I. Practical Task
The Light Microscope
The cells, the microorganisms and the fine tissue structures are tiny enough (with rear
exception) to see them with bare eyes. The light microscope is such optical instrument
which helps the examination and the characterization of the cells and tissues. The
“microscope” naming comes from Greek language, composed from the words:
“micros” (tiny) and “skopeo” looking. By the help of light microscope not only the
eukaryotic (7-30 µ m) and the prokaryotic (1-8 µ m) cells, but even the lager cell
organelles can be observed.
The most important terms of the microscope and the image creation:
Light:
multiplane waves spreading in space and consist of elementary
particles (photons)
Monochromatic light: light ray possessing one single wavelength
Complex light:
mixture of light rays with more different wavelengths. The
wavelengths range of visible light: 400-700 nm.
Characteristics of light waves:
Wavelength: The distance between two waves in the same phase (?, expressed in nm).
Frequency: The oscillation within a minute.
Amplitude: The distance between the maximums of waves being in positive and
negative phase.
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B
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Figure 1. The build-up of a complex light microscope
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1. Construction of the microscope
The main parts of the light microscope are the following (Fig. 1.):
Mechanical parts:
1. Stand
2. Slide tray
3. Tube
Parts for mechanical adjustment
4. Macrometer screw
5. Micrometer screw
The optical parts:
The parts for object illumination
6. Light source
7. Diaphragm
8. Condenser
9. Color filter
The parts of the optical magnification system
A. Ocular lens
B. (Objective revolver)
C. Objective lens
2. The basic principles of microscope operation
Incorporated in a metal tube, the microscope has two separate lens systems, sharing a
common optical axe (Fig. 2.). The lens close to the examined specimen (object) is the
objective lens, the lens close to our eyes is the ocular lens. The light is reflected to the
specimen on the slide tray through an adjustable mirror, a light filter and condenser lens
system. The objective first creates a real, reversed and magnified image of the object.
This image is magnified further with the objective. The final result is a magnified
virtual image with the same orientation as the object.
ocular
prism
objective
object
condenser
lightsource
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Figure 2. Light pathway in the microscope
The magnification of the microscope
Beams from the object go through the objective and the ocular lens, creating a
magnified image of the object that depends on the lens magnification. The
magnification of the microscope (Nm) can be obtained by the multiplication of the
magnifications of the objective (Nob) and the ocular (Noc) lens.
Nm = Mob · Moc
In a binocular microscope the incorporated prism extends the light route. In order to get
the final magnification the obtained (Nm) value must be multiplied with the prism factor of
the intercalated prism (engraved on the tube).
The objective lens has 100x and the ocular lens has 25x of maximal magnification, whereas
(in principle) the magnification of the microscope is 2500x. However in practice, because of
the resolution limitation of the microscope, larger magnification, than 1500x cannot be
applied. Over than 1000-1500 times of magnification, the details of the image is impossible
to enhance: only the separated points of the image become larger and the number of the
distinct points does not grow. For that reason the applicability of the microscope is limited by
the resolution, not by the magnification.
The resolution of the microscope:
The resolution ( ) of a microscope at optimal exposure:
wavelength
( )=
,
A numeric aperture
where A = n·sin?. The letter n is the refraction index of the media between the cover
slip and the objective (air n=1, distilled water n=1.33, cedar oil n=1.51). ? labels the angle
closed by the main optical axe and the outermost light beam (half angle of the objective on
Fig. 3.) The resolution limit of a microscope means the distance between two object points,
at which still they can be observed as distinct points. The resolution of our optical equipment
is better as closer points can be seen as separate ones. In order to understand the resolution,
Abbe introduced the term of numeric aperture. From the equation shown above, there are
two different way to increase the resolution.
(i)
Reduction of the numerator, i.e. application of light beam with a shorter
wavelength. With UV light the resolution can be reduced to 0.1 µm, but special
quartz lenses and UV-light detector are needed, therefore the light microscope
with UV light source is only a principal possibility.
