Microscope Notes

• Microscope – an instrument that produces an enlarged image of an object.

• Biologists use microscopes to study cells, cell parts, and organisms that are too small to be seen with the
naked eye.
• Microscopes magnify and show details of the image.
• Two types of microscopes:
o Light Microscope – light passes through one or more lenses to produce an enlarged image of a
specimen
o Electron Microscope – forms image of a specimen using a beam of electrons rather than light
• Some early microscopes:

• Zacharias Janssen – Dutch spectacle maker who tested several lenses in a tube and discovered that
nearby objects appeared significantly enlarged.

• Robert Hooke – used a microscope to observe a thin slice of cork. The spaces he saw reminded him of
the small rooms where monks lived, so he called them “cells,” which he used to describe the smallest

units of life.
 o He used microscopes with two and three lenses, but they didn’t produce very detailed images.
o Drawings made by Robert Hooke:

• Anton van Leeuwenhoek - Dutch merchant who learned how to grind lenses to make simple
microscopes (have only one lens). These produced clearer and more enlarged images than Hooke’s.
o He is considered “The Father of Microscopy” and built over 500 different microscopes.
o He was the first to discover microorganisms under a microscope by observing a drop of pond
water filled with life. He called them “tiny animalcules.”
o He also saw and studied bacteria and the blood flow through capillaries in the tail of a fish.
o Drawings made by Anton van Leeuwenhoek:

Parts of a Compound Light Microscope:

1. Body tube: Keeps the two sets of lenses a set distance apart.
2. Rotating nosepiece: Allows one to
change between the objective lenses.
3-5. Objective lens: The second set of lenses in a
compound microscope (usually 4x, 10x, 40x).
6. Stage clips: Hold the slide in place.
7. Diaphragm: Adjusts the amount of light
that hits the slide from the light source.
8. Light source: Where light comes from to see image.
9. Ocular lens: The first lens in a compound microscope
(usually 10x).
10. Arm
11. Stage: Where one puts the slide to view.
12. Coarse Adjustment Knob: Moves the stage up and
down very quickly.
13. Fine Adjustment Knob: Moves the stage up and
down very slowly.
14. Base
 • Compound Light Microscope
o Light passes through the specimen on the slide and uses two lenses to form its image.
o Capable of two things (below) that vary in different microscopes:
▪ Magnification: a measure of how much the image is enlarged
Total magnification = (ocular lens)(objective lens being used)
[The ocular lens usually has a 10x magnification, but that can vary.]

4x objective lens = (10x)(4x) = 40 times total magnification

10x objective lens = (10x)(10x) = 100 times total magnification
40x objective lens = (10x)(40x) = 400 times total magnification

▪ Resolution: a measure of the clarity of an image; how clear the details are

The Electron Microscope

• Resolution is the limiting factor to a light microscope since the greater the magnification is, the less it is
able to resolve the image. At magnifications beyond 2,000x, the image becomes blurry, but electron
microscopes can be used at greater magnifications.

• Characteristics of the Electron Microscope

o A beam of electrons is used to produce an enlarged image of the specimen (it does not use light).
o This electron beam and the specimen must be placed inside a vacuum chamber so that the
electrons in the beam do not bounce off gas molecules in the air.
▪ Since living things cannot survive in a vacuum, the electron microscope cannot be used to
view living cells.
 o Much more powerful than light microscopes.

pollen Dust mite mitochondrion

o There are two types of electron microscopes:

1. Transmission Electron Microscope (TEM)

▪ Uses a beam of electrons transmitted through a very thinly sliced specimen. Magnets guide
the beam of electrons toward the specimen, and the image is produced to view.
▪ Magnification to 200,000 times.
2. Scanning Electron Microscope (SEM)

▪ Provides detailed 3-D images.

▪ The specimen is sprayed with a fine metal coating (it is not sliced to view as in the TEM).
▪ As the beam of electrons is passed over the specimen’s surface, the metal coating emits a
shower of electrons, and a 3-D image of the specimen’s surface is produced to view.

Rules for Using the Compound Microscope

1. ALWAYS carry the microscope with one hand holding the arm and your other hand supporting
under the base.
2. Plug in the cord and turn on the light source of the microscope.
3. Place your slide on the stage and arrange the stage clips to hold the slide in place.
o Keep the stage dry and ALWAYS make sure that your slide is dry, especially the bottom,
before putting it on the microscope.
4. Always start with the 4x objective lens (should already be on this from when the microscope was
put away). Focus this objective lens using the coarse adjustment knob.
 5. Once the image is in focus, carefully swing the 10x objective lens in place and focus this objective
lens using the coarse adjustment knob.
6. Once the image is in focus, VERY carefully swing the 40x objective lens into place – BE SURE
TO NOT TOUCH THE SLIDE. Focus this objective lens using ONLY the fine adjustment knob.
o NEVER use the coarse adjustment knob while using the high power objective lens (40x).
7. Make observations and take notes as needed before preparing to put the microscope away (#8-11).
8. Lower the stage using the coarse adjustment knob.
9. Swing the objective lens back to low power (4x).
10. Turn off light source, unplug and neatly wrap cord around microscope (NOT around lenses or
light source).
11. Place protective cover over microscope before you put the microscope away.

