Study of

Microscopes
 KEY
CONCEPTS
 History of Microscopy
 Common terminology used in
microscopy
 Basic Principle of Light microscopy
 Handling of microscope
 Applications of light microscope
 Principle, working and applications
of Phase contrast microscope
 Principle, working and applications
of Electron microscope
 Introduction to
Microscopes
• Microscopes are used both in classrooms and in
making important evaluations in medical
laboratories and other microtechnologies.
• The different types of microscopes are designed
for these different uses, and therefore will vary
based on their resolution, magnification, depth
of field, field of view, illumination method,
degree of automation, and type of image they
produce..
Four categories of microscopes:

• Dissection
• Compound.
• Electron
• Phase Contrast
• Confocal
 HISTORY OF MICROSCOPES

• Hans and Zacharias Janssen

invented the first microscope
between 1590 and 1610.
• The first well known users were
van Leeuwenhoek and
Hooke.
• Van Leeuwenhoek used a
single lens microscope, Hooke
used a compound microscope.
 History (Contd.)

• The real improvement of the microscope came with the invention

of the achromatic lens.
• Achromatic lenses for spectacles were developed by Chester
Moore Hall, in 1729.
• Jan and Harmanus van Deyl were the first to make these lenses,
at the end of the eigteenth century and Harmanus van Deyl started
the commercial fabrication of achromatic objectives in 1807.

An achromatic lens can be defined as a lens which is made by a combination of two

different types of lenses carrying different focal powers in a manner such that the
images formed by the light of both the combined lenses are free from chromatic
aberration or achromatism.
 History (Contd.)
Further developments followed…..
…..improvement of the microscope stand
……… the development of methods to increase the contrast.

The Dutchman Zernike invented the phase contrast

microscope in 1934.
• Other contrast enhancing methods were developed,
modulation contrast devised by Hoffman and Gross and
differential interference contrast (DIC) , all with several
variants.
• A well known DIC sytem was patented by Nomarsky in
1953.
• Fluorescence microscopy, in principle already seen by
Köhler in 1904, has become a very valuable addition to
light microscopy since about 1970
 MICROSCOPY:
•
INTRODUCTION
Microscopy is defined as the use of a microscope to
magnify and study the small objects that are too small to
be visualized with the naked eye.

• Three main types of microscopes:

i. Optical or light microscope,

ii. Scanning probe microscope, and
iii. Electron microscope.
• Power - Naked eye ~ 0.1 mm
- Light microscope ~ 0.1 μm
- Electron microscope ~ 2.5 nm
 The modern day
Microscope A microscope is an array of two
lenses.
• Ocular lens/ Eye piece and
• Objective lens.
Lenses combine to enlarge the objects.
Focal plane Image plane

Image plane

Condenser Eyepiece
lens Objective Tube lens
lens lens

Modern microscope with ICS

(Infinity Colour corrected System)

Classic compound microscope

Always carry a microscope with one hand holding the arm and
one hand under the base.
 MICROSCOPY – BASIC
PRINCIPLE
• Light passes through specimen through a single or
a series of magnifying lenses to allow a magnified
view of the sample.

• Important factors in light microscope include:

– Magnification

– Resolution

– Contrast
 MICROSCOPY – BASIC
PRINCIPLE
• Magnification:
Total magnification (M) achieved is the product of
the magnification power of the lenses used.
- M (Microscope) = M (Objective lens) X M
(Ocular lens)
• Simple microscopes, have a magnification power of
1000- 1500 whereas electron microscopes have
magnification power greater than 250,000.
Comparing Powers of Magnification
 Calculating Magnification

• Find the power of the lens. It is found on the side of the lens.
Magnification power of a lens is always identified by the label
of x (10x, 1000x)
• Multiply the power of the eyepiece by the power of the
objective lens.
Examples:
eyepiece obj. lens Magnification
10x times 100x 1000x
10x 50x 500x
10x times 40x 400x

times
 MICROSCOPY – BASIC
• PRINCIPLE
Resolution:
- Resolving power is the ability of a lens to separate or distinguish
small objects that are close together.
- Depends on the quality of lens and the wavelength of illuminating
light.
- shorter wavelength  greater resolution
• Contrast:
- Reflects the number of visible shades in the specimen.
- Is needed to make objects stand out from the background.
- Achieved through various staining techniques.
- Microorganisms are essentially transparent and must be stained
for bright-field microscopy.
 MICROSCOPY – BASIC
Absorption
PRINCIPLE
When light passes through an object the intensity is reduced depending upon the color
absorbed. Thus the selective absorption of white light produces colored light.

