The Microscopic Techniques

Introduction to the Microscope

What Is Microscope?
It is an instrument which deals with too small
organisms that may cannot be seen distinctly
with the naked eye.
Microscopes provide the observer with

* Enhanced resolution: ability to observe two nearby

objects as distinct objects
Contrast : ability to detect different regions of the
specimen on the basis of intensity or color
* Magnification ability to make small objects visible
 The human eye can resolve objects of the order of 0.1
mm, while the light microscope can resolve objects on the
order of 0.2 μm (200 nm) with a magnification of 1000.
The transmission electron microscope, can resolve objects
on the order of 0.1nm (100 A ˚ units).
 Microscope History
•1000AD – The first vision aid was •1590 - Italian, Salvino
invented (inventor unknown) D'Armate is credited with
called a reading stone. It was ;a inventing the first wearable
glass sphere that magnified when eyeglasses
laid on top of reading materials.
1590 - Two Dutch eye glass •1665– English physicist, Robert
makers, Zaccharias Janssen and Hooke looked at a sliver of cork
son Hans Janssen experimented through a microscope lens and
with multiple lenses placed in a noticed some "pores" or "cells" in
tube. The Janssens observed that
viewed objects in front of the
tube appeared greatly enlarged,
creating both the forerunner of
the compound microscope and
the telescope.
•1674 – Anton van Leeuwenhoek built a simple microscope with
only one lens to examine blood, yeast, insects and many other tiny
objects. Leeuwenhoek was the first person to describe bacteria.
• 18th century – Technical innovations improved microscopes,
leading to microscopy becoming popular among scientists. Lenses
combining two types of glass reduced the "chromatic effect" the
disturbing halos resulting from differences in refraction of light.

• 1830 – Joseph Jackson Lister: reduces spherical aberration or

the "chromatic effect" by showing that several weak lenses used
together at certain distances gave good magnification without
blurring the image. This was the prototype for the compound
microscope.

• 1872 – Ernst Abbe wrote a mathematical formula called the

"Abbe Sine Condition". His formula provided calculations that
allowed for the maximum resolution in microscopes possible.
• 1903 – Richard Zsigmondy developed the ultra microscope that
could study objects below the wavelength of light. He won the
Nobel Prize in Chemistry in 1925.

• 1932 – Frits Zernike invented the phase-contrast microscope

that allowed for the study of colorless and transparent biological
materials for which he won the Nobel Prize in Physics in 1953.
• 1931 – Ernst Ruska co-invented the electron microscope for
which he won the Nobel Prize in Physics in 1986. An electron
microscope depends on electrons rather than light to view an
object, electrons are speeded up in a vacuum until their
wavelength is extremely short, only one hundred thousandth that
of white light. Electron microscopes make it possible to view
objects as small as the diameter of an atom.

• 1981 – Gerd Binnig and Heinrich Rohrer invented the scanning

tunneling microscope that gives three-dimensional images of
objects down to the atomic level. Binnig and Rohrer won the
Nobel Prize in Physics in 1986.
 Microscope Types
1- Compound Microscope 2- Dissecting Microscope
Compound microscopes are light (also called stereo microscope)
illuminated. The image seen with this type A dissecting microscope is light illuminated.
of microscope is two dimensional. This The image that appears is three dimensional.
microscope is the most commonly used. It is used for dissection to get a better look at
You can view individual cells, even living the larger specimen. You cannot see individual
ones. It has high magnification. However, it cells because it has a low magnification
has a low resolution.
3. Scanning Electron Microscope (SEM) 4. Transmission Electron Microscope
SEM use electron illumination. The image (TEM)
is seen in 3-D. It has high magnification TEM is electron illuminated. This gives a
and high resolution. The specimen is 2-D view. Thin slices of specimen are
coated in gold and the electrons bounce obtained. The electron beams pass through
off to give you an exterior view of the it. It has high magnification and high
specimen. The pictures are in black and resolution.
white.
 The Light Microscope (Optical Microscope)
Our eyes cannot focus on objects nearer than about 25 cm

The light microscope is an optical

instrument which operates on the
principal that light energy will pass
through and around a suitably thin
object, and with the aid of lenses
form a magnified impression on the
visual sensory layer of the eye
 How lenses bend and focus light to form images
 When light energy passes from one medium to another (i.e. AIR and
GLASS) the light rays are bent at the point of interface. This process is
called refraction.
. The measure of how greatly a substance slows the velocity of light
is called the refractive index

 The direction and magnitude

of bending is determined by
the refractive indexes of the
two media forming the
interface
  As parallel rays of light encounter a convex lens, they are slowed and
bent towards the normal path.
 The point at which these rays converge is called the focal point
 The distance between the center of the lens and the focal point is
called the focal length.
 The strength of a lens is directly related to its focal length. A lens with
a short focal length has a greater capacity for magnification than a lens
with a longer focal length