Preparation of a Wet Mount Slide

• Wet mount slides are used to view living organisms, such as ones that need to be kept moist, and any
liquid substances.

1. Using the appropriate tool, put your specimen in the center of a clean and dry slide.
2. Add one large solid drop of water over your specimen (water should not move on slide).
3. Hold a clean and dry coverslip at a 45° angle over your specimen/water drop. Let one edge of
the coverslip touch an edge of the water drop.
4. Gently drop the rest of the coverslip into place – want to avoid getting air bubbles (the whole
slip should touch water). *Remember to keep the rest of the slide [and microscope stage] dry!*

How to Measure Under the Microscope

• The micrometer (m) is the unit of measurement used to measure things under the microscope.
o One micrometer = 0.000001 meter (10-6).
• To estimate the size of each cell, use the diameter of each objective lens shown below.

The 10x objective lens has a field of view The 40x objective lens has a field of view
with a diameter of 1,500 m. with a diameter of 375 m.

Cell Cell

The size of the cell would be about 500 m. The size of the cell would be about 100 m.
 Phase contrast microscopy
Unstained living cells absorb practically no light. Poor light absorption results in extremely small
differences in the intensity distribution in the image. This makes the cells barely, or not at all,
visible in a bright field microscope. Phase-contrast microscopy is an optical microscopy
technique that converts phase shifts in the light passing through a transparent specimen to
brightness changes in the image. It was first described in 1934 by Dutch physicist Frits Zernike.

 Principle of Phase contrast Microscopy

When light passes through cells, small phase shifts occur, which are invisible to the human eye.
In a phase-contrast microscope, these phase shifts are converted into changes in amplitude,
which can be observed as differences in image contrast.
 The Working of Phase contrast Microscopy

1. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a

collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in
the substage condenser front focal plane.

2. Wavefronts passing through the annulus illuminate the specimen and either pass through
undeviated or are diffracted and retarded in phase by structures and phase gradients present
in the specimen.

3. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane
by a phase plate and focused at the intermediate image plane to form the final phase-contrast
image observed in the eyepieces.

 Parts of Phase contrast Microscopy

Phase-contrast microscopy is basically a specially designed light microscope with all the basic
parts in addition to which an annular phase plate and annular diaphragm are fitted.

The annular diaphragm

 It is situated below the condenser.

 It is made up of a circular disc having a circular annular groove.

 The light rays are allowed to pass through the annular groove.

 Through the annular groove of the annular diaphragm, the light rays fall on the specimen
or object to be studied.

 At the back focal plane of the objective develops an image.

 The annular phase plate is placed at this back focal plane.

The phase plate

 It is either a negative phase plate having a thick circular area or a positive phase plate
having a thin circular groove.
  This thick or thin area in the phase plate is called the conjugate area.

 The phase plate is a transparent disc.

 With the help of the annular diaphragm and the phase plate, the phase contrast is obtained
in this microscope.

 This is obtained by separating the direct rays from the diffracted rays.

 The direct light rays pass through the annular groove whereas the diffracted light rays
pass through the region outside the groove.

 Depending upon the different refractive indices of different cell components, the object to
be studied shows a different degree of contrast in this microscope.

 Applications of Phase contrast Microscopy

To produce high-contrast images of transparent specimens, such as

 living cells (usually in culture),

 microorganisms,

 thin tissue slices,

 lithographic patterns,

 fibers,

 latex dispersions,

 glass fragments, and

 subcellular particles (including nuclei and other organelles).

 Advantages of Phase contrast Microscopy

 Living cells can be observed in their natural state without previous fixation or labeling.

 It makes a highly transparent object more visible.

  No special preparation of fixation or staining etc. is needed to study an object under a
phase-contrast microscope which saves a lot of time.

 Examining intracellular components of living cells at relatively high resolution. eg: The
dynamic motility of mitochondria, mitotic chromosomes & vacuoles.

 It made it possible for biologists to study living cells and how they proliferate through
cell division.

 Phase-contrast optical components can be added to virtually any bright field microscope,
provided the specialized phase objectives conform to the tube length parameters and the
condenser will accept an annular phase ring of the correct size.

 Limitations of Phase contrast Microscopy

 Phase-contrast condensers and objective lenses add considerable cost to a microscope,

and so phase contrast is often not used in teaching labs except perhaps in classes in the
health professions.

 To use phase-contrast the light path must be aligned.

