Page 1
Electron Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Electron Microscopy
Lesson Developer: Anuradha Sharma
College/Department: Botany Dept., Hindu College, University of Delhi
Page 2
Electron Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Electron Microscopy
Lesson Developer: Anuradha Sharma
College/Department: Botany Dept., Hindu College, University of Delhi
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Electron Microscopy
Introduction
? Principle of microscopy
? Comparative account of different types of
microscopes
? Basic components of an electron microscope
Types of Electron Microscope
? Transmission Electron Microscope (TEM)
? Scanning Electron Microscope (SEM)
? Scanning Transmission Electron Microscope (STEM)
? Environmental Scanning Electron Microscope (ESEM)
Techniques for electron microscope
? Negative Staining
? Freeze -Fracture and Freeze –Etch
? Shadow Casting
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Page 3
Electron Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Electron Microscopy
Lesson Developer: Anuradha Sharma
College/Department: Botany Dept., Hindu College, University of Delhi
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Electron Microscopy
Introduction
? Principle of microscopy
? Comparative account of different types of
microscopes
? Basic components of an electron microscope
Types of Electron Microscope
? Transmission Electron Microscope (TEM)
? Scanning Electron Microscope (SEM)
? Scanning Transmission Electron Microscope (STEM)
? Environmental Scanning Electron Microscope (ESEM)
Techniques for electron microscope
? Negative Staining
? Freeze -Fracture and Freeze –Etch
? Shadow Casting
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 2
Introduction
Principle of Microscopy
The prokaryotic and eukaryotic cells fall within the size range of 1-100 µm. Unaided human eye
cannot resolve objects smaller than 100 µm size. Therefore, microscopes are needed for
visualization of subcellular architecture. Microscope not only magnifies the image of objects but
also increases the resolution, which refers to ability to distinguish closely adjacent objects as
separate entities. The greater is the resolving power of the microscope, the greater is the clarity
of the image produced.
The lower limit of resolution for any optical system can be calculated from the following
relationship.
r = 0.61?/ n sin a
where r, or resolving power, is the minimum distance between two points that can be
recognized as separate, ? is the wavelength of light (or other radiation) used to illuminate the
object, n is the refractive index of the medium in which the object is placed, and sin a is the
sine of half the angle between the specimen and the objective lens. The entire term n sin a is
often referred to as the numerical aperture.
Page 4
Electron Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Electron Microscopy
Lesson Developer: Anuradha Sharma
College/Department: Botany Dept., Hindu College, University of Delhi
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Electron Microscopy
Introduction
? Principle of microscopy
? Comparative account of different types of
microscopes
? Basic components of an electron microscope
Types of Electron Microscope
? Transmission Electron Microscope (TEM)
? Scanning Electron Microscope (SEM)
? Scanning Transmission Electron Microscope (STEM)
? Environmental Scanning Electron Microscope (ESEM)
Techniques for electron microscope
? Negative Staining
? Freeze -Fracture and Freeze –Etch
? Shadow Casting
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 2
Introduction
Principle of Microscopy
The prokaryotic and eukaryotic cells fall within the size range of 1-100 µm. Unaided human eye
cannot resolve objects smaller than 100 µm size. Therefore, microscopes are needed for
visualization of subcellular architecture. Microscope not only magnifies the image of objects but
also increases the resolution, which refers to ability to distinguish closely adjacent objects as
separate entities. The greater is the resolving power of the microscope, the greater is the clarity
of the image produced.
The lower limit of resolution for any optical system can be calculated from the following
relationship.
r = 0.61?/ n sin a
where r, or resolving power, is the minimum distance between two points that can be
recognized as separate, ? is the wavelength of light (or other radiation) used to illuminate the
object, n is the refractive index of the medium in which the object is placed, and sin a is the
sine of half the angle between the specimen and the objective lens. The entire term n sin a is
often referred to as the numerical aperture.
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 3
Frequently asked question
What do you understand by numerical aperture?
The numerical aperture of the objective a microscope is a measure of its resolving power. The
value of numerical aperture is given by NA = n sin a.
