TEJASVI NAVADHITAMASTU

“Let our (the teacher and the taught) learning be radiant”

Let our efforts at learning be luminous and filled with joy, and endowed with the force of purpose

Dr. Prabhakar Singh

Department of Biochemistry
Faculty of Science, V.B.S. Purvanchal University, Jaunpur
PIN-222 003, (U.P.), INDIA.
Mobile No.: +91-9454695363
E-Mail: [email protected]
 VEER BAHADUR SINGH PURVANCHAL UNIVERSITY JAUNPUR-222003

E –content
Course: M.Sc.
Subject: Biochemistry; Biotechnology, Microbiology, Environmental Science
Topic: Instrumentation and Analytical Techniques
Subtopic: MICROSCOPY
Prepared by: Dr. Prabhakar Singh
Department : Biochemistry
Faculty : Science
Email: [email protected]
Contact: +91-9454695363

Note-This E-content has been prepared as a reading material for students without any
commercial interest. Original contributors are acknowledged.
MICROSCOPY
• A microscope (Greek: micron = small and scopos
= aim)
• MICROSCOPE - 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
microscopy
Microscopy is to get a magnified image, in which structures may be
resolved which could not be resolved with the help of an unaided eye.
Magnification
• It is the ratio of the size of an object seen
under microscope to the actual size
observed with unaided eye.
• The total magnification of microscope
is calculated by multiplying the magnifying
power of the objective lens by that of eye piece.
Resolving power
• It is the ability to differentiate two close points as separate.
• The resolving power of human eye is 0.25 mm
• The light microscope can separate dots that are 0.25µm apart.
• The electron microscope can separate dots that are 0.5nm apart.
Limit of resolution
It is the minimum distance between two points to identify them
separately.
It is calculated by Abbé equation.

Limit of resolution is inversely proportional to power or resolution.

If the wavelength is shorter then the resolution will be greater.
Working distance
• It is the distance between the objective
and the objective slide.
• The working distance decreases with
increasing magnification.
In most areas of optics, and especially
in microscopy, the numerical aperture
of an optical system such as
an objective lens is defined by

where n is the index of refraction of the

medium in which the lens is working
(1.00 for air, 1.33 for pure water, and
typically 1.52 for immersion oil;[1] see
Due to Snell's law, the numerical aperture
also list of refractive indices), and θ is
remains the same:
the maximal half-angle of the cone of
light that can enter or exit the lens. In
general, this is the angle of the
real marginal ray in the system.
Angular aperture
The angular aperture of a lens is the apparent angle of the lens aperture as
seen from the focal point:

In a medium with an index of refraction close to 1, such as air, the angular

aperture is approximately equal to twice the numerical aperture of the
lens.[1]

f-number

In optics, the f-number (sometimes called focal ratio, f-ratio, f-stop,

or relative aperture[1]) of an optical system is theratio of the lens's focal
length to the diameter of the entrance pupil.[2] I
Light microscope
The optical microscope, often referred to as light microscope, is a type
of microscope which uses visible light and a system of lenses to magnify
images of small samples. Optical microscopes are the oldest design of
microscope and were possibly invented in their present compound form in
the 17th century. Basic optical microscopes can be very simple, although
there are many complex designs which aim to improve resolution and
sample contrast.
SIMPLE MICROSCOPE

A simple microscope is a
microscope that uses a lens
or set of lenses to enlarge an
object through angular
magnification alone, giving
the viewer an erect
enlarged virtual
image.[3][4] Simple
microscopes are not capable
of high magnification. The
use of a single convex lens
or groups of lenses are still
found in simple magnification
devices such as
the magnifying glass.
COMPOUND MICROSCOPE

A compound microscope is a
microscope which uses a lens close to
the object being viewed to collect light
(called the objective lens) which
focuses a real image of the object
inside the microscope (image 1). That
image is then magnified by a second
lens or group of lenses (called
the eyepiece) that gives the viewer an
enlarged inverted virtual image of the
object (image 2).[5] The use of a
compound objective/eyepiece
combination allows for much higher
magnification, reduced chromatic
aberration and exchangeable objective
lenses to adjust the magnification. A
compound microscope also enables
more advanced illumination setups,
such as phase contrast.
OPTICAL PATH IN COMPOUND MICROSCOPE
Parts of microscope

• Illuminator - This is the light source located below the specimen.