 Generally, more light is needed for phase contrast than for corresponding bright-field
viewing, since the technique is based on the diminishment of the brightness of most
objects.
 Freeze Fracture and Etching - a brief introduction
Freeze fracture describes the technique of breaking a frozen specimen to reveal internal
structures. Freeze etching is the sublimation of surface ice under vacuum to reveal details of the
fractured face that were originally hidden. A metal/carbon mix enables the sample to be imaged
in a SEM (block-face) or TEM (replica). It is used to investigate for instance cell organelles,
membranes, layers and emulsions. The technique is traditionally used for biological applications
but started to develop significance in physics and material science. Recently, freeze fracture
electron microscopy, particularly freeze replica immune labeling (FRIL), has provided new
insights into the roles of membrane proteins in dynamic cellular processes.

Fit for the environment of an electron microscope

The chamber of an electron microscope is evacuated to a very low pressure. The structure of a
living cell placed into this environment cannot be preserved due to the extremely quick
evaporation of the water which makes most part of the cell.

There are a number of preparation possibilities for biological samples. The material could be
preserved (fixed) so the subsequent dehydration produces minimal damage to the in vivo
structure, an environmental SEM could be used or the water could be frozen. High pressure
freezing is the only way to observe hydrated structures in their natural state. The ice built by high
pressure freezing is not hexagonal ice, which shows an increase of volume from water to ice but
amorphous ice, where the volume stays constant. Therefore structures which are sensitive to
osmotic and temperature change are preserved.

To observe structures such as cell organelles, membranes, emulsions or surface interfaces of

liquids, freeze fracture is the only way to do that. The frozen sample is broken by force of a knife
(or similar) or a released spring load and breaks along the lines of least resistance.
Sublimation and condensation of water - Freeze etching and contamination

To reveal details of the fractured surface, ice has to be removed. This needs to be done by
sublimating the ice to preserve the structure of the specimen. Ice is directly transformed into
water vapour without going through the liquid state which would lead to a change in volume and
structural damage.

The sublimation/condensation process of water depends on the saturation pressure at a particular

temperature and the effective partial water pressure of water or ice in the chamber. Note: a good
vacuum reduces the partial water pressure.

For example: Ice or frozen specimens with a temperature of -120°C have a saturation pressure of
about 10-7 mbar. If this pressure is established in the chamber, condensation and evaporation are
in equilibrium. The amount of evaporated molecules is equal to the amount of condensed
molecules. At a higher pressure the condensation rate is higher than the sublimation rate – ice
crystals grow on the specimen’s surface. This has to be avoided by all means. A colder (than the
specimen) plate above the specimen reduces the local pressure and works as a condensation trap.
Water molecules driven up from the specimen preferentially attach to the colder surface. At a
lower pressure than the saturation pressure more molecules sublimate than condensate and freeze
etching takes place.

Performing freeze etching until the sample is completely ice free, is called freeze drying. This
process only works for small samples to be performed in a reasonable time. It is done in several
steps by heating up from around -120°C to -60°C maintaining the temperature of each step for a
certain time. This can take up to days.

At specimen temperatures below -120°C the etching rate is very low, etching times increase to
impractical durations. If the pressure of the vacuum chamber is fixed, it is possible to increase
the etching rate by raising the specimen temperature. Careful with temperatures higher than -
90°C for biological samples. Etching rates increase tremendously. Additionally hexagonal ice
forms from the vitrified ice and causes dehydration arte facts.

The theoretical sublimation rates of pure water are reduced because:

• Water from the depth of a specimen sublimates slower than water from the surface.

• Solvents of salt and macromolecules reduce the sublimation speed.

• The bound water which is a considerable part of biological samples have a lower sublimation
rate.
Freeze fracture to generate images

Freeze-fracture and freeze etching techniques require ultrathin heavy metal and carbon films
deposited under vacuum on the fractured surface.

Freeze fractured samples are coated under an angle with metal followed by a carbon backing up
film (Leica EM ACE600 freeze fracture or Leica EM BAF060 with Leica EM VCT100) to
produce either a replica to be imaged in a TEM or a block face for the SEM.

For both methods the fractured surface is coated after a certain amount of etching time the same
way. First a thin (2- 7nm) heavy metal coating under an angle to produce topographic contrast
(shadowing). Second a thick carbon layer (15- 20nm) coated under 90° to stabilize the ultra-thin
metal film. At this point the etching process is stopped. To image very small structures the heavy
metal is applied in a very low angle (2-8°) and the sample is rotated during coating. This adds
contrast to filamentous and small structures. Such technique is called low angle rotary
shadowing.

E-beam evaporation should be used for the heavy metal film. This is the coating technique giving
a very fine and directional deposition. The supporting layer of carbon stabilizes the structures
which are uncovered by metal. Those structures would change their contour during the increase
of temperature, the sample would not be completely conductive and a replica would not stick
together.

Replica for TEM

After the coating process the biological structure is washed away with acids and cleaned in
distilled water. Only the replica made of metal and carbon is left. It is placed on a TEM grid and
transferred into the microscope.

Block-face for SEM

After the coating process the sample is kept under cryogenic conditions and transferred into a
cryo- SEM. It is very important to keep the sample under constant temperature otherwise
artefacts can be introduced in this last step.