A Brief Overview
Dr Saleh M.Y.
Practical Lab Experiment -1-
- Historical Background
- Varibles Used In Microscopy
- Compound Microscope - Structure And Function
- Use Of Microscope
- Various Types Of Microscopes
- Care Of Microscope
- Understanding the
optical principles and
- Role of microscopy
- A microscope (Greek: micron = small and scopos =
- MICROSCOPE: Is an instrument for viewing
objects that are too small to be seen by the naked
or unaided eye
- MICROSCOPY: The science of investigating
small objects using such an instrument is called
1590 - Hans Janssen and his son Zacharias Janssen,
developed first microscope.
1609 - Galileo Galilei - occhiolino or compound
1620 - Christian Huygens, another Dutchman,
developed a simple 2-lens ocular system that was
Antony van Leeuwenhoek
- Anton van Leeuwenhoek is
generally credited with bringing
the microscope to the attention of
- A tradesman of Delft, Holland.
1661 - He discovered bacteria, free-living and
parasitic microscopic protists, sperm cells, blood
cells, microscopic nematodes etc.
Microscope used by
Anton von Leeuwenhoek
An old pocket Microscope
Degree of enlargement
No of times the length, breadth or diameter,
of an object is multiplied.
MAGNIFICATION VS SHARPNESS
USEFUL MAGNIFICATION AND EMPTY
RESOLUTION: Ability to reveal closely
adjacent structural details as separate and
LIMIT OF RESOLUTION (LR): The min
distance between two visible bodies at
which they can be seen as separate and
not in contact with each other
LR = 0.61 x W W = Wavelength
NA NA = Num aperture
Types of microscope Resolving power
Compound Microscope 200 nanometers
Scanning Electron Microscope 10 nanometers
Transmission Electron Microscope 0.2 nanometers
- Ratio of diameter of lens to its focal length
- NA = n Sin θ/2
n = refractive index,
θ = angle of aperture (CAD) B
n of air = 1
n of oil = 1.5
- Capacity of an objective to render outline
of the image of an object clear and distinct
- Depends of elimination of Spherical and
- Chromatic aberration
- Correction of aberration – Achromatic
objective and Apochromatic objectives.
Red focus 14
Focus of axial rays
Focus of marginal rays 15
TYPES OF MICROSCOPE
- Simple microscope
- Compound microscope
- Phase Contrast
- Dark Ground Microscope
- Fluorescent Microscope
- Electron Microscope
Compound microscope made
by John Cuff 1750
PARTS OF COMPOUND MICROSCOPE
- Ocular (Eye piece)
- Body or Tube
- Coarse focusing knob
- Fine focusing knob
- Objective Lens
- Movable stage
- Condenser Lenses
- Field (Iris) Diaphragm
- Mirror and light source
• Mounted on Nose piece
It forms magnified real image.
• Magnification of objective
= Optical Tube length
• Scan - 4X
• Low Power - 10X
• High Power - 40X
• Oil immersion - 100X 19
OIL IMMERSION OBJECTIVE
- Highest magnification
- Oil prevents refraction of light outwards and
allows it to pass straight in to objective
FBEG - OIL D
ABCD - AIR
What’s the power of this lens?
To calculate the power of magnification, multiply the power of the
ocular lens by the power of the objective, e..g.: 10x40=400 times
What are the powers of
magnification for each
the objectives we have
on our microscopes?
Fill in the table on
Comparing Powers of Magnification
We can see better details with higher the powers of
magnification, but we cannot see as much of the image.
Which of these images
would be viewed at a
higher power of
Let’s give it a try ...
1 – Turn on the microscope and then rotate the nosepiece to click the
red-banded objective into place.
2 – Place a slide on the stage and secure it using the stage clips. Use
the coarse adjustment knob (large knob) to get it the image into view
and then use the fine adjustment knob (small knob) to make it
3 – Once you have the image in view, rotate the nosepiece to view it
under different powers. Draw what you see on your worksheet!
