by base.
• Use coarse adjustment knob to lower stage as low as
possible.
•
Place dust cover over the microscope.
•
Carry the microscope as described previously; gently put
scope on the shelf, matching shelf# with microscope#.