Refraction
Direction change of a ray of light passing from one transparent medium to another with
different optical density. A ray from less to more dense medium is bent perpendicular to
the surface (towards the normal), with greater deviation for shorter wavelengths.

ra Short wavelengths are “bent”

c more than long wavelengths

Light is “bent” and the resultant colors separate (dispersion).

Red is least refracted, violet most refracted.
 MICROSCOPY – BASIC
PRINCIPLE
Control

We see the color that is reflected by the object

Absorption

No blue/green light
red filter
 MICROSCOPY – BASIC
PRINCIPLE
Refractive index
It is a measure of how greatly a substance slows the velocity of light, and the direction
and magnitude of bending is determined by the refractive indexes of the two media
forming the interface. When light passes from air into glass, a medium with a greater
refractive index, it is slowed and bent toward the normal.

Focal point & Focal length

When the light source is distant so that parallel rays of light strike the lens, a convex lens
will focus these rays at a specific point, the focal point. The distance between the center
of the lens and the focal point is called the focal length.
 Reflection and
Refraction
Snell’s Law: The angle of
reflection (Ør) is equal to the angle
Transmitte
of incidence (Øi) regardless of the
Reflected Beam d
(refracted)Beam
surface material
The angle of the transmitted
r t beam (Øt) is dependent upon the
composition of the material
i

Incident Beam

n1 sin Øi = n2 sin Øt

The velocity of light in a material

of refractive index n

is c/n
 Numerical
Aperture
Resolving power is Resolving power is
directly related the ability of an
to numerical aperture. A objective to resolve
two distinct lines
The higher the NA the very close together
greater the resolution

Light cone

NA=n(sin )

(n=the lowest refractive index between the

object and first objective element) (hopefully 1)
 is 1/2 the angular aperture of the objective
 Numerical
• The wider the angleAperture
the lens is capable of receiving light at, the greater
its resolving power
• The higher the NA, the shorter the working distance

Images reproduced from:

http://micro.magnet.fsu.edu/
 MICROSCOPY – BASIC
TERMS

Field of View
It is the diameter of the circle of light that you see when looking into a
microscope and it is measured in millimeters. The lowest powers have the
widest field of view. As you increase power, the field of view gets smaller.
Some wide angle or super wide angle eyepieces increase the field of view
over standard eyepieces.

Depth of Field
It is a function of the objective lenses and means the farthest and nearest
points in the field of view are in simultaneous sharp focus. Low magnification
objectives have more depth of field than high magnification objectives.
 Field of
View
Field of view is the area (circle) that you see when
looking through the eyepiece
 MICROSCOPE
TYPES

Light microscope Electron microscope

Compound microscope
Bright-field microscope Transmission
electron microscope
Dark-field microscope (TEM)
Phase-contrast microscope Scanning electron
Fluorescence microscope microscope
(SEM)
 MICROSCOPE
TYPES
COMPOUND MICROSCOPE DISSECTION MICROSCOPE
Compound microscopes are light A dissection microscope is light
illuminated. The image seen illuminated. The image that
with this type of microscope is appears is three dimensional. It
two This is used for dissection to get a
dimensional.
microscope is the better look the larger
commonly used. You can view
most at specimen.You cannot see
individual cells, even living individual cells because it has a
ones. It has high magnification. low magnification.
However, it has a low resolution.
 MICROSCOPE
TYPES
SCANNING ELECTRON MICROSCPE TRANSMISSION ELECTRON MICROSCPE

SEM uses electron illumination. TEM is also electron

The image is three illuminated. This gives a two
dimension. seen in dimensional view. Thin slices of
magnification It high specimen are obtained. The
has
resolution. The specimen is electron beams pass through
coated in gold andand high
the electrons this. It has high magnification
bounce off to give you and and high resolution.
exterior view of the specimen.
The pictures are in black and
white.
 Light Microscopes

Light source

Lens: to focus
light on sample

Sample
Transmitt
ed light

Stacking
of lenses
to
increase
magnifica
eye, objective,
tion
camera
Lens: to
focus light
COMPOUNDLIGHT
MICROSCOPY

(Atlas RM: Principles of microbiology, St Louis, 2006, Mosby.)

 Ray Diagram of Image
formation in Compound
microscope

Fo,- Focal length of objective length (L1).

I1 - Magnified image (I1) of the object.
Fe,- Focal length of eye piece (L2).
I2- Magnified image (I2) of the object.