Be careful with the largest objective! Sometimes there is
not enough room and you will not be able to use it!
4 – When you are done, turn off the microscope and put up the
slides you used.
How to make a wet-mount slide …
1 – Get a clean slide and coverslip from your teacher.
2 – Place ONE drop of water in the middle of the slide. Don’t use
too much or the water will run off the edge and make a mess!
3 – Place the edge of the cover slip on one side of the water drop.
4 - Slowly lower the cover slip on top of the drop.
Cover Lower slowly
5 – Place the slide on the stage and view it first with the red-banded
objective. Once you see the image, you can rotate the nosepiece to
view the slide with the different objectives.
You do not need to use the stage clips
when viewing wet-mount slides!
Multiple step operation employed to attain
1. Remove any diffusing filter.
2. Put a slide on the stage and focus.
3. Completely close the field diaphragm.
4. Move the condenser until the border
or the iris hexagon is neat and clear.
5. Center if necessary.
6. Open the field diaphragm until the tip
of the hexagon touches the field limit
The Parts of a Microscope
The Parts of a Microscope
Clips Coarse Adj
Diaphragm Fine Adjustment
The body tube holds the objective lenses
and the ocular lens at the proper distance
The Nose Piece holds the objective lenses
and can be turned to increase the
The Objective Lenses increase
magnification (usually from 10x to 40x)
These 2 clips hold the slide/specimen in
place on the stage.
The Diaphragm controls the amount of light
on the slide/specimen
Turn to let more light in or to
Projects light upwards through the
diaphragm, the specimen and the lenses
Some have lights, others have mirrors
where you must move the mirror to reflect
Magnifies the specimen image
Used to support the microscope when
carried. Holds the body tube, nose piece
and objective lenses
Supports the slide/specimen
Coarse Adjustment Knob
Moves the stage up and down (quickly) for
focusing your image
Fine Adjustment Knob
This knob moves the stage SLIGHTLY to
sharpen the image
Supports the microscope
To determine your magnification…you
just multiply the ocular lens by the
Ocular 10x Objective 40x:10 x 40 = 400
So the object is 400 times “larger”
Objective Lens have
written on them.
Ocular lenses usually magnifies by 10x
HOW A MICROSCOPE WORKS ?
OPTICAL PATH IN COMPOUND
Method of using Compound
1. Grasp the microscopes arm with
one hand and place your other
hand under the base.
2. Place the microscope on a bench.
arm Adjust seat
3. Clean Lenses.
4. Turn the coarse adjustment knob
to raise the body tube
5. Revolve the nose piece to set low-
power objective lens.
6. Adjust the Condenser lenses and
7. Place a slide on the stage and secure
with stage clips.
8. Switch on the light at low intensity and
then increase intensity.
9. Turn the coarse
adjustment knob to lower
the body tube until the low
power objective reaches
its lowest point.
10. Looking through the eyepiece, very slowly move
the coarse adjustment knob until the specimen
comes into focus.
11. Adjust distance between eye piece.
12. Switch to the high power objective lens only
after adjusting condenser and iris diaphragm.
13. Place a drop of oil over specimen before using
oil immersion objective.
14. Lower the objective until oil makes contact with
15. Looking through the eyepiece, very slowly
focus the objective away from the slide i.e by
raising the objective lens.
How to observe a slide ?
Causes of Error in Focusing
- Revolving Nose Piece is off centre
- Preparation is upside down
- Thick cover slip
- Dirt or Dried oil over Lens
- Air bubble in immersion oil
- Poor illumination – Condenser not fully
PHASE CONTRAST MICROSCOPE
Phase Contrast Microscope
- First described in 1934 by Dutch physicist
- Produces high-contrast images of
- Advantage - Living cells can be examined
in their natural state
Principle Of Phase Contrast
- Unstained bacteria have constituents of
different refractive index .
- Diffraction of light
- Phase contrast microscope employs an
optical mechanism to translate minute
variations in phase into corresponding
changes in intensity of image.
