Download Lab 2 The Microscope

Lab 3: The Microscope
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With the development and modernization of light optics and the microscope along with
increasing availability of these instruments to biologists over the next 200 years it
became clear that living organisms had a basic unit - the cell.
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In the mid 1800’s the cell theory was developed (Schleiden & Schwann), which simply
states that all living organisms are composed of cells. As the idea of spontaneous
generation (life arising from non-life) was disproved, the cell theory was expanded to
include the latin phrase omnis cellule e cellula which means that cells arise only from
pre-existing cells.
Theory:
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In describing the theory of microscopy and image formation there are three important
concepts that must be understood - resolution, magnification and contrast.
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Resolving Power/Resolution - resolution is defined as the ability to distinguish fine
details. A better definition is the ability to distinguish 2 points as two separate and
distinct points!
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The human eye is quite limited in resolving power. One reason is due to the fact that you
can only focus on an object a certain distance from the eye - this is known as the near
point. To bring an object closer to the eye with proper focus you could use a simple
microscope/magnifying glass. The resolving power of the naked human eye is
approximately 0.2mm.
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Using a modern compound light microscope (LM), the resolving power increases to
0.2µm (down at the level of bacterial cells). This represents a 1,000X increase in
resolving power over the naked eye.
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A transmission (meaning that the electron beam passes through the specimen) electron
microscope (TEM) has a resolving power of about 2.0Å (at the molecular/atomic level).
The TEM resolving power is 1,000X greater than the LM, making it 1,000,000X better at
resolving fine details than our naked human eyes.
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The limit of resolving power is based on the nature of the source employed for imaging.
All sources used for microscopy (LM, EM, x-ray, UV) have one thing in common. They
all exhibit wave motion.
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Visible light/white light consists of the collective colors of the rainbow (ROYGBIV).
The different colors are actually differing wavelengths which range between 380nm
(violet) to 750nm (red). In converting metric units this range can also be expressed as
between 0.38µm to 0.75µm.
© B. Woelker, 2008
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There is an inverse relationship between wavelength and energy. The shorter the
wavelength, the higher the energy level (violet is highest and red the lowest energy of the
visible spectrum).
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Wavelength is defined as the distance from the crest of one wave to the crest of the next
wave (or trough to trough).
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X-rays and ultraviolet (UV) are parts of the electromagnetic spectrum, which also move
as waves, however, their wavelengths are shorter than for visible light. Electrons can also
move as waves as proposed by the French physicist de Broglie who stated that small,
high velocity particles (such as electrons) exhibit wave motion - referred to as particle
waves.
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Ernst Abbe working for Zeiss in the late 1800’s developed the inverse relationship
between resolving power and source wavelength. Essentially, the relationship is that the
ultimate resolving power of an instrument can be no better than one-half the source
wavelength.
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RP = 0.5 x source wavelength
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For visible light the shortest wavelength possible is close to 0.4µm. One-half of 0.4µm is
0.2µm (the resolving power of the LM. Obviously electrons have a much shorter
wavelength in order to achieve a resolving power of 2Å, which approaches the atomic
level and is definitely at the molecular level.
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Why would molecular resolution be important to the biologist? Eukaryotic cells (contain
a true nucleus) possess a number of different organelles (such as mitochondria,
ribosomes, lysosomes and the nucleus itself). These organelles are composed of
molecules that we are able to visualize using an electron microscope!
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An understanding of (cell) structure leads to an understanding of (cell) function
(physiology). The successful use of the electron microscope to examine biological
structures revolutionized our knowledge of cell biology (cytology) beginning in the mid
1950’s.
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Magnification - is defined as the product (multiplication) of the lenses. The main
magnifying lenses of a LM are the objective and ocular (eyepiece) lenses. The
magnification relationship follows:
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Magnification = Ocular X Objective
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The typical ocular lens of a LM is capable of 10X magnification. The objective lenses
(located on the rotating nosepiece) are typically 4X, 10X, 40X and 100X. The ultimate
total magnifications of the LM are calculated below.
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Ocular Lens Magnification x Objective Lens Magnification = Total Magnification
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10X (ocular lens) x 4X (scanning power) = 40X
© B. Woelker, 2008
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10X x 10X (low power) = 100X
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10X x 40X (high power) = 400X
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10X x 100X (oil immersion lens - never use without oil!) = 1,000X
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Useful vs. Empty Magnification:
The resolving power defines the useful magnification of the LM which is about 1,000X.