(ii)
To increase the value of the numeric aperture; in a light microscope visible light
source is applied. The value cannot be reduced below 400 nm, because, the
resolution of the microscope can only be improved by changing the value of the
numeric aperture. Using cedar oil the value of numeric aperture is 1.43 (sin72°=
0.95; than 0.95x1.51= 1.43, thus the limit of resolution is 0.28). In case of the
„dry” objectives, there is air between the object and the front objective. In this
case the value of numeric aperture is less then 1, since the principal maximum of
the sin? is=1. The half angle of a lens can be increased only until 72°, since at
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lager angle than this the light beams became totally reflected. The refractive
index of the air is 0.95, therefore numeric aperture of the dry objective with the
best resolution is 0.95 (the value of the numeric aperture is engraved on every
objective; see Figure 5.)
objective
eeeeee
object
Figure 3. Half angle of the objective
The resolution of the microscope can be enhanced by dropping a solution with higher
refractive index between the front lens and the cover slip. Those lenses (mainly objectives
with 100x magnification) are named as immersion objectives. The immersion liquid
mentioned above is cedar oil, thus these lenses are objectives with oil immersion (they are
labeled with HI). For the WI labeled objectives distilled water is the liquid which should be
used. The name of homogenous immersion means that the refractive index of the immersion
liquid is the same as the refraction of the objective and the cover slip, i.e. after leaving the
object, the light passes through an optically homogenous media (Fig. 4).
immersion
objective
dry objective
frontal lens
immersion liquid
cover slip
slide glass
Figure 4. Light path in dry objective (left side) and in immersion objective (right side)
Usually there are four objectives with different magnification in the objective revolver of the
light microscope: the 10-times, 20-times, 40-times and 100-times magnification. (Fig. 1.).
By rotating the objective revolver, the objectives and in parallel can the magnification and the
resolution can be changed easily. Except the product number, there are three numbers on
small magnifying and four numbers on larger magnifying objectives: e.g. 40/0.65 and
below 160/0.17 (Fig. 5.). The first number (40) shows the magnification of the objectives, the
second (0.65) indicates the numeric aperture. The third number (160) gives the necessary
tube length, the four (0.17) shows the largest thickness of the cover slip. The last number is
shown only on larger magnifying objectives, where between the objective and the object the
distance is small.
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label of color correction
immersion labeling
value of numerical apperture
suggested thickness of cover slip
magnification of the
objective
phasecontrast labeling
length of the tube (mm)
Immersion stripe
frontal lense of objective
Figure 5. Labels on the objective
3. Handling the microscope
The microscope should be positioned on desktop so, that the examination could be continued
conveniently for an extended period. The distance between the two tubes of the binocular
microscope can be adjusted suitably to eyes’ pupils distance. Finally one clear field of vision
will be seen. The adjustment of the microscope starts with the projection of the light from its
source into the optical axe of the microscope. Care should be taken, that the field of vision
should enlightened equally, and must not glare the observer. A object should be placed on the
slide tray, than is must be fastened by clips. In examination of the permanent specimens, the
cover slip must be in top position. First, the object can be searched by adjustment of
macrometer screw, then sharpen the view with the micrometer screw.
Important: During lowering the objective the rough adjustment should be done by watching
the object from aside. Then raise the objective until the sharp picture of the object can be
seen. This precaution should be mainly kept at the usage of objective with larger
magnification, because the slide with the specimens made by tiresome work could easily be
broken by objectives (needless to say that a small object can more easily be found by smaller
magnifying objective!). First raise the objective with the macrometer screw, then do the fine
adjustment with the micrometer screw (the micrometer screw should not be turned more than
the maximal 180 degree). By rotation of the revolver an objective with larger magnification
could be adjusted to the optical axe of the light microscope. The contrast can be enhanced by
lowering the condenser lens and by narrowing the diaphragm. The microscope can be easily
set up with the application of the Köhler’s basic exposure principles: (i) the object should
be reached by beams in right angle. (ii) The vision field should be exposed equally (iii) The
light beams that do not contribute to image creation should be excluded (iv) Set the
condenser to a position, that the picture of the diaphragm should be sharp.
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4. Cleaning and storage of the microscope
Before and after use, please clean the microscope lenses and the accessories with a soft cloth
or with fine deerskin. The occasional fingerprints or grease on the ocular lens must also be
wiped with soft cloth. The dye and Canada balsam contamination can be cleaned by a fast
evaporating solvent (alcohol, ether, benzene). Protect the lenses from dust contamination.
5. Objects that disturb the microscopic examination
The air bubbles are the most frequent contaminants in the specimens. The air bubble round
shaped object with thick shiny wall (The smaller bubbles have thicker wall). When the slide
glasses are cleaned with soft tissue, fine, invisible filament may stay behind as contaminants
on the glass surface. The main rule: as cleaner materials we work as more enjoyment we shall
have during the work!
6. Practical tasks
6. 1. Microscope handling
Please, identify the components of the microscope by looking Fig. 1, learn and practice, how
to use them.
