TYPES OF MICROSCOPES

Light Microscopy

Bright field Microscopes—the most common general use microscopes. Bright field
microscopes are named because the microscopic “field” is bright, while the object being viewed
is dark.
Features
 Simple design
 Light directed at specimen is absorbed to form image
 Unstained specimens have poor contrast
 Stained specimens show excellent contrast
 Ideal for stained bacteria, cells, tissues
 High N.A., good resolution
 Bright background, dark specimen tungsten or halogen light source

Fig 1: Bright Field Microscope

Dark field Microscopes (DF)

The dark field microscope creates a dark background to allow viewing of small unstained
objects, such as motile bacteria, that would be difficult to view in a bright field. The central
portion of the light is blocked so that only oblique light strikes the specimen, scattering light rays
that then enter the objective to form the image.
Features
 A method from the 19th century
 Bright specimen, dark background
 Light not scattered by the specimen bypasses the objective, therefore making the
“field” dark.
 Can see very small objects but resolution is variable.
 High contrast, good for unstained, live, and motile specimens
 Fig 2: Dark Field Microscope

Phase Contrast— This is a microscope that uses the differences in the phase of light transmitted
or reflected by a specimen to form distinct, contrasting images of different parts of the specimen.
The central portion of the light source is blocked, creating a ring of light from the condenser that
illuminates the specimen. The light waves refracted by the specimen are slowed by a phase
retardation plate (in phase objectives) increasing the difference in wavelength between refracted
and unrefracted rays (which do not pass through the phase plate).
When the refracted and and unrefracted waves are focused, they produce an interference due to
the difference in wavelengths--this is seen as differences in brightness in the specimen.
Features
- Technology from 1940’s
- Provides high contrast, good resolution
- Good for bacteria, flagella, cilia, organelles such as mitochondria
- Good for unstained or live mounts
- Phase halos (artifacts) occur

Fig 3: Phase contrast Microscope

Polarization Microscopy: Detects specimens that are birefringent (have the characteristic of
double refraction, i.e. the velocity of light refracted by a substance is not the same in all
directions). The specimen is placed between two polarizers crossed at 90o to each other (one in
condenser and one in objective).
Features
- Bright image, dark background
- Used for substances with highly organized molecular structure, such as crystals, minerals
- Can be quantitative

Fig. 4: Polarizing Microscopes

Fluorescence microscope: These allows the detection of molecules and ions within cells.
Fluorescent dyes absorb short wavelengths of light and emit longer wavelengths. Barrier filters
and a dichroic prism select the excitation wavelength that strikes the specimen and exclude the
excitation wavelength from the detector, allowing only emitted light to reach the detector
(oculars).
Features
- Uses ultra violet light source = mercury or xenon arc lamp.
- High contrast, high resolution image
-Special fluorescent dyes used to locate“molecules” in a specimen
- Black background, bright-stained specimen
- No condenser required, light comes from above (“epi”) specimen
- Multiple fluorescent probes available
- Detects small quantities, molecules; can use antibody staining techniques, imaging structural
components of cells, conducting viability studies on cell populations, imaging the genetic
material within a cell (DNA and RNA), and viewing specific cells within a larger population
with techniques.
 Fig. 5: Fluorescence Microscope

Electron Microscopy

Electron microscopy is a technique for obtaining high resolution images of biological and non-
biological specimens. It is used in biomedical research to investigate the detailed structure of
tissues, cells, organelles and macromolecular complexes.

Electron microscopes use beam of electron in place of light. The image produced by electron
microscopes is perceived by CRT or X-ray plates. Electron microscopes were developed due to
the limitations of light microscopes which are limited by the physics of light. The limitation of
light was first observed in 1930, when the fine details of the internal structure of organic cells
such as mitochondrion and nucleic could not be on observed. This required10,000x plus
magnification which was not possible using current optical microscopes.

This is of two types:

Scanning Electron Microscopy (SEM)
Transmission Electron Microscopy (TEM)

Scanning Electron Microscopy (SEM): This works be scanning the surface of an object with a
focused beam of electrons and detecting electrons that are reflected from and knocked off the
sample surface. At low magnifications, entire objects (such as insects) viewed on the SEM can
be in focus at the same time.
Working principle is as follows: Fixed, dehydrated specimens are mounted on stubs and
surface-coated with gold, palladium or rhodium. The specimen is placed in a vacuum and an
electron beam scans back and forth over it. Electrons that bounce off the metal-coated specimen
surface are collected, converted to a digital image and displayed on a TV-like monitor (Fig. 6).
Features
- Electron beam is focused using a magnetic field
- SEM provides a 3-D image
-Gives information about external topography of specimen
-Much higher resolution and magnification than possible in low magnification