n refers to the refractive index (1 for air)
a is half the angle subtended by the rays entering into the objective lens
Higher the NA higher the resolving power
Low NA= Low resolving power High NA= High resolving power
Source: http://www.doitpoms.ac.uk/tlplib/optical-microscopy/images/diagram6.gif
There are only a small number of variables affect the resolving power of a microscope. The
refractive index can be increased by immersing the sample in oil (n = 1.5) rather than air (n =
1.0), and moving the lens closer to the specimen to increase a. The upper theoretical limit
of a is 90 °, meaning that the value of sin a cannot exceed 1. Hence the maximum numerical
aperture of an optical system employing an oil immersion lens will be 1.5 X 1 = 1.5. A
microscope using white light, which has an average wavelength of about 550 nm, will therefore,
have a resolving power of 550/1.5, or about 220 nm. This means that objects closer to one
another or smaller than 220 nm cannot be distinguished. A resolving power of 220 nm is
adequate to see some details of subcellular structure, but many organelles, such as ribosomes,
cellular membranes, microtubules, microfilaments, intermediate filaments, and chromatin fibers,
cannot be resolved at this level .The wavelength of an electron is much shorter than that of
Page 5
Electron Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Electron Microscopy
Lesson Developer: Anuradha Sharma
College/Department: Botany Dept., Hindu College, University of Delhi
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Electron Microscopy
Introduction
? Principle of microscopy
? Comparative account of different types of
microscopes
? Basic components of an electron microscope
Types of Electron Microscope
? Transmission Electron Microscope (TEM)
? Scanning Electron Microscope (SEM)
? Scanning Transmission Electron Microscope (STEM)
? Environmental Scanning Electron Microscope (ESEM)
Techniques for electron microscope
? Negative Staining
? Freeze -Fracture and Freeze –Etch
? Shadow Casting
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 2
Introduction
Principle of Microscopy
The prokaryotic and eukaryotic cells fall within the size range of 1-100 µm. Unaided human eye
cannot resolve objects smaller than 100 µm size. Therefore, microscopes are needed for
visualization of subcellular architecture. Microscope not only magnifies the image of objects but
also increases the resolution, which refers to ability to distinguish closely adjacent objects as
separate entities. The greater is the resolving power of the microscope, the greater is the clarity
of the image produced.
The lower limit of resolution for any optical system can be calculated from the following
relationship.
r = 0.61?/ n sin a
where r, or resolving power, is the minimum distance between two points that can be
recognized as separate, ? is the wavelength of light (or other radiation) used to illuminate the
object, n is the refractive index of the medium in which the object is placed, and sin a is the
sine of half the angle between the specimen and the objective lens. The entire term n sin a is
often referred to as the numerical aperture.
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 3
Frequently asked question
What do you understand by numerical aperture?
The numerical aperture of the objective a microscope is a measure of its resolving power. The
value of numerical aperture is given by NA = n sin a.
n refers to the refractive index (1 for air)
a is half the angle subtended by the rays entering into the objective lens
Higher the NA higher the resolving power
Low NA= Low resolving power High NA= High resolving power
Source: http://www.doitpoms.ac.uk/tlplib/optical-microscopy/images/diagram6.gif
There are only a small number of variables affect the resolving power of a microscope. The
refractive index can be increased by immersing the sample in oil (n = 1.5) rather than air (n =
1.0), and moving the lens closer to the specimen to increase a. The upper theoretical limit
of a is 90 °, meaning that the value of sin a cannot exceed 1. Hence the maximum numerical
aperture of an optical system employing an oil immersion lens will be 1.5 X 1 = 1.5. A
microscope using white light, which has an average wavelength of about 550 nm, will therefore,
have a resolving power of 550/1.5, or about 220 nm. This means that objects closer to one
another or smaller than 220 nm cannot be distinguished. A resolving power of 220 nm is
adequate to see some details of subcellular structure, but many organelles, such as ribosomes,
cellular membranes, microtubules, microfilaments, intermediate filaments, and chromatin fibers,
cannot be resolved at this level .The wavelength of an electron is much shorter than that of
Electron Microscopy
Institute of Lifelong Learning, University of Delhi 4
visible light, the electron microscope has a theoretical limit of resolution much lower that of the
light microscope—about 0.1-0.2 nm instead of 200-300 nm. Because of problems of specimen
preparation of biological samples, the practical limit of resolution is almost about 2 nm which
means 100 times more resolution than that of light microscope. Electron microscopes thus
offers the possibility of increasing the resolving power many folds. There are two types of
electron microscopes:
? Transmission electron microscope
? Scanning electron microscope
The electrostatic and electromagnetic lenses are used in an electron microscope to control the
electron beam and focus it to form an image. In Transmission electron microscope (TEM), the
electrons are transmitted through an object and then focused by the lenses to form the image.
In Scanning electron microscope (SEM), the electrons are reflected by the object in a scanned
pattern which are then used to form the image. SEM is becoming increasingly popular with cell
biologists because of its remarkable ability to study surface topography, along with improved
resolution (30-100 Å) and its ability to show 3D structure.
Table: Comparative account of different types of microscopes
Source: Author, Images courtsey: Dr Mani Arora
Description Compound
Confocal Microscope Scanning Electron
Microscope (SEM)
Transmission Electron
Microscope (TEM)
Source of
illumination
for Image
Formation
visible light laser light electrons electrons
Types of cells
visualized
Individual
cells can be
visualised,
even living
ones.
Individual cells can
be visualised, even
living ones.
The specimen is
coated with gold
and the electrons
are reflected back
and give the details
of surface
topography of the
specimen.
Thin sections of the
specimen are obtained. The
electron beams pass
through the sections and
form an image with high
magnification and high
resolution.
Image Two
dimensional
3-D 2-D
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