Lecture 12 - Electron Microscopy | Cell Biology- Botany PDF Download

 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