• Condenser - Focuses the ray of light through the specimen.
• Stage - The fixed stage is a horizontal platform that holds the specimen.
• Objective - The lens that is directly above the stage.
• Nosepiece - The portion of the body that holds the objectives over the
stage.
• Iris diaphragm - Regulates the amount of light into the condenser.
• Base – Base supports the microscope which is horseshoe shaped.
• Coarse focusing knob - Used to make relatively wide focusing
adjustments to the microscope.
• Fine focusing knob - Used to make relatively small adjustments to
the microscope.
• Body - The microscope body.
• Ocular eyepiece - Lens on the top of the body tube. It has a
magnification of 10× normal vision.
 Objective
PROPERTY LOW POWER HIGH POWER OIL IMMERSION
Magnification of objective 10x 40-45x 90-100x

Magnification of eyepiece 10x 10x 10x

Total magnification 100x 450 – 450x 900 – 1000x

Numerical aperture 0.25 – 0.30 0.55 – 0.65 1.25 – 1.4

Mirror used Concave Concave Plane
Focal length (Approx) 16 mm 4 mm 1.8 – 2 mm
Working distance 4 – 8 mm 0.5 – 0.7 mm 0.1 mm
Iris diaphragm Partially closed Partially opened Fully opened
Position of condenser Lowest Slightly raised Fully raised
Maximum resolution(Approx) 0.9 µm 0.35µm 0.18µm
 Bright Field Microscopy
• Simplest optical microscopy illumination technique
• Uses visible light as source of illumination
• > the shorter the wavelength, the greater the
resolution (blue is the best)
• Contrast comes from absorbance of light in the
sample, or from staining.
• When the diaphragm is wide open the image is
brighter and contrast is low.
• Sources of illumination: Lamp on the base
• Types of image produced: Relatively large
internal structures and outline can be seen
• Total Magnification: (if 10x ocular
magnification is used)
Range: 10x-1000x
• Resolution: Up to 200nm (white light)
Source: http://www.austincc.edu/biocr/1406/labm/ex3/prelab_3_8.htm
 Dark Field Microscopy
Dark field microscopy (dark ground microscopy) describes microscopy
methods, in both light and electron microscopy, which exclude the
unscattered beam from the image. As a result, the field around the
specimen (i.e., where there is no specimen to scatter the beam) is
generally dark.

• Type of microscopy which is the exact opposite of a bright field

microscope
• Dark background/field with the specimen being the only one
illuminated.
• Used in observing unstained specimens
• Most microscopes have the potential to do dark field
microscopy such as compound or stereomicroscopes.
• 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 directions, resulting to a
“cone of light”
• Image formed: Dark background with illuminated
specimen; may be inverted or not depending on
microscope used
• Total Magnification: Can range from those of
compound microscopes (10x to 1000x)
 Phase Contrast Microscopy
Phase-contrast imaging is a method of imaging that has a range of different
applications. It exploits differences in the refractive index of different materials to
differentiate between structures under analysis. In conventional light microscopy,
phase contrast can be employed to distinguish between structures of similar
transparency, and to examine crystals on the basis of their double refraction.

Phase-contrast microscopy is an optical-microscopy technique that

converts phase shifts in light passing through a transparent specimen to
brightness changes in the image.
PRINCIPLE
• A unique part of the phase-contrast microscope,
called the phase-plate, amplifies this change in
phase to one-half wavelength
• When both the direct (undiffracted) and reflected
(diffracted) types of light waves converge at the
ocular lens, constructive and destructive
interference occurs
In 1935 F.Zernike produced the phase contrast microscope. Phase-contrast
microscope is also called as zernike microscope.

Phase-contrast microscope uses a special condenser and objective lenses.

This condenser lens on the light microscope splits a light beam and throws the
light rays slightly out of phase.

The separated beams of light then pass through and around the specimen, and
small differences in the refractive index within the specimen show up as different
degrees of brightness and contrast.

