DOROTHY D.

SILVA
SCHOOL OF TEACHER EDUCATION
SAINT LOUIS UNIVERSITY
Purposes

1. To become familiar with the history and

diversity of microscope instruments.

2. To understand the components, use, and care

of the compound microscope.

3. To learn the correct use of the microscope for

observation and measurement of microorganisms.
A microscope (Greek: micron = small and scopos = aim)
MICROSCOPE - An instrument for viewing objects that
are too small to be seen by the naked or unaided eye
History:
1665 - Robert Hooke discovered
and described the fundamental
unit of all living things (cells) by
examining thin slices of cork

UNSWFYBIO/2008JK
1674 - The first man to
witness a live cell under a
microscope was Anton Van
Leeuwenhoek, describing
the algae Spirogyra and
named the moving
organisms animalcules,
meaning "little animals"
 This instrument contains two lens systems for magnifying
specimens: the ocular lens in the eyepiece and the objective
lens located in the nose-piece. The specimen is illuminated by
a beam of tungsten light focused on it by a sub-stage lens
called a condenser, and the result is that the specimen
appears dark against a bright background. A major limitation
of this system is the absence of contrast between the
specimen and the surrounding medium, which makes it
difficult to observe living cells. Therefore, most brightfield
observations are performed on nonviable, stained
preparations.
 This is similar to the ordinary light microscope; however, the
condenser system is modified so that the specimen is not
illuminated directly. The con-denser directs the light
obliquely so that the light is deflected or scattered from the
specimen, which then appears bright against a dark
background. Living specimens may be observed more readily
with darkfield than with brightfield microscopy.
 Observation of microorganisms in an unstained state is
possible with this microscope. Its optics include special
objectives and a condenser that make visible cellular
components that differ only slightly in their refractive
indexes. As light is transmitted through a specimen with a
refractive index different from that of the surrounding
medium, a portion of the light is refracted (bent) due to slight
variations in density and thickness of the cellular
components. The special optics convert the difference
between transmitted light and refracted rays, resulting in a
significant variation in the intensity of light and thereby
producing a discernible image of the structure under study.
The image appears dark against a light background.
 This microscope is used most frequently to visualize specimens
that are chemically tagged with a fluorescent dye. The source of
illumination is an ultraviolet (UV) light obtained from a high-
pressure mercury lamp or hydrogen quartz lamp. The ocular lens is
fitted with a filter that permits the longer ultraviolet wavelengths
to pass, while the shorter wavelengths are blocked or eliminated.
Ultraviolet radiations are absorbed by the fluorescent label and the
energy is re-emitted in the form of a different wavelength in the
visible light range. The fluorescent dyes absorb at wavelengths
between 230 and 350 nanometers (nm) and emit orange, yellow,
or greenish light. This microscope is used primarily for the
detection of antigen-antibody reactions. Antibodies are conjugated
with a fluorescent dye that becomes excited in the presence of
ultraviolet light, and the fluorescent portion of the dye becomes
visible against a black background.
 This instrument provides a revolutionary method of microscopy, with
magnifications up to one million. This permits visualization of
submicroscopic cellular particles as well as viral agents. In the electron
microscope, the specimen is illuminated by a beam of electrons rather
than light, and the focusing is carried out by electromagnets instead of a
set of optics. These components are sealed in a tube in which a complete
vacuum is established. Transmission electron microscopes require
specimens that are thinly prepared, fixed, and dehydrated for the
electron beam to pass freely through them. As the electrons pass through
the specimen, images are formed by directing the electrons onto
photographic film, thus making internal cellular structures visible.
Scanning electron microscopes are used for visualizing surface
characteristics rather than intracellular structures A narrow beam of
electrons scans back and forth, producing a three-dimensional image as
the electrons are reflected off the specimen's surface.
Stage
 allows for the passage of light from an illuminating source below to the
lens system above the stage; provides a surface for the placement of a
slide with its specimen over the central opening
Illumination
 The light source is positioned in the base of the instrument. Some
microscopes are equipped with a built-in light source to pro-vide direct
illumination. Others are provided with a mirror; one side flat and the
other concave.
 An external light source, such as a lamp, is placed in front of the mirror to
direct the light upward into the lens system. The flat side of the mirror is
used for artificial light, and the concave side for sunlight.
Abbe Condenser
 collect and concentrate light passing upward from the light source into
the lens systems; equipped with an iris diaphragm, a shutter controlled by
a lever that is used to regulate the amount of light entering the lens
system.

Body Tube
 houses the lens system that magnifies the specimen. The upper end of
the tube contains the ocular or eyepiece lens. The lower portion consists
of a movable nosepiece containing the objective lenses. Rotation of the
nosepiece positions objectives above the stage opening. The body tube
may be raised or lowered with the aid of coarse-adjustment and fine-
adjustment knobs that are located above or below the stage, depending
on the type and make of the instrument.
 MAGNIFICATION

 Degree of enlargement
 No of times the length, breadth or diameter of an object is
multiplied.