Fig. 6: Working principle of SEM

TRANSMISSION ELECTRON MICROSCOPY (TEM)

This was the first electron microscope to be developed. The TEM helps researchers to look in
very high resolution at a thin section of a sample.
Principle of operation:
Fixed, dehydrated specimens are embedded in a resin, hardened, sectioned, stained with heavy
metals such as uranium and lead, and inserted into the electron column in the microscope. The
electron beam is absorbed or deflected by the heavy metal stains and shadows are cast onto film
or a phosphorescent plate (image is a shadow) at the bottom of the column (Fig. 7).
Features
- 2-D image
- Reveals internal cell structure
- High resolution, high magnification
- Electron beam is focused by magnetic field
 Fig. 7: Principle of operation of TEM

OPERATING THE MICROSCOPE

 The proper way to operate your microscope is to start it focus with the lowest power
objective lens while you look through the side and move the lens down as close to the
slide without touching it.
 Next, you look through the eyepiece lens and focus upward only until the image is sharp.
If you cannot get it in focus, repeat the process over.
 When you have a sharp image at low power lens, you turn the revolving nosepiece to
click the next higher power lens and do a minor adjustments with the fine focus knob.
Continue with subsequent objective lenses and fine focusing each time.

HANDLING AND CARE OF THE MICROSCOPE

1. Always carry your microscope with one hand on the Arm and one hand on the Base, and close
to your body.
2. Plug the microscope in and place excess wire on the table.
3. Always start and end focusing with low power Objectives lens.
4. Always make sure the stage and lenses are clean before you put away the microscope.
5. Always use good quality lens tissue to clean before you put away the microscope.
6. Always cover your microscope with a dust jacket when not in use.
DISSECTION
Dissection is the cutting open of animal in order to ascertain the structure of its parts, so as to
define their boundaries and display clearly their mutual relations. Dissection consists mainly in
removing the connective tissue which binds the several parts together. It requires lots of
preparations beforehand.

Preparation for Dissection

In preparing for dissection, some material must be obtained before the dissection is
initiated. Materials required are: dissection trays, Petri dishes Dissection kit, Anesthetics (e.g.
Chloroform, Ethane, Formalin, Urethane) Microscope, and Animals.

Procedures for Dissection

1. Procuring Animals: Order for animals to be used for dissection should be placed with
an animal supplier or sourced for according to the number of students in a class. This
exercise should be done two-three days before dissection is to be performed.
2. Anaesthesizing the Animals: Before dissection, animals are given anaesthesia.
Chloroform and ether are used as anaesthesing agents. Rats, frogs and pigeons are
generally freshly chloroformed for the dissection, though for some of the dissections e. g.
cranial nerves in rat and internal ear in frog, preserved specimens are required.
Preservatives commonly used are formalin (5% or 8% or 10%) and 70% alcohol.
3. Theoretical Knowledge: You should have a well-labelled diagram of the dissection to
be done. You should have some theoretical knowledge of the dissection to be performed.
Example: if students have to dissect male or female reproductive organs of rat, frog or
other animals, then you should be able to differentiate them morphologically, just by
looking at them externally.
4. Set- up of dissection Trays/boards: Large animals like frog, rat, and fish other animals
are dissected in dissection trays/boards whereas small animals such as cockroach and
small insects can be dissected in petri plates. It should be seen that wax is spread over
boards uniformly and water does not drip form the boards.
5. Pithing: Some of the experiments like muscle twitch and heart profusion in frog do not
require chloroform-anaesthetized animals. Before dissection is started, you have to
anesthetise the frog by injecting 2.5 ml of 20% urethane intramuscularly. This would
quite the frog. Alternatively you could immobilize the frog by pithing. Pithing is a
technique used to immobilize or kill an animal by inserting a needle or metal rod into the
animal’s brain. It is regarded as a humane means of immobilizing small animals being
observed in experiments.
6. Opening up:
 After immobilizing animals using anesthetics or pithing, you open up the
animal.
 Invertebrates are better opened up from their dorsal side while vertebrates from
their ventral side.
 In both cases, you pick the skin up with a pair of forceps (if the animal is big), if it
is small, you will have to hold the animal with your left hand while with a pair of
 scissors you make your incision with the right hand on the mid-line of the animal
with the scissors pointing upwards to avoid damage to lower internal structures.
 This incision should go along the line of the animal as much as possible.
 Then make side slits in the same manner, with the scissors pointing upwards to
avoid damage to underlying tissues.
 With the scapel, gently scrape the inside layer of the skin to separate skin from the
underlying tissues by cutting through subcutaneous tissue.
 Once the skin has been removed the animal must be laid down (even if it is small)
and pinned securely to the dissecting board.
 It is important to pin specimen securely down so that both hands will be free for
use. Seekers could then be used to probe parts, when other walls have to be
opened up to reach internal organs, the same cautions as we took for opening up
the skill will apply.
 You have to be very gentle in dissecting so as to avoid damage to internal organs.
 If the dissection involves cutting through tissues (especially blood vessels) then
you must take steps to ensure that the blood flow or the contents of the cut tissues
or organs do not interfere with your studies. Usually, you may have to wash, and
soak away blood with cotton wool or blotting paper.
7. Safe Disposal of Dissected Animals
Dissected animals should be buried deep in the soil. Simply placing the material in a
plastic bag and putting it in the dustbin is not good enough.