- The condenser annulus or annular
diaphragm is opaque flat-black (light
absorbing) plate with a transparent
- Produces hollow cone of light.
- Placed in back focal plane of objective.
1. Enhances phase difference by retarding
diffracted wave front by one quarter of
2. Reduces intensity of direct rays and
equalizes it with diffracted rays
Images of Phase Contrast
Spirilium volutans Clostridium botulinum
Comparision of Images of Bright Field
and Phase Contrast Microscopy
Uses of Phase Contrast Microscopy
- Phase contrast enables visualization of
internal cellular components.
- Diagnosis of tumor cells .
- Examination of growth, dynamics, and
behavior of a wide variety of living cells in
Dark Ground Microscope
- Optical system to enhance the contrast of
unstained bodies .
- Specimen appears gleaming bright against
PRINCIPLE OF DGI
Optical path in Dark Ground
Requisites for Dark Ground
- Dark ground
- High intensity
- Funnel stop
Uses of Dark Ground Microscopy
Useful in demonstrating
- Campylobacter jejuni
FITC EX - 495 nm EM - 520nm
TRITC EX – 540 nm EM – 590 nm
Texas Red Ex – 600 nm EM – 615 nm
- UV rays passes through exciter filter
- Dark ground condenser
- Micro organisms stained with fluorescent
dye, when examined under microscope
with ultraviolet light are seen as bright
object against dark background
Use of Fluorescence Microscopy
- Auramine Rhodamine – Yellow
Fl - Tubercle bacilli
- Acridine Orange R - gives
orange red Fl with RNA and
yellow green Fl with DNA
-Electron Microscopes uses a beam of highly
energetic electrons to examine objects on a
very fine scale. This examination can yield the
- Crystallographic structure
TYPES OF EM
- Transmission Electron Microscope (TEM)
- Scanning Electron Microscope (SEM)
- Stream of electrons is formed
- Accelerated using a positive electrical
- Focused by metallic aperture and Electro
-Interactions occur inside the irradiated sample
which are detected and transformed into an
image . 76
-Projector Lens forms
image on Fluorescent
- 2D Image
10,000 X to 100,000 X
Scanning Electron Microscope
- Scan a gold-plated specimen to give a 3-D
view of the surface of an object which is
black and white.
- Used to study surface features of cells and
- Scanning Electron microscope has resolution
1000 times better than Light microscope .
Working of SEM
Vibrio cholerae with
Treponema pallidum polar flagella 80
- Used in metallurgy
- Examination of cultures in flat bottom
- Micro dissection
- Examination of parasites
- Observation of agglutination in serology
- Double Microscope
- Produces 3D images
- Uses two Polariser
- Gives info about Birefringence of a body
- Used in Crystallography, Urine
- Apple Green Birefringerence in
- Uses a laser beam to illuminate
a specimen whose image is
then digitally enhanced for
viewing on a computer monitor.
- Laser beam scans single plane
of 1µm thickness.
Comparison of Depth of Light
Collection and Image clarity
Light Microscope Confocal Scanning
PRINCIPLE OF CONFOCAL MICROSCOPY
USES OF CONFOCAL
- Observing cellular morphology in
- Eg. used in diagnosing Ca cervix
- Evaluation and diagnosis of basal cell
carcinoma of skin
What is the advantage of using a
- By having a confocal pinhole, the
microscope is really efficient at rejecting
out of focus fluorescent light so that very
thin section of a sample can be analyzed.
- By scanning many thin sections through a
sample, one can build up a very clean
three-dimensional image .
- SCANNING PROBE MICROSCOPE -
Class of Microscope that measures surface
features by moving a sharp probe over
object surface. Used to visualize atoms and
- Scanning Tunneling Microscope
- Atomic Force Microscope (AFM)
CARE OF THE MICROSCOPE
- Proper storage
- Care of Lenses
- Care of oil emersion objective
- Care of lamp