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This useful magnification figure of 1,000X is based on the relationship between the
resolving power of the unaided human eye (0.2mm) and the actual resolving power of the
instrument (LM=0.2µm). Since there are 1,000µm per millimeter (mm), the maximum
useful magnification of the LM is 1,000X.
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Magnifications beyond this are termed empty since you are magnifying without a
corresponding increase in resolution (detail). The (transmission) electron microscope’s
useful magnification is approximately 1,000,000X based on its 2.0Å resolving power.
The useful magnification is simply a ratio of the resolving power of the human eye to the
resolving power of the instrument.
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Contrast - even with the highest resolving power imaginable, without contrast, image
formation is impossible.
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Contrast is defined as differences in optical density (blacks vs. whites vs.
grays/halftones). For example, if a black object were on a white table (or visa versa), the
object stands out from the background and is easily recognized and studied.
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Contrast is especially important in electron optics since the source electrons are of a
single wavelength. There is no spectrum of wavelengths (colors/rainbow), therefore, all
electron images and micrographs are black and white.
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In the preparation of samples for the LM, contrast is enhanced by using stains
(pigments/dyes). We can take advantage of the visible spectrum and enhance color
differences and contrast. In sample preparation for TEM (transmission meaning that the
electron source passes through the specimen), heavy metal stains are used.
Parts of the Microscope:
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You will be responsible for knowing all the parts of the microscope and their functions.
A labeled image of your Olympus LM is shown on the next slide.
A. Coarse/Fine Focus (controls the distance between the objective lens and the actual
specimen).
B. Ocular(s) (serve to magnify and focus the final real image on the retina (sensory layer) of
the eye).
C. Objective Lenses (main magnifying and focusing lens in the microscope - 4X, 10X, 40X,
100X on the rotating nosepiece).
© B. Woelker, 2008
D.
E.
F.
G.
H.
I.
Specimen (usually mounted on a glass slide).
Mechanical (X, Y) Stage (supports the specimen).
Condenser lens (focuses the source at the specimen level-refraction).
Iris Diaphragm (regulates the amount of light to the specimen).
Illuminator (provides the source of photons (light).
Base (provides stability and dampens vibrations).
B
D
C
A
E
G, F
H
I
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Care of the Microscope:
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The compound LM you will be using is an expensive, precision instrument which must
be properly cared for.
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Firstly, carry the LM upright with two hands. Note the number painted on the back of
the LM, which corresponds to the cabinet location it is stored in (please put it back in the
correct location).
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Once at your table, the LM lenses should be cleaned for the best image quality - use only
lens paper! The use of anything else can permanently damage (scratch) the lenses!
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When you are finished with the LM, rotate the short 4X objective into place, clean the
lenses and return it to the storage cabinet.
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Proper LM Usage:
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Once the LM lenses are clean, plug in the instrument and turn on the light source
(switch). Always start at low magnification (40X total/4X objective lens) and low light
© B. Woelker, 2008
using the iris diaphragm lever and not the dimmer/brightness control knob (unless the
source is excessively bright).
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Place the slide (the label of a prepared slide should be oriented to the left) in the frame of
the mechanical stage and lower the sickle arm on the upper corner of the slide. Use the
X,Y controls to position the center of the slide (or specimen if you can see one with the
naked eye) over the condenser lens.
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Coarse focus to locate the specimen and then fine focus. Move the stage to locate areas
of interest and magnify on those areas.
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In order to magnify, center (lenses are parcentric) the area of interest and then rotate the
next higher magnification objective lens into place. The image should be nearly focussed
after the change in magnification since the lenses are parfocal - you will probably have to
fine focus. You may need to increase the light intensity as you increase magnification
using the iris diaphragm!
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To use the 100X oil objective lens you must firstly focus at 400X and then swing the
objective turret half-way between the 40X and 100X objective lenses. Place a central
drop of immersion oil on the slide using the glass/plastic wand in the oil bottle. Finally,
slowly rotate the 100X objective so that it contacts the oil drop (it should never hit the
slide if your were properly focussed at 400X). You should only have to fine focus and
increase the light intensity with the iris. Lenses and slides must be cleaned of oil before
the LM is put away. Role of oil & index of refraction.