Focal point

Specimen
Convex lens

Inverted, reversed, and enlarged image

Anatomy of a bright-field, compound light microscope-overview
 Microscope Care

• Always carry with 2 hands

• Never touch the lenses with your fingers.
• Only use lens paper for cleaning
• When you are finished with your "scope",
rotate the nosepiece so that it's on the low
power objective, roll the stage down to
lowest level
 Using the Microscope
1- start with the lowest power objective lens first and while
looking from the side, put the lens down as close to the
specimen as possible without touching it.
2- Look through the eyepiece lens and focus upward only
until the image is sharp.
If you can't get it in focus, repeat the process again.
3- Once the image is sharp with the low power lens, you
should be able to simply click in the next power lens and do
minor adjustments with the focus knob.
4- If your microscope has a fine focus adjustment, turning it
a little bit should be all that's necessary.
5- Continue with subsequent objective lenses and fine focus
each time.
 Using High Power
1- Rotate to 40x objective, locate desired portion of specimen in the
center of the field.
2- Refocus very carefully so that the specimen is focused as sharply as
possible. (Do not alter focus for the Following steps) Partially rotate
so that 40x and 100x objectives include the specimen.
3- Place a small drop of oil on the slide in the center of the lighted
area. (Take care not to drop on the stage.).
4- Rotate so that the 100x oil immersion objective touches the oil and
clicks into place.
5- Focus only with fine focus. Hopefully, the specimen will come into
focus easily. Do not change focus dramatically.
Clean up: When you have finished for the day, wipe the 100x oil
immersion objective carefully with lens paper to remove all oil. Wipe
oil from the slide thoroughly with a Kim wipe.
 Types of Light Microscope
There are a variety of light microscopes mostly
employed in Microbiology:

1. Bright-field

2. Dark-field

3. Phase-contrast

4. Fluorescence
1-The Bright-field Microscope
Called the ordinary microscope because it forms a dark
image against a brighter background. Image should remain
in focus when objectives are changed.
The objective lens forms an enlarged real image within the
microscope and the eyepiece lens further magnifies this
primarily image. Upon looking in the microscope, the
enlarged specimen image (Virtual image) appears to lie
just beyond the stage about 25 cm away.

Total magnification is calculated by multiplying the

objective and eyepiece magnification together.
Three factors determine the quality of an optical
image:

a. Magnification

b. Resolution

c. Contrast.
a. Magnification:
is the apparent increase in size affected by a convex lens.
A compound microscope uses two sets of lenses, with
differing focal lengths, to facilitate magnification.
The total magnification achieved by the lens array is the
product of each individual lens.
Magnification (total) = mag.(obj. lens) x mag. (ocu. lens)
Example: Mag (obj) = 40X and Mag (ocular) = 10X
Then Mag (total) = (40X) (10X) = 400X.
It is much easier to make two lenses with average
magnifying powers and put them together in a compound
microscope than to make a single lens with a very high
magnifying power.
Compound microscopes are usually designed to give a
highest possible magnification of only 1,000-1,500X.
b. Resolution
is not the same thing as magnification.
One of the ways to increase the resolution of an image is to increase the
amount of light that enters the objective lens by using immersion oil.
Immersion oil (with a density
closer to glass) can be used to
reduce the refraction of light rays
(compared to air) and allows more
light to enter the objective. This
improves resolution. Only the
highest power (100X) objective on
the microscope is designed for use
with immersion oil.

Another way to increase resolution of an image is to decrease the

wavelength of light that is used to illuminate the specimen (use blue
light instead of white light).
c. Contrast
Microbes are composed of water, nucleic acids,
proteins, and lipids.
Most appear colorless against a colorless
background when observed using bright field
microscopy.
Therefore in order to see them, we must devise a
way to increase the contrast.
Direct staining of the microorganisms and Indirect
(negative) staining of the background In order to
stain a specimen, it must first be fixed to the slide
and chemically altered. This results in the death of a
specimen.
2- Dark-field microscopy
A technique that is often used to observe living, unstained cells and
organisms.
Dark field microscopes illuminate the sample in such a way that
unreflected, and unrefracted light does not enter the objective, only
light that has been reflected/refracted by the specimen passes through
the objective and forms the image. This results in a specimen that is
brilliantly illuminated on a dark field, The field surrounding the
specimen appears black.
 3- Phase-contrast microscopes
Convert slight differences in refractive index and density
into variations in light intensity. This allow organelles of
the living cell to become visible with fair contrast in them
4- Fluorescence microscopy

Specimens are treated with dye molecules called

Fluorochromes which brightly fluoresce when
exposed to light of a specific wavelength.