Microscopy

MEERA VORA
Dr. Mahnaz M. Kazi
Dr. Pina J.Trivedi
Cytogenetics Laboratory
The Gujarat Cancer & Research Institute,
Ahmedabad -380 016
 Outline
 Introduction
 Parts of microscope
 Types of microscope
 Light microscope
 Subtypes of microscope
 Electron microscope
• The word “microscope” is formed of two words:
“micros” means small and “skipein” means to look.

• It is an optical instrument consisting of lens or

combination of lenses for making enlarged or
magnified image of minute objects.

• The science dealing with all aspects of a microscope

is called microscopy.
Principle of microscope
• Light from a mirror is reflected up
through the specimen, or object to
be viewed, into the powerful
objective lens, which produces the
first magnification.

• The image produced by the

objective lens is then magnified
again by the eyepiece lens, which
acts as a simple magnifying glass.
 Parts of microscope

eyepiece Adjustment knobs

This is the part used to look through These are knobs that are used to focus the
the microscope. Its found at the top of microscope. two types; fine knobs, coarse
the microscope. knobs.

Objective lenses diaphragm

These are the major lenses used for it’s known as the iris. Its primary role
specimen visualization. They have a is to control the amount of light that
magnification power of 40x-100X.
reaches the specimen.

stage illuminator
This is the section on which the This is the microscopes light source,
specimen is placed for viewing. located at the base. It is used instead of
a mirror. it captures light from an
external source of a low voltage.
 Magnification
• Magnification is the ability of a microscope to produce an image
of an object at a scale larger than its actual size.

• Magnification is related to scaling up visuals or images to be

able to see more detail, increasing resolution, using microscope.

• To find the total magnification of the microscope, multiply

magnification of the objective lens by magnification of the ocular
lens.
40x (objective lens) ✕ 10x (ocular lens) = 400x magnification.
 Resolution
• In microscopy, the term 'resolution' is used to describe the ability
of a microscope to distinguish detail.

• This is the minimum distance at which two distinct points of a

specimen can still be seen as separate entities.
 Numerical Aperture
• The numerical aperture of a microscope objective is a
measure of its ability to gather light and resolve fine
specimen detail at a fixed object distance.
Na = n ✕ sinμ
• n= the refractive index of the medium between the object and
the lens.
• μ =the angle of cone of the light admitted by the lens
• Sin μ can not exceed 1
 Objective lenses and its types

• The objective lens gathers light from the specimen, magnifies

the image of the specimen, and projects the magnified image
into the body tube.
• Type of objective lenses:-
• Plan apochromatic objectives lenses
These objectives produces a flat image across the field of view
• Apochromatic objective lenses
Apochromatic lens, is a photographic or other lens that has
better correction of chromatic and spherical aberration than the
much more common achromat lenses
• Semi apochromatic objective lenses
These lenses are chromatically corrected for red and blue, and
the green focus is also close. They are spherically corrected for
blue and green. This objective is better suited for color viewing or
recording than achromatic objectives.
• Achromatic objective lenses
This objective brings red and blue light to a common focus, and
is corrected for spherical aberrations for green. It is excellent for
black and white viewing. If an objective is not labeled, it is
achromatic.
• Quartz lenses
Quartz has a high optical transparency for a wide range of
wavelengths such as UV and infrared.
 Köhler illumination
• Köhler illumination is a method of specimen illumination used for
transmitted and reflected optical microscopy.
• Köhler illumination acts to generate an even illumination of the
sample and ensures that an image of the illumination source is
not visible in the resulting image.
 Köhler illumination requires several optical components to
function:
• Collector lens and/or field lens
• Field diaphragm
• Condenser diaphragm
• Condenser lens
• method of providing the optimum specimen illumination
• Closing or opening the condenser diaphragm controls the
angle of the light rays emerging from the condenser and
reaching the specimen
 Microscope types
There are three basic types of microscopes: optical
microscopy, charged particle (electron and ion)
microscopy, and scanning probe microscopy.
01 02 03

Light microscopy Electron microscopy Scanning probe microscopy

Scanning probe
commonly uses visible An electron
microscopy (SPM) is a branch
light and a system of microscope is uses a
of microscopy that forms
lenses to generate beam of
images of surfaces using a
magnified images of accelerated electrons as
physical probe that scans the
small objects a source of illumination.
specimen.
Types of light (optical) microscopy
The types of Light Microscopes include:

 Light (optical) microscopy

 Bright field Light Microscope

 Dark-Field Light Microscope

 Phase Contrast Light Microscope

 Fluorescence Light Microscope

Bright field light microscopy
• Simplest optical microscopy illumination
technique
• Uses visible light as source of
illumination
• the shorter the wavelength, the greater
the resolution
• It is the standard microscope that is
used in Biology, Cellular Biology, and
Microbiological Laboratory studies.
 Bright-field illumination,
sample contrast comes from absorbance of light in the sample

[Morphology of cells (brightfield

microscopy). showing tumor cell islands
surrounded by fibroblasts.]
 Dark-field light microscopy
• Dark background/field with the specimen being the only one
illuminated.
• Used in observing unstained specimens
• Light source: light bulb from a microscope
• Condenser type : specialized to block most light from the
source; contains an annular/patch stop which disperses the
light in various direction , resulting to a “cone of light”
• Image formed: dark background with illuminated specimen
Application of dark field microscope
• Is used to identify bacteria like
thin and distinctively shaped.
• Used to identify algae.
• A metallurgical darkfield
microscope is used to observe
hairline metal fractures.
• It is more useful in examining
external details, such as
outlines, edges, grain
boundaries and surface
defects than internal structure.
 Advantages and disadvantages of dark
field light microscopy
Advantages Disadvantages
• Viewing live organisms. • Air bubbles in the slide can cause
problems.
• More detailed view of external • Can easily mistake dust for an
features. organism.
• Adding fluorescent dye increase • Needs intense amount of light
the ability to see specimen. which can hurt the eyes and
cause glare.
• Used to view unstained
specimens more clearly.
 Dark-field illumination,
sample contrast comes from light scattered by the sample