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Imaging Features:
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There are two important parameters to keep in mind when viewing samples with the LM.
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Image inversion - images appear upside down and backwards. To assist in your
comprehension of this you will look at a slide of the letter “e” in its typical orientation at
40X total. How does the letter “e” appear when viewed under the LM?
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Depth of focus - everything you observe has depth to it (3-D objects). You need to fine
focus through the depth of a sample to observe all features of the sample. To assist in this
parameter you will observe the slide of crossed silk threads at 100X.
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The Scanning Electron Microscope (SEM) combines high resolution (40Å) with high
depth to produce virtually 3-D images. We will look at this instrument next week when
the EM facility tour takes place.
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Comparison of LM to TEM:
The primary differences between the LM and the TEM are:
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Source (photons versus electrons which are emitted from a tungsten filament by
application of 50,000 volts and higher),
Lenses (glass versus electromagnetic since glass cannot refract electrons),
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Operating environment (normal atmospheric pressure of 760 mm Hg versus 10-5 mm
Hg or high vacuum - this high vacuum environment prevents electron collisions with air
molecules, which would cause unwanted scattering, however, the observation of living
samples is not possible),
Stains (pigments/dyes vs. heavy metals such as osmium - Os).
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In addition, TEM samples must be sectioned ultrathin (600-900Å) to permit electrons to
pass through them.
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Calibration of the Light Microscope:
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When you look through the ocular of a LM you will observe a circular field of view. This
circle has a diameter (d) which can be crudely measured using a metric ruler mounted on
a glass slide. Alternatively, an ocular and stage micrometer may be used.
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At mag (40X) locate the dark ruler lines and place one on the exact edge of the field of
view. Now count the number of spaces (1mm) that run across the field. The results are
shown below.
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Knowing the diameter of the field allows us to approximate the size of objects we will
observe using the LM. For example, if ten cells are lined up across the center of the entire
field of view at 400X, the length of an individual cell is 400µm divided by 10 or 40µm.
Calibration
d = diameter of field of view
40X
100X
400X
1000X
d= 4.0 mm (4000µm)
d= 1.6 mm (1600µm)
d= 0.4 mm (400µm)
d= 0.16 mm (160µm)
y = sp x d / magnification of objective lens
(sp = scanning power or 4)
Calibration of the of the ocular micrometer
Ocular micrometers are calibrated by comparing the ocular micrometer scale with a calibrated
stage micrometer. The stage micrometer is a microscope slide that has a carefully calibrated
scale which is divided into 0.1 mm and 0.01 mm units.
© B. Woelker, 2008
Procedure:
1. Install the 10x ocular containing the ocular micrometer disc in the microscope.
2. Place the calibrated stage micrometer slide on the stage and focus on the scale.
3. Adjust the field so that the zero line of the ocular disc scale is exactly superimposed
upon the zero line of the stage micrometer scale.
4. Without moving the stage micrometer, locate the point as far to the extreme right as
possible where any two lines are exactly superimposed upon each other.
5. Count the number of divisions (mm) on the stage micrometer between the zero line and
the superimposed line to the far right (about 1mm under scanning power).
6. Count the number of ocular divisions between the zero line and the superimposed line to
the far right (about 40 divisions under scanning power).
7. Divide the distance determined in step 5 by the number of ocular divisions in step 6 and
multiply by 1000 to give the ocular micrometer units in µm.
stage micrometer divisions (mm) x 1000 µm = µm per ocular unit
ocular micrometer divisions
8. Repeat steps 3 through 7 for each objective on the microscope. If the ocular micrometer
is moved to a different scope, the calibration procedure must be repeated. If a new
objective is added to the microscope, the calibration procedure must be done for the
objective.
Example:
Using an ocular micrometer in the eye piece of a phase contrast scope and its 40X
objective, it is found that 100 ocular units are equal to 1 mm on the stage micrometer
scale.
One ocular unit =
1 mm x 1000 µm = 10 µm
100 units mm
1 MM
© B. Woelker, 2008
The ocular micrometer has to be calibrated for every magnification and for every light
microscope. Once the calibration is finished, you can proceed measuring a variety of
cells (Paramecium caudatum, Elodea, cheek cells). Determine the size of these cells
under various magnifications.
How long is Paramecium caudatum?
Paramecium
Field of view under Scanning Power
© B. Woelker, 2008