Image
 PARTS
OFACOMPOUNDMICROSCOPE The lens of microscope
that you look through

holds the eyepiece and the

objective lenses

The large knob on the microscope

that you turn to bring the object into
Support the body tube focus
Bottom of the body tube that
hol the objective lenses
for fine adjustment

Specimen is put here

Holds the slides

Source to capture daylight/

Always carry a microscope Mostly replaced by electric
with one hand holding the bulbs
arm and one hand under the
Supports the rest of
base. the
PARTS OFASTEREO
MICROSCOPE
holds the eyepiece and
the
objective lenses

Always carry a microscope with one hand holding

r the arm and one
hand under the base.
 PARTS
OFACOMPOUNDMICROSCOPE
Head (Body)
• It is the upper part of the microscope that connects the eyepiece
to the nosepiece or turret. Some heads are fixed in place and
allow you to tilt them from angles of 0° up to 60° or even 360
°.
• Monocular –with a single eyepiece. Economical models and
are very satisfactory for their usage. A monocular head with a
second vertical viewing port is called a teaching head (dual
view head) which can be used by a second person (or teacher)
to observe the same image as the first person or to be used with Monocular Head
various cameras.
• Binocular –with two eyepieces. They are generally used on
high power compound microscopes and all low power stereo
microscopes and are generally more comfortable.
• There are different types of heads for adjusting the
interpupillary distance (IPD)
• (1) seidentoff – IPD is adjusted by twisting the eyepieces
similar to most binoculars
• (2) slider – IPD is adjustable side-to-side by sliding
the eyepieces toward and away from each other.
 PARTS
OFACOMPOUNDMICROSCOPE
Objective Lenses
• The objective lenses are the most important
components of microscopes ranging from 4X
to 100X magnification in common
microscopes.

• Their basic function is to gather the light

passing through the specimen and then to
project the image up into the body of the
microscope.

• Then, the eyepiece lens system further

magnifies the image for your eye to see.

• Most quality microscopes use glass for

the objectives
Objectives

PLAN-APO-40X 0.25 N.A. 160/1.2

Flat field Apochromat Magnification Numerical Tube Coverglass

Factor Aperture Length Thickness
 PARTS
OFACOMPOUNDMICROSCOPE
Condenser
• This is made up of two simple lenses and it condenses
light on to the object.

Light/ Illumination system

• For day light illumination, a mirror is fitted which is plane
on one side and concave on the other side.
• Plane mirror is used in sunlight while concave in
artificial light.
• Currently, most of the microscopes have in-built electrical
illumination varying from 20 to 100 watts.
Koehler illumination system
• It is a technique to optimize light quality and sharpness by
aligning and adjusting each component of the optical
system starting with a focusing illuminator.
• The light quality will be even and bright.
• The Koehler is the best form of illumination possible,
offered
 PARTS
OFACOMPOUNDMICROSCOPE
Diaphragm
• It is also called the sub-stage diaphragm or aperture
diaphragm.
• The diaphragm is normally located under the stage and
adjusts the amount of light passing into the slide or specimen.
• It is most useful at high powers.
Most compound microscopes have one of two types :
1.Disc Diaphragm –simple and less expensive. It is located
between the light source and the slide or specimen. It contains a
rotating disk (usually fixed) with 5-10 openings of differing
diameters to control the amount of light passing through to the
specimen.
2.Iris Diaphragm – is the better but expensive. It has a
continuously variable diameter (like the iris of an eye or a camera
shutter) which has a function to limit the size of the opening
through which light passes from the light source to optimize
resolution, contrast, and sharpness. It is usually controlled by a
lever.
 PARTS
Stage
OFACOMPOUNDMICROSCOPE

• The platform beneath the objectives on which the slide or object to be observed is placed
is called a stage.
• It has a smooth, flat surface and can be rectangular or circular.
• It can be stationary or can move up and down.
•It has an opening for passing light. Plain
stage – Simple, mostly stationary
Mechanical stage - The more sophisticated
 It makes it much easier to center specimen slides (standard slide size is 1”x 3”) with
precise movements in two axes with knobs that slides the stage to fix the specimen in
field of view.
 A vernier type scale on the stage allows for making measurements of the specimen.
 The “X” axis moves a slide forward and back (north/south) and the “Y” axis moves the
slide side-to-side (east/west or left/right)
 COMPOUND LIGHT
MICROSCOPY
• The light microscopes use visible light or ultraviolet
rays to illuminate specimens.
• Two lenses in the compound microscope:
- Ocular lens (10x, sometimes 20x)
- Objective lens (4x, 10x, 20x, 40x, 100x)
• Resolution and contrast are controlled by condenser
and iris diaphragm.
• The condenser functions to focus the light source on
the specimen and provides uniform illumination.
• An iris diaphragm controls the amount of light
reaching the specimen.
 Applications of Compound
Microscope
 A compound microscope is used for viewing
small specimens.
 Used to visualize different samples at high
magnification (40 – 1000x).
 Used to observe both prokaryotic and
eukaryotic cells.
 To study cells and cell structures.
 Plays a pivotal role in the Clinical
laboratory.
- To detect the microorganisms directly in
clinical specimens.
 USING THE MICROSCOPE