Uses:
Phase-contrast microscopy is especially useful for studying microbial motility,
studying eukaryotic Cells, determining the shape of living cells, and detecting
bacterial components such as endospores and Inclusion bodies that contain
poly--hydroxyalkanoates (e.g., poly-hydroxybutyrate), polymetaphosphate,
sulfur, or other substances.
• Constructive interference corresponds to bright spots in the
field of view
• Destructive interference corresponds to dark spots
 A fluorescence microscope is basically a conventional light
microscope with added features and components that extend its
capabilities.

conventional microscope fluorescence microscope

 uses light to illuminate the sample and

 uses a much higher intensity


light to illuminate the sample
produce a magnified image of the sample.
This light excites fluorescence
species in the sample, which then emit


light of a longer wavelength.

A fluorescent microscope also

produces a magnified image of the
sample, but the image is based on the
second light source
 Preparation of Specimen
Techniques
•taken up by the cells
•incorporated and
concentrated in specific
subcellular compartments

Fluorescent •living cells are then mounted

Fluorescent Dyes on a microscope slide and
Dyes examined in a fluorescence
microscope.
•modify cells so that they create
their own fluorescing molecules
•the location of that protein can
be studied. It is also possible to
watch the movements of the
Tagging of Proteins proteins and its interactions with
other cellular components inside
the cell
Tagging of Proteins
• use of antibodies to which a
fluorescent marker has been
attached.
Immunofluorescence • recognize and bind selectively
to specific target molecules in
the cell
Immunofluorescence
TYPES OF FLUOROPHORES USED
•fluorescein,
•DAPI,
•propidium iodide
•green fluorescent protein (GFP)
•Texas Red
Electron Microscopy
Scanning Electron Microscope
• Illumination: electrons
• Magnification: ~100,000x
• How it works: Detect electrons back-scattered by
the sample.
• Image: Monotone (but may be color enhanced),
3-D surface of specimen
Pros Cons
• High magnification • Needs specimen to be in
• High resolution vacuum
• Shows the surface of • Needs living cells and
specimen tissues and whole, soft-
bodied organisms to be
treated, usu. coated w/
gold film
• No color
• Cannot examine live
specimen
• Really. Big. And
Expensive. Equipment.
Transmission Electron Microscope
• Illumination: electrons
• Magnification: ~100,000x
• How it works: Detect electrons scattered as they
move through the sample.
• Image: Monotone (but may be color enhanced),
2-D structure of specimen
Source:
http://www.lab.anhb.uwa.
edu.au/hb313/main_pages
/timetable/lectures/Image
6.gif
Pros Cons
• High magnification • Needs specimen to be
• High resolution in vacuum
• Shows small structures • Needs specimen to be
that cannot be seen covered in gold film
under light microscopes • Specimen <100nm
thick (obviously cannot
observe live specimen)
• No color
• Really. Big. And.
Expensive. Equipment
 COATING OF SAMPLES

• Coating of samples is required in the field of electron

microscopy to enable or improve the imaging of samples.

• Creating a conductive layer of metal on the sample inhibits

charging, reduces thermal damage and improves the
secondary electron signal required for topographic examination
in the SEM.

• Fine carbon layers, being transparent to the electron beam but

conductive, are needed for x-ray microanalysis, to support films
on grids and back up replicas to be imaged in the TEM.

• The coating technique used depends on the resolution and

application.
Light Vs Electron microscope
CONFOCAL MICROSCOPY
Confocal microscopy is an optical imaging technique for
increasing optical resolution and contrast of a micrograph by means
of adding a spatial pinhole placed at the confocal plane of the lens to
eliminate out-of-focus light.[1]
It enables the
reconstruction of three-
dimensional structures
from the
obtained images. This
technique has gained
popularity in the
scientific and industrial
communities and
typical applications are
in life
sciences, semiconducto
r inspection
and materials science.
Basic concept of confocal Microscopy

The principle of confocal imaging was patented in 1957 by Marvin

Minsky[2][3] and aims to overcome some limitations of traditional wide-
field fluorescence microscopes.

In a conventional (i.e., wide-field) fluorescence microscope, the

entire specimen is flooded evenly in light from a light source.