30
Resolution is the minimum distance
apart that 2 objects can be in order for
them to appear as separate items.
The greater the resolution the clearer
the image.
d = 1.22
 
Where d is the resolving power, λ is the
Resolution
wavelength, NA is the numerical
aperture.
NA = n sinu
 N = the lowest refractive index between the
object and first objective element
 u is 1/2 the angular aperture of the objective
LIMIT OF RESOLUTION (LR) – The minimum distance between
two visible bodies at which they can be seen as separate and
not in contact with each other
Types of microscope Resolving power

Compound Microscope 200 nanometers

Scanning Electron 10 nanometers

Microscope

Transmission Electron 0.2 nanometers

Microscope
Estimating Specimen Size

 The area of the slide that you see when you look through a
microscope is called the "Field of View". If you know how wide
your field of view is, you can estimate the size of things you see
in the field of view. Figuring out the width of the field of view is
easy --- all you need is a thin metric ruler.
 By carefully placing a thin metric ruler on the stage (where a
slide would usually go) and focusing under low power, we can
measure the field of view in millimeters. Through the
microscope it would look something like what you see here on
the left. The total width of the field of view in this example is
less than 1.5 mm. A fair estimate would be 1.3 or 1.4 mm.
(Relax, it's an estimate).
Estimating Specimen Size

 Now millimeters is a nice metric unit, but when we use a

MICROscope we tend to use MICROmeters. To convert from
millimeters to micrometers, move the decimal 3 places to the
right. Our 1.3 mm estimate becomes 1300 micrometers.
 Now we can get the ruler out of the way, prepare a slide, focus,
and estimate the size of things we see !
 For example, if something we were looking at took up half of
the field of view, its size would be approximately 1/2 x 1300
micrometers = 650 micrometers. If something appeared to be
1/5 of the field of view, we would estimate its size to be 1/5 x
1300 = 260 micrometers.
Calculating Specimen Size

Because the high power objective is so close to the

stage, we can't measure the width of the field of view
under high power directly. The ruler just doesn't fit
between the objective & the stage. No problem. We
can use the width of the field of view under low
power (which we measure using the steps above) and
the relationship between the low & high
power magnifications to mathematically calculate the
width of the field of view under high power.
Calculating Specimen Size

First of all memorize this :

When switching from low to high power, the area in
the field of view gets smaller & darker. (You see a
smaller area of the slide under high power.) This is
why centering what you want to see prior to switching
to high power is so important.
The fraction of the area seen under high power is the
same as the ratio of the low & high power
magnifications.
For example : if the low power objective is 20x and
the high power objective is 40x, then under high
power we will see 20/40 or 1/2 of the area of the slide
we saw under low power.
 ocular power = 10x
low power objective = 20x
high power objective = 50x

a) What is the highest magnification you

could get using this microscope ?

b) If the diameter of the low power field is 2

mm, what is the diameter of the high power
field of view in mm? in micrometers ?

c) If 10 cells can fit end to end in the low

power field of view, how many of those cells
would you see under high power ?
 ocular power = 10x
low power objective = 20x
high power objective = 50x

a) What is the highest magnification you could get using this

microscope ? 500x
Ocular x high power = 10 x 50 = 500. (We can only use 2 lenses
at a time, not all three.)
b) If the diameter of the low power field is 2 mm, what is the
diameter of the high power field of view in mm ? .8 mm
The ratio of low to high power is 20/50.
So at high power you will see 2/5 of the low power field of view
(2 mm). 2/5 x 2 = 4/5 = .8 mm
in micrometers ? 800 micrometers
To convert mm to micrometers, move the decimal 3 places to
the right (multiply by 1000). .8 mm x 1000 = 800 micrometers

c) If 10 cells can fit end to end in the low power field of view,
how many of those cells would you see under high power ?
4 cells.
We can answer this question the same way we go about "b"
above. At high power we would see 2/5 of the low field. 2/5 x
10 cells = 4 cells would be seen under high power.
 ocular power = 10x
low power objective = 10x
high power objective = 40x

a) What is the approximate width of the

field of view in micrometers ?

b) What would be the width of the field of

view under high power ?

c) If 5 cells fit across the high power field of

view, what is the approximate size of each
cell ?
 ocular power = 10x
low power objective = 10x
high power objective = 40x

a) What is the approximate width of the field of view in

micrometers ? 3500 - 3800 micrometers

Each white space is 1 mm. We can see approximately 3 1/2 (or

so) white spaces. That is equivalent to 3.5 mm, which converts
to 3500 micrometers. Any answer in the range above would be
OK.
b) What would be the width of the field of view under high
power ?
875 micrometers
The ratio of low to high power for this microscope is 10/40 or
1/4. So, under high power we will see 1/4 of the low power field
of view. 1/4 x 3500 micrometers (from "a" above) = 875
micrometers.