• To carry the microscope grasp the microscopes

arm with one hand. Place your other hand under
the base.
• Place the microscope on a table with the arm
toward you.
• Turn the coarse adjustment knob to raise the body
tube.
• Revolve the nosepiece until the low-power
objective clicks into place.
• Adjust the diaphragm for proper light .
• While looking through the eyepiece, also adjust the
mirror until you see a bright white circle of light.
 HANDLING &
CARE
• DON'T SWING THE MICROSCOPE
• Never touch the lenses with your fingers. Your body
produces an oil that smudges the glass. Use only lens
paper to clean the glass.
• When you are finished with your "scope" assignment,
rotate the nose piece so that it's on the low power
objective, roll the nose piece so that it's all the way down
to the stage, then replace the dust cover
• USE PROPER TRANSPORTING TECHNIQUES
• Dust is an enemy to microscope lenses; always keep the
microscope covered when not in use.
 THE DARK-FIELD
MICROSCOPY
• Produce a bright image against a dark background.
• This microscopy is used to examine transparent or semi-transparent
specimens which cannot be distinguished from the background. It
shuts out background light and allows only scattered light to reach the
specimen in order to heighten textural detail.
• Dark-field microscopy removes the unscattered beam from the
image. Only light that has been reflected or refracted by
the specimen forms the image.
• This is accomplished through the use of an annular aperture that
will produce a hollow cone of light that does not enter the
objective lens.
APPLICATIONS:
 Used to observe living, unstained samples.
 Used for visualizing different types of algae.
 Used for viewing blood cells and bacteria. (dark-field microscope,
combined with phase contrast)
 THE BRIGHT-FIELD
MICROSCOPY
• Bright field microscopy is the conventional technique.
• Produce a dark image against a brighter background.
• Bright-field microscopy uses light from the lamp source
to illuminate the specimen.
• This light is gathered in the condenser, then shaped into a
cone where the apex is focused on the plane of the
specimen.

APPLICATIONS:
 Used for observing stained or naturally pigmented or
highly contrasted specimens.
 Generally used with compound microscopes.
Comparison of Light Pathways of bright field and dark
field Microscopy

Light Field Dark Field

http://www.tutorsglobe.com/homework-help/microbiology/dark-field-microscope-72650.aspx
https://www.slideshare.net/shamawan/dark-field-microscopy-1-by-ehtisham-ul-haq
 THE PHASE-CONTRAST
MICROSCOPY
Frits Zernike was awarded the Nobel Prize in Physics in 1953
for the invention of Phase Contrast Microscope.
 THE PHASE-CONTRAST
MICROSCOPY
Principle of phase contrast
• Light is also an oscillation and the
phase changes, when passing
through an object.
• Even if the object is colorless and
transparent, there is still a
change in phase when light pass
through it.
• When light passes through an object
that is more optically dense than its
environment (background), the
wavefronts are retarded with respect to
the unaffected, bypassing background
light.
 THE PHASE-CONTRAST
M ICROSCOPY
Principle (Contd.)
• There is a difference in the phase of the
light that has passed through
(diffracted light) and the remaining
light (direct light).
• The phase shift of the object light is
retarted by ¼ of the uninterrupted
light, in most biological samples.
• The visualization of phase shifts in the
object light is to change the phase of
the background light is done on the
image plane.
• The object would appear dark against
the background due to the difference in
the phases of the background and the
object.
 THE PHASE-CONTRAST
M ICROSCOPY
Principle (Contd.)

Image dark and background bright Image bright and background dark
 Working Of PHASE-CONTRAST
M ICROSCOPE
It has a special type of condenser,
objective and a special
magnifier.