All parts of the specimen in the optical path are excited at the same time
and the resulting fluorescence is detected by the microscope photodetector
or camera including a large unfocused background part.

In contrast, a confocal microscope uses point illumination (see Point Spread

Function) and a pinhole in an optically conjugate plane in front of the
detector to eliminate out-of-focus signal - the name "confocal" stems from
this configuration. As only light produced by fluorescence very close to
the focal plane can be detected.
 Principle of confocal microscopy

In confocal microscopy two pinholes are typically used:

• A pinhole is placed in front of the illumination source to allow
transmission only through a small area
• This illumination pinhole is imaged onto the focal plane of the
specimen, i.e. only a point of the specimen is illuminated at
one time
• Fluorescence excited in this manner at the focal plane is
imaged onto a confocal pinhole placed right in front of the
detector
• Only fluorescence excited within the focal plane of the
specimen will go through the detector pinhole
• Need to scan point onto the sample
Dye in the specimen is
excited by the laser light
& fluoresces. The
fluorescent (green) light
is descanned by the
same mirrors that are
used to scan the
excitation (blue) light
from the laser beam
then it passes through
the dichoric mirror
then it is focused on to
pinhole the light
passing through the
pinhole is measured by
the detector such as
photomultiplier tube.
For visualization,
detector is attached to
the computer, which
builds up the image at
the rate of 0.1-1 second
for single image.
Alexander Jablonski Diagram
• Light from the
excitation filter excites
the fluorochoromes to
a higher energy state
• From the high state it
declines slowly
releasing energy
• Transition between
absorption & emission
 Better resolution
Importance Cells can be live or fixed
Serial optical sections can be collected

A confocal microscope creates sharp images of a specimen that would appear

otherwise blurred with the conventional microscope –this is achieved by excluding
most of the light from the specimen, but not from the microscope’s focal plane.

The image obtained has better contrast & less hazy .

In confocal microscopy, a series of thin slices of the specimen is assembled to

generate a 3-dimensinal image.

The specimen is everywhere illuminated axially, rather than at different angles,

thereby avoiding optical aberrations--entire field of view is illuminated uniformly.

The field of view can be made larger than that of the static objective by controlling
the amplitude of the stage movements.
HOW DOES A CONFOCAL MICROSCOPE WORK

Confocal microscope incorporates 2 ideas :

1. Point-by-point illumination of the specimen.

2. Rejection of out of focus of light.

Light source of very high intensity is used—Zirconium arc lamp in

Minsky’s design & laser light source in modern design.

1. Laser provides intense blue excitation light.

2. The light reflects off a dichoric mirror, which directs it to an
assembly of vertically and horizontally scanning mirrors.
3. These motor driven mirrors scan the laser beam across the
specimen.
4. The specimen is scanned by moving the stage back & forth in
the vertical & horizontal directions and optics are kept
stationary.
LIMITATIONS OF CONFOCAL MICROSCOPY

1.Resolution : It has inherent resolution limitation due to diffraction.

Maximum best resolution of confocal microscopy is typically about
200nm.
2.Pin hole size : Strength of optical sectioning depends on the size of
the pinhole.
3.Intensity of the incident light.

4.Fluorophores :
a)The fluorophore should tag the correct part of the specimen.
b)Fluorophore should be sensitive enough for the given excitation wave
length.
C)It should not significantly alter the dynamics of the organism in the
living specimen.
5.Photobleaching
References

1. Satyanarayana, U. “Biochemistry” 5th Edition, Elsevier India 2017,

ISBN: 9788131249406
2. Voet D, Voet J. .G “Biochemistry”, 4th Edition, John Wiley & Sons, Inc.,2010.
ISBN: 978-0-470-57095-1
3. David L. Nelson; Michael M. Cox “Lehninger Principles of Biochemistry”
Seventh Edition, Macmillan 2017, ISBN:9781464187957
4. Wilson, K., Walker, J. (eds.); Cambridge University Press, Cambridge, 2000, 784
pp., ISBN 0‐521‐65873‐X (paperback)

All the original contributors of the concept and findings published are
gratefully acknowledged while preparing the e-content for the
students of Biochemistry and allied sciences