c) If 5 cells fit across the high power field of view, what is the
approximate size of each cell ?
175 micrometers
If 5 cells fit in the high power field of view (which we determined
is 875 micrometers in "b"), then the size of 1 cell = 875/5 = 175
micrometers.
ocular = 10x
low power objective = 20x
high power objective = 40x

a) What is the approximate size of the

cell in micrometers ?

b) What would be the high power field

of view ?

c) How many cells like the one in the

picture could fit in the high power field
of view ?
 ocular = 10x
low power objective = 20x
high power objective = 40x

a) What is the approximate size of the cell in micrometers ?

500 micrometers
First, we have to visualize how many of those cells could fit across
the field --- about 4. So 2 mm (the width of the field) / 4 = .5 mm,
which converts to 500 micrometers.
b) What would be the high power field of view ?
1000 micrometers
The ratio of low to high power for this scope is 20/40, or 1/2. So
we will see 1/2 of the low power field under high power. 1/2 x 2
mm = 1mm, which converts to 1000 micrometers.

c) How many cells like the one in the picture could fit in the high
power field of view ?
2 cells
Again the ratio of low to high power is 20/40, or 1/2. If we can see
4 cells across the low field of view we will see 1/2 as many in the
high field of view. 1/2 x 4 = 2 cells.
 1.Remove all unnecessary materials such as books, papers, purses, and
hats from the laboratory bench.
 2.Uncoil the microscope's electric wire and plug it into an electrical
outlet.
 3.Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will
decrease the efficiency of the microscope. The ocular; scanning, low-
power, and high-power lenses may be cleaned by wiping several times
with acceptable lens tissue. Never use paper toweling or cloth on a lens
surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper
moistened with methanol is used to wipe it clean. If the lens is very dirty
it may be cleaned with xylol however the xyol cleansing procedure should
be performed only by the instructor, and only if necessary. Consistent use
of xylol may loosen the lens.
 1. Place the microscope slide with the specimen within the
stage clips on the fixed stage. Move the slide to center the
specimen over the opening in the stage directly over the light
source.
 2. Rotate the scanning lens or the low power lens into
position. While watching from the side to insure that the lens
doesn't touch the specimen, turn the coarse focus knob to
move the stage as close as it can get to the lens without
touching the lens. (Always watch from the side whenever you
move a specimen towards any objective lens to make sure
the lens doesn't crash through the specimen and get
damaged!)
 3. Now, while looking through the ocular lens, turn the coarse focus knob
carefully, and slowly move the stage away from the lens until the specimen comes
into vague focus. Then, use the fine focus knob to bring the specimen into sharp
focus.

 4. If this is the first specimen of the day, you should Kohler your microscope at this
point (while it is in focus). Otherwise, if your microscope has already been
Kohlered you won't need to do it again

 5. Routinely adjust the light source by means of the light source transformer
setting, and/or the iris diaphragm, for optimum illumination for each new slide
and for each change in magnification.

 6. Our microscopes are parfocal, which means that when one lens is in focus,
other lenses will also have the same focal length and can be rotated into position
without further major adjustment. In practice, however; usually a half-turn of the
fine-adjustment knob in either direction is necessary for sharp focus.
 7. Once you have brought the specimen into sharp focus with a low-
powered lens, preparation may be made for visualizing the specimen
under oil immersion. Place a drop of oil on the slide directly over the area
to be viewed. Rotate the nosepiece until the oil-immersion objective locks
into position. Care should be taken not to allow the high-power objective
to touch the drop of oil. The slide is observed from the side as the
objective is rotated slowly into position. This will ensure that the
objective will be properly immersed in the oil. The fine-adjustment knob
is readjusted to bring the image into sharp focus.

 8. During microscopic examination of microbial organisms, it is always

necessary to observe several areas of the preparation. This is
accomplished by scanning the slide with-out the application of additional
immersion oil. This will require continuous, very fine adjustments by the
slow, back-and-forth rotation of the fine adjustment knob only.
1.Clean all lenses with dry, clean lens paper. If you need to, you can use a
drop or two of methanol to help clean the lens. Use xylol to remove oil
from the stage only.

2. Place the low-power objective in position and bring the stage and
objectives close 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 in the manner

previously described.
 Lubey, Steve. Lubey's Biohelp! – Using the
Microscope. Aug. 26, 2005
http://www.borg.com/~lubehawk/mscope.htm
 Microscope image. http://www.hc-sc.gc.ca/ewh-
semt/contaminants/person/impact/index_e.html
 Microscopy.
http://www2.hawaii.edu/~johnb/micro/m140/sylla
bus/week/handouts/m140.2.4.html
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