 Condenser annulus: It is an an
opaque disk with a thin
transparent ring that produces a
hollow cone of light.
 Condenser: Condenses the light
cone which passes through the
specimen.
 Working Of PHASE-CONTRAST
MICROSCOPE

• As this cone passes through a

cell some light rays are bent
due to variation in density and Undiffracte
d light
refractive index within the Diffracted
light
specimen and are retarded by
1/4 wavelength.

• The deviated light is

focused
to form an image of the object.
 Working Of PHASE-CONTRAST
MICROSCOPE
• The un-deviated light rays
strike a phase ring in the phase
plate.

• It is a special optical disks

located in the objective, while
the deviated rays miss the
ring and passed through the
rest of the plate.

• The undeviated light which

strikes the phase ring gets
advance by 1/4 wavelength
while passing through this ring.
THE PHASE-CONTRAST
MICROSCOPY

Optical Path of Phase Contrast Microscope

THE PHASE-CONTRAST
MICROSCOPY
 Working Of PHASE-CONTRAST
•
M ICROSCOPE
The deviated and un-deviated
waves become 1/2 wavelength
different from each other.

• These diffracted rays from the

specimen and the undiffracted
rays combine and the phase
difference is converted into
difference in light intensity.

• The background formed by

un-deviated light is bright
while the unstained object
appears dark and well-
defined.
 Working Of PHASE-CONTRAST
MICROSCOPE
• The phase plate is designed and positioned in such a way that the
retarded object light will through the area of phase plate which
is coated with phase retarding material.
• Therefore 1/4th (λ/4) retarded phase of specimen further retards by
1/4th (λ/4)
Final retardation of specimen light = (λ/4) +(λ/4) = (λ/2)
• On the other hand, the undiffracted background light passes
through a
channel and remains unaltered.
• When the two lights combine at the focal length, a
negative/destructive interference is created.

Specimen appears darker in a bright background

 Working Of PHASE-CONTRAST
MICROSCOPE
• If the undiffracted background light passes the area of phase plate
which is coated with phase retarding material.
• Therefore phase of background retards by 1/4th (λ/4)
• On the other hand, the retarded object light when passes through the
channel, remains λ/4, from the previous retardation.
• When the two lights combine at the focal length, a
positive/constructive interference is created.

Specimen appears brighter in a dark background

 Working Of PHASE-CONTRAST
M ICROSCOPE
A combination of destructive and constructive interference create
high contrast in the final image with fine details.
 Applications of Phase-contrast
Microscope
• Used to study prokaryote and eukaryote cells.
• Used to observe living cells, bacterial
components such as endospores,
inclusion bodies.
• Transparent cells can be observed without
staining them.
• Because it is not necessary to stain cells, cell
division and other processes can be observed in a living
state.

Phase Contrast Microscopy- https://youtu.be/vr4tYUnaHNQ

https://en.wikipedia.org/wiki/File:Dark_field_and_phase_contrast_mi
 ELECTRON
MICROSCOPE
Invented by two German scientists, Ernst Ruska and Max Knoll in
1931,
 ELECTRON
MICROSCOPE
The electron microscope is an optical instrument which utilizes
electrons as source of illumination for observing objects at a great
magnification.
Wavelength of electron beam is much shorter than light, resulting
in much higher resolution.
 The resolution is about 0.01 nanometers (magnification up to
300,000X).

 Two different types of EMs are:

 Transmission Electron Microscope (TEM): TEM allows one
the study of the inner structure and contours of objects (tissues,
cells, virusses)
 Scanning electron Microscope (SEM): SEM is applied to
visualize the surface of tissues, macromolecular aggregates and
materials.
 Type of Electron
Microscope