THE DISSECTING KIT

1. Dissecting scissors: used for cutting skin, muscles and bone

2. Scalpel: used for cutting and shaving off back skin and muscle.

3. Forceps: Used for gently holding and moving and or removing.

4. Dissecting Probe/seeker: Used for gently moving things around when dissecting and
pointing to structures.

5. Pins: used to pin back skin and muscles.

6. Magnifying glass: Enlarges and makes bigger specimen viewed by the observer.
 BIOLOGICAL DRAWINGS
Definition: Biological drawing is the ability to draw, label and annotate biological specimens.

Why Biological Drawings?

1. Biological drawing is a type of data collection because drawings help to record data from
specimens.
2. It is important because it aids in remembering what was observed from the specimen.
3. It provides permanent record of what has been observed.

What equipment is needed?

Sharp pencil - HB is generally preferred, but H, 2H or B (for emphasis) can all be used
according to preference.
Pencil sharpener - A nail file may also be useful to keep the point really sharp.
Eraser
Ruler- For label lines
Plain paper

General Principles
When assessing biological drawing, marks are awarded for both quality of drawing and
labeling. The latter may include annotation. The general principles described below apply to all
types of biological drawing:

a. Use a sharp pencil only. Don’t use pens or coloured pencils.

b. Use clear, continuous lines. A line which encloses a shape, such as a circle, should join
up neatly without obvious overlap. Overlapping lines is a common error in hastily drawn
sketches and is easily spotted and penalised by examiners.
c. Don’t use any form of shading. This includes stippling, cross-hatching and shading.
Students find this is a hard instruction to follow, and it is sometimes difficult to justify.
Although shading may help to make the drawing look more realistic and/or to
discriminate between areas of the specimen, it does not represent a permanent structural
feature. Artistic impression is certainly not what is required.
 d. Accuracy is paramount. It shows good observation. Remember that observation is
assisted by understanding, so a good knowledge of theory goes alongside good drawing.
Pay particular attention to the outlines of structures and to the relative proportions of
different parts of the specimen. Don’t draw what you think you should see, for example
text book style drawings. Draw what you observe.
e. Guidelines can help. Faint sketching of the main areas of the specimen which can later
be erased may help. Some students find a simple grid helps them.
f. Magnification and illumination. To help in the drawing process it is often useful to use
a hand lens or a magnifying glass for larger specimens and, for microscopy, both low and
high power lenses when making preliminary observations. Field biologists usually carry a
hand lens as standard equipment. Dissection, and drawing from a dissection, is greatly
aided by good illumination of the specimen by a lamp and by a tripod lens placed over
the material where possible.
g. Drawing: All drawings should be done with sharp pencil on white, unlined paper.
Drawing should be at the center of the page. Do not draw in a corner. Make a large, clear
drawing. It should occupy at least half a page.
h. Correct mistakes. If you make a mistake, use a good quality eraser to rub out the lines
completely.
i. Include a title. Include a title stating what the specimen is. This is usually below the
drawing.
j. Include a scale. Include a scale if required. If you are drawing from a microscope, it is
useful to state the combined magnification of the eyepiece plus objective lenses used
when making the drawing, e.g. x100 (low power) or x400 (high power).

Labeling
When labeling biological drawings, follow the guidance below:
 Use a sharp pencil.
 Label all relevant structures, including all tissues in the case of microscopy.
 Use a ruler for label lines and scale bars.
 Label lines should start exactly at the structure being labelled; don’t use arrowheads.
 Arrange label lines neatly and make sure they don’t cross over each other. It is visually
attractive, though not essential, if the length of the label lines is adjusted so that the actual labels
are right or left justified, i.e. line up vertically above each other on either side of the drawing.
 Labels should be written horizontally, as in a textbook, not written at the same angle as
the label line.
 As previously mentioned, a title, stating what the specimen is, should be added at the
bottom of the drawing.