6.2. Identification of contaminants
Please, put a drop of water on a slide and cover with a cover slip so, that an air bubble should
remain under the cover. Examine the image of the air bubble, distinguish them from cells
(e.g. red blood cells). Examine the elementary filaments of cotton, wools and synthetic cotton
and synthetic fibers.
6.3. Microscopic measurements: measuring the distance with ocular micrometer
The measurement in microscope can be done by applying a tool, called as the ocular
micrometer. The ocular micrometer is glass disk with a fine scale, that fits into the ocular lens
holder of the microscope (Fig, 6.). In the microscope clear and magnified picture of the fine
scale on the objective micrometer can be seen. The units of the scale must be calibrated with
an objective micrometer for each of the objectives. The objective micrometer is a special
slide where 1 mm distance divided up precisely to 100 unit. Thus, the distance between two
neighborhood lines are 10 µm (Fig. 6.). The ocular micrometer has to be calibrated in the
following way: Place the objective micrometer onto the slide tray and focus it. Turn the
ocular micrometer so, that it should be in parallel with the scale of the objective micrometer.
(Fig, 7.). From the overlaps of the two scale define that how many of the objective units are
equal with how many of the ocular units. By establishing a proportion the unit size (in µm)
of the ocular micrometer can be defined. Before measurement, the ocular micrometer must be
calibrated at every objectives.
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Figure 6. The objective (left side) and the ocular micrometer (right side)
hair
scale of objective micrometer
scale of
ocular
micrometer
scale of ocular micrometer
Figure 7. The calibration of the ocular micrometer scale with the objective micrometer scale
Task
Prepare a smear from sheep red blood cell on a slide. After finishing the careful calibration of
the ocular micrometer, put the dried smear onto the slide tray. First at 200x of magnification
adjust a sharp focus of the smear, and measure the diameter of a sheep red blood cell. Repeat
this at 400x of magnification.
Measure the thickness of a hair.
6.4. Determining the resolution
For one of the objectives define the resolution limit by using the equation above,
(supposing, that the = 500nm).
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6.5. Finding a specific point of your object
There are two scales, placed rectangular of each other on the slide tray of a modern
microscope. These scales are to help you to retrieve an object part in view field. Adjust a
given point of a specimen and then record the scale values. Take out the slide, than put it
back on the slide tray. Adjust both scale to previous registered values. If you worked
precisely than you will see your desired object point in the view field.
6.6. Examination of cell organelles with light microscope
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Examination of the nucleus
- Prepare epidermis layer of the onion squama. Drop water on it and cover with a
coverslide. Examine the specimen with microscope and make notes about what you see.
Drip 10% acetic acid to the edge of the coverslide and intake the fluid under the
coverslide with filter paper. Watch the fixative effect of the acetic acid and how the nuclei
appear.
- Stain epidermis layer of the onion squama with methyl-green. Treat the epidermis cells in
the staining dishes by the following way: Fix them in Carnoy fixative liquid for 20 min.
(60 ml alcohol, 30 ml chloroform and 10 ml glacial acetic acid. Wash in tap water for 1-2
min and satin with methyl-green for 10 min (composed from: 0.5% methyl-green in 0.1
M acetic buffer, pH=4). Wash it again, then rinse it to 50% alcohol for 5 min. Cover it,
then examine the stained specimen. Examine it at an adequate magnification. Examine the
nuclei in the stained specimen. Check the images of nucleus. Solve the tasks of the
cytological album and write the answer in your practical exercise book.
- Measure 4-5 onion cells and their nuclei, determine the average size.
Figure 8. The Buerker chamber
Figure. 9. The scale of the Buerker chamber. The gap between the cover slip an the slide is
0.1 mm.
6.7. Cell counting with Buerker chamber
We can count the cell number in a suspension with light microscope. For the cell counting we
need a specially scaled slide, like the Buerker chamber (Fig. 8)
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- Put 2-3 glass tube in the rack. Pipette with an automatic pipette 990 µl PBS (Phosphate
buffer) into the first tube, and 900-900 µl into the next ones. Prepare a serial dilution: Shake
up the sheep red blood cell (SRBC) suspension and add 10 µl to the first tube. Mix it and
transfer 100 µl into the next tube. After a profound mixing of the diluted SRBC suspension,
drip a small amount under the Buerker chamber coverslide (If the cell suspension show to
high density in the microscope then continue the dilution in a further tube). Depending on the
cell density, count the cells in the 25 squares, or in 10 rectangles of the Buerker chamber
(Fig. 9.) Depending on the dilution rate determine cell number/ml in the original from the
obtained average value.
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