TEM images are 2D projections of the sample while SEMs

provide a 3D image of the surface of the sample
Type of Electron
Microscope
 Principle of Electron
Microscope
• The overall design of an electron microscope is similar to that of a
light microscope.
• In the electron microscope, the light is substituted with electrons and the
glass lenses are substituted with electromagnetic/electrostatic lenses.
 Principle of Electron
Microscope
• The electrons are accelerated
with high voltages (60 – 1200
kV depending on the type of
TEM) and are guided through
the electron microscope column
by electromagnetic lenses.
• The beam penetrates and
interacts with the specimen and
leads to an image.
• The image is monitored on a
phosphorescent screen or
specially designed CCD camera
and recorded.
 Parts of Electron
Microscope
Electron Gun:
• It is the source of electrons and is
located at the top of microscope body.
• It consists of hot, tungsten filament,
which forms the beam of electrons.
Microscope Column:
• Consists of an evacuated metal tube.
houses the electron gun,
electromagnetic lenses, viewing screen
and photographic plate.
• It provides shield to the operator from
x-rays that are generated
when
electrons strike the metal surface.
Electromagnetic lenses or coils:
• The coils correspond to the condenser, objective and ocular lenses of the
light microscope.
• Each coil has coils of electric wire wound on a hollow metal cylinder.
 Parts of Electron
Microscope
Fluorescent Screen:
• The magnified image is viewed on a
fluorescent screen.
• The screen is coated with a chemical
which, by its excitation, forms the image
as on the television screen.
Transformers:
High voltage current is required for the
electron gun provided by high voltage
transformers which can boost the voltage
from 220V to 50- 100KV.
Vacuum Pumps:
Vacuum inside the microscope columns
which is maintained with the help of high
vacuum pumping by diffusion/iron pumps
Water Cooling Systems: The water cooling systems is required to prevent
overheating of the various parts by the pumps and coils.
 Working of Electron
Microscopes
The electrons are used for magnification and image formation.
 In TEM, a tungsten filament is used to make an
electron beam in a vacuum chamber.
 The beam is condensed with the help of positively
charged anode rays and a condenser to fall on the
surface of the sample.
 The beam passes through the thin slice of sample, less
than 100nm and scatter the electrons.
 Denser regions in specimen, scatter more electrons
and appear darker.
 Magnetic (and electric) field of the lenses control path
of electrons.
 Transmitted electrons (those that do not scatter) pass
through the objective coil, which magnifies the object,
 The projective coil further magnifies the image
and projects it on the fluorescent screen.
 The contrast of biological material can be
increased by
using salts of high atomic number.
 Working of Electron
Magnification:
Microscopes
 The objective and projector coils
help in magnifying the image
formed in the electron microscope.
 In order to have maximum
magnification, an intermediate coil
is fitted between objective
projector coils.
and
Resolving Power:
 The electrons can be used with much
shorter wavelength for finer details.
 At 100 KV voltage the wavelength of
electrons is 0.037 Ao and the limit of
resolution is less than 0.018 Ao.
 The resolving power of a good electron
microscope is 4-10 Ao.
 Working of Electron
Microscopes
Scanning Electron Microscope
 The SEM is used in studying
surface
structure of thick specimens.
 The electrons do not form the image by being
transmitted but by getting emitted from the
surface of the specimen.
 Generally, the image resolution of an SEM
poorer than that of a TEM.
 As the SEM image relies on surface
emissions rather than transmission, it is able
to image bulk samples up to many
centimeters in size
 It has a great depth of field, and so can
produce 3 dimensional images.
Working of Electron
Microscopes
 Applications of Electron
Microscopes
 Electron microscopes are used to investigate the ultra structure
of a wide range of biological and inorganic specimens including
microorganisms, cells, large molecules, biopsy samples, metals,
and crystals.
 Industrially, the electron microscope is often used for quality
control and failure analysis.

https://www.youtube.com/watch?v=PmfjYkKeVEA
https://www.youtube.com/watch?v=sCJxxkeOaGw
 Light Microscopes Vs Electron
1
Microscope
Light Microscope Electron Microscope (EM)
Light rays are used to illuminate the specimen. Electron beam produced by the cathode is used

2 Condenser, objective and eye piece lenses are made of glass. All lenses are electromagnets.

Electron microscope ha high resolving power i.e.,

Light microscope has low resolving power i.e., below 0.25 μm to about 250 times more than light microscope. Thus, it
3. 0.30 μm. can resolve down to 0.0001 μm.

EM has direct magnification as high as 160,000x and

4 It has a useful magnification of 500x to 1500x. photographic magnification is 1000,000x or more

In light microscope the image formation depends upon of light In electron microscope, it is due to electron
5 absorption in different zones of the objects. scattering.

The image is received on zinc sulphate fluorescent

6 Image is seen by eyes through ocular lens. No screen is needed. screen or photographic plate
7 No vacuum is needed EM operates at high vacuum
8 Cheap and negligible running cost Very costly and heavy running costs

9. For viewing, the object /cell/section is placed on glass slide For viewing, the object /cell/section is placed on the
grid
10. Specimen may be living, dead or Non-living Specimen may be dead or non-living

11.. Colored Dyes are used as stains Heavy Metals are used as stains

12. Colored image is formed Black and White image is formed

13. Wavelength of radiation used: 400 to 700nm Wavelength of radiation used: 0.005 nm
Light Microscopes Vs Electron
Microscope

Image formed by a light Image formed by an electron

microscope microscope