[Images dark field microscopy of

breast cancer cells BT-747 recognized
by the complex GNS-anti HER2 ]
Phase contrast light microscopy
• Enhances contrast in micrographs by
converting phase shifts of light waves into
brightness
• Differences in density or refractive index
within the specimen or cell causes light
waves to be diffracted at different degree
• Diffraction of light waves implies a change
in the phase of their wavelength
• A unique part of the phase contrast
microscope, called the phase-plate,
amplified this change in phase to one – half
wavelength
• When both the direct and reflected types
of waves converge at the ocular lens,
constructive and destructive interference
occurs
• Constructive interference corresponds to
bright spots in the field of view
• Destructive interference corresponds to
dark spots
• The end result is a magnified and highly
contrasted view of a living, unstained,
normally transparent specimen.
• Source of illumination: visible light from
an illuminator
 Application of phase contrast microscope
To produce high-contrast images of transparent specimens, such
as
1.living cells (usually in culture),
2.microorganisms,
3.thin tissue slices,
4.lithographic patterns,
5.fibers,
6.latex dispersions,
7.glass fragments, and
8.subcellular particles (including nuclei and other organelles).
Advantages Disadvantages
Phase-contrast optical components can be Phase-contrast condensers and objective lenses
added to virtually any brightfield microscope, add considerable costly to a microscope
provided the specialized phase objectives
It makes a highly transparent object more To use phase-contrast the light path must be
visible. aligned.
No special preparation of fixation or staining etc. Generally, more light is needed for phase
contrast than for corresponding bright-field
viewing.
Examining intracellular components of living
cells at relatively high resolution.
It made it possible for biologists to study living
cells and how they proliferate through cell
division.
 Phase-contrast illumination,
sample contrast comes from interference of different path lengths of light
through the sample.

[Phase contrast microscopy of Rat1

cells.]
 Fluorescence light microscopy
• The technique is used to study specimen,
which can be made to fluorescence
• Fluorescence is a phenomenon that takes
place when a substances absorbs light at a
given wavelength and emit light at another
wavelength.
• The sample to be analyzed is placed on a
lens and the sample is coated with a
fluorescence materials
• The light is illuminated through the lens with
the higher energy source.
• The illumination light is absorbed by the
• The sample cause them to emit a
longer lower energy wavelength light
• This light can be separated from the
surroundings radiation with filters.
• The light from the light source is
passed through the excitation
• The specific wavelength of light is
passed through the sample via
dichromatic filter
• Objective lens focuses the light to the
specimen
• Light emitted from the specimen is
filtered by barrier filter [structure of a fluorescence microscope]
• Fluorescence dye: use of fluorescence dye has thus increased
the specificity for protein or other molecules in a cell as well as
provide high contrast.
• Two type of fluorescence dyes are commonly used,
1. Fluorescein
2. Rhodamine

Application of fluorescence microscopy

• To identify structures in fixed and live biological samples.
• It allows the use of multicolor staining, labeling of structures
within cells, and the measurement of the physiological state of
a cell.
Advantages Disadvantages
Fluorescence microscopy is the most popular Fluorophores lose their ability to fluoresce as they
method for studying the dynamic behavior are illuminated in a process
exhibited in live-cell imaging called photobleaching.

Different molecules can now be stained with fluorescent molecules have a tendency to
different colors, allowing multiple types of the generate reactive chemical species when under
molecule to be tracked simultaneously. illumination which enhances the phototoxic
effect.
The sensitivity is high enough to detect as Cells are susceptible to phototoxicity, particularly
few as 50 molecules per cubic micrometer. with short-wavelength light.
fluorescence microscopy only allows observation
of the specific structures which have been labeled
for fluorescence.
Fluorescence light microscopy

[Colon cancer cells, fluorescence light

micrograph]
 Electron microscope
• An electron microscope is a microscope that uses a beam of
accelerated electrons as a source of illumination.
• The wavelength of an electron can be up to 1,00,000 times
shorter than that of visible light photons.
• Electron microscope have a higher resolving power than light
microscope and can reveal the structure of smaller objects.

 It offers unique possibilities to gain insight into;

1. Structure
2. Topology
3. Morphology
4. Composition of materials
Advantages of Electron microscope
• Very high magnification, Incredibly high resolution
• Material rarely distorted by preparation
• It is possible to investigate a greater depth of field
• Diverse applications
• To study objects of >0.2 micrometer.
• For analysis of subcellular structure
• For study of intracellular pathogens and viruses
Types of electron microscope
Analytical Scanning Scanning electron Transmission
electron transmission microscope (SEM) electron
microscope electron microscope (TEM)
(AEM) microscope(STEM)
 Transmission electron microscope(TEM)
• TEM is a microscopy technique
in which a beam of electrons is
transmitted through an ultra-
thin specimen, interacting with
the specimen as it passes
through it.
• A transmission electron
microscope can achieve better
than 50pm resolution and
magnification of up to about
10,000,000x
 Scanning Electron Microscope (SEM)
● A scanning electron
microscope (SEM) is a type
of electron microscope that
produces images of a sample by
scanning the surface with a
focused beam of electrons.

● The electrons interact

with atoms in the sample,
producing various signals that
contain information about the
surface topography and
Thank you…..