Lecture 6 - Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy | Cell Biology- Botany PDF Download

 Page 1


Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy 
Lesson Developer: Sunita Yadav 
College/Department:Hansraj College, Department of Botany, Hindu 
College, University of Delhi 
Page 2


Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy 
Lesson Developer: Sunita Yadav 
College/Department:Hansraj College, Department of Botany, Hindu 
College, University of Delhi 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 1 
 
Table of Contents       
 
Chapter: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy                                             
? Introduction 
? Principles of microscopy 
? Illuminating wavelength 
? Resolution 
? Practical limit of resolution is roughly 200 nm for light 
microscopy 
? Light Microscopy 
? Magnifying lens 
? Bright field microscopy 
? A light source 
? Stand 
? Specimen stage 
? Objective lens 
? Eyepiece lens 
? Stereomicroscopes 
? Phase contrast Microscopy 
? Principle 
? Setting up a microscope for phase contrast 
? Applications 
? Polarized Light Microscopy 
? Sample preparation for light microscopy 
? Whole mount 
? Sections 
? Fixation 
Page 3


Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy 
Lesson Developer: Sunita Yadav 
College/Department:Hansraj College, Department of Botany, Hindu 
College, University of Delhi 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 1 
 
Table of Contents       
 
Chapter: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy                                             
? Introduction 
? Principles of microscopy 
? Illuminating wavelength 
? Resolution 
? Practical limit of resolution is roughly 200 nm for light 
microscopy 
? Light Microscopy 
? Magnifying lens 
? Bright field microscopy 
? A light source 
? Stand 
? Specimen stage 
? Objective lens 
? Eyepiece lens 
? Stereomicroscopes 
? Phase contrast Microscopy 
? Principle 
? Setting up a microscope for phase contrast 
? Applications 
? Polarized Light Microscopy 
? Sample preparation for light microscopy 
? Whole mount 
? Sections 
? Fixation 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 2 
? Embedding 
? Sectioning 
? Staining 
 
? Summary  
? Exercise/ Practice 
? Glossary 
? References/ Bibliography/ Further Reading 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 4


Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy 
Lesson Developer: Sunita Yadav 
College/Department:Hansraj College, Department of Botany, Hindu 
College, University of Delhi 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 1 
 
Table of Contents       
 
Chapter: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy                                             
? Introduction 
? Principles of microscopy 
? Illuminating wavelength 
? Resolution 
? Practical limit of resolution is roughly 200 nm for light 
microscopy 
? Light Microscopy 
? Magnifying lens 
? Bright field microscopy 
? A light source 
? Stand 
? Specimen stage 
? Objective lens 
? Eyepiece lens 
? Stereomicroscopes 
? Phase contrast Microscopy 
? Principle 
? Setting up a microscope for phase contrast 
? Applications 
? Polarized Light Microscopy 
? Sample preparation for light microscopy 
? Whole mount 
? Sections 
? Fixation 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 2 
? Embedding 
? Sectioning 
? Staining 
 
? Summary  
? Exercise/ Practice 
? Glossary 
? References/ Bibliography/ Further Reading 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 3 
 
Introduction 
Microscopes constitute the very basic requirement for cell biologists, thereby facilitating the 
deciphering the fine details of intracellular components. The microscope is an obligatory tool 
for this purpose because most cellular structures are too small to be seen by the unaided 
eye. The beginnings of cell biology can be traced to the invention of the light microscope, 
which made it possible for scientists to examine enlarged images of the cells and thus to 
analyze cellular structure and its dynamics. The first light microscope was developed in 
1590 by Z. Janssen and H. Janssen. During the next century, many microscopic 
observations were reported, notably those of Robert Hooke (who observed the first cells) 
and Antonie van Leeuwenhoek (who provided first glimpse of internal cell structure through 
improved microscopes). Since then, the light microscopes have undergone numerous 
improvements and modifications till the present time. Light microscopy has experienced a 
renaissance in recent years through specialized technological improvements that allow 
researchers to explore aspects of cell structure and behaviour. Most images produced by 
microscopes are now recorded electronically using digital imaging techniques, like digital 
cameras, digital image acquisition software, digital printing and digital display methods. 
Additionally, vast improvements have been made in the biological aspects of specimen 
preparation. These advancements have fostered many more applications of the microscope 
in biomedical research. These advances have involved the merging of technologies from 
physics, engineering, chemistry and molecular biology, that have greatly expanded the 
ability to study cells using light microscope. Biochemical analyses is frequently accompanied 
by microscopic examination of tissue, cell or organelle preparations. Such examinations are 
used in many different applications, for example: 
1. to evaluate the integrity of samples during an experiment;  
2. to map the fine details of the spatial distribution of macromolecules within cells;  
3. to directly measure biochemical events within the living tissues.  
There are fundamentally two different types of microscopes:  
? the light microscope and  
? the electron microscope.  
Light microscopes use a series of glass lenses to focus light in order to form an image 
whereas electron microscopes use electromagnetic lenses to focus a beam of electrons. 
Page 5


Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy 
Lesson Developer: Sunita Yadav 
College/Department:Hansraj College, Department of Botany, Hindu 
College, University of Delhi 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 1 
 
Table of Contents       
 
Chapter: Principles of Microscopy: Light Microscopy and Phase 
Contrast Microscopy                                             
? Introduction 
? Principles of microscopy 
? Illuminating wavelength 
? Resolution 
? Practical limit of resolution is roughly 200 nm for light 
microscopy 
? Light Microscopy 
? Magnifying lens 
? Bright field microscopy 
? A light source 
? Stand 
? Specimen stage 
? Objective lens 
? Eyepiece lens 
? Stereomicroscopes 
? Phase contrast Microscopy 
? Principle 
? Setting up a microscope for phase contrast 
? Applications 
? Polarized Light Microscopy 
? Sample preparation for light microscopy 
? Whole mount 
? Sections 
? Fixation 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 2 
? Embedding 
? Sectioning 
? Staining 
 
? Summary  
? Exercise/ Practice 
? Glossary 
? References/ Bibliography/ Further Reading 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 3 
 
Introduction 
Microscopes constitute the very basic requirement for cell biologists, thereby facilitating the 
deciphering the fine details of intracellular components. The microscope is an obligatory tool 
for this purpose because most cellular structures are too small to be seen by the unaided 
eye. The beginnings of cell biology can be traced to the invention of the light microscope, 
which made it possible for scientists to examine enlarged images of the cells and thus to 
analyze cellular structure and its dynamics. The first light microscope was developed in 
1590 by Z. Janssen and H. Janssen. During the next century, many microscopic 
observations were reported, notably those of Robert Hooke (who observed the first cells) 
and Antonie van Leeuwenhoek (who provided first glimpse of internal cell structure through 
improved microscopes). Since then, the light microscopes have undergone numerous 
improvements and modifications till the present time. Light microscopy has experienced a 
renaissance in recent years through specialized technological improvements that allow 
researchers to explore aspects of cell structure and behaviour. Most images produced by 
microscopes are now recorded electronically using digital imaging techniques, like digital 
cameras, digital image acquisition software, digital printing and digital display methods. 
Additionally, vast improvements have been made in the biological aspects of specimen 
preparation. These advancements have fostered many more applications of the microscope 
in biomedical research. These advances have involved the merging of technologies from 
physics, engineering, chemistry and molecular biology, that have greatly expanded the 
ability to study cells using light microscope. Biochemical analyses is frequently accompanied 
by microscopic examination of tissue, cell or organelle preparations. Such examinations are 
used in many different applications, for example: 
1. to evaluate the integrity of samples during an experiment;  
2. to map the fine details of the spatial distribution of macromolecules within cells;  
3. to directly measure biochemical events within the living tissues.  
There are fundamentally two different types of microscopes:  
? the light microscope and  
? the electron microscope.  
Light microscopes use a series of glass lenses to focus light in order to form an image 
whereas electron microscopes use electromagnetic lenses to focus a beam of electrons. 
Principles of Microscopy: Light Microscopy and Phase Contrast Microscopy 
 
Institute of Lifelong Learning, University of Delhi 4 
Light microscopes are able to magnify to a maximum of approximately 1500 times whereas 
electron microscopes to a maximum of approximately 200000 times. Standard light 
microscopes have a lateral resolution limit of about 0.5 micrometers for routine analysis. In 
contrast, electron microscopes have a lateral resolution of up to 1 nanometer. Both living 
and dead specimens are viewed with a light microscope, and often in real colour, whereas 
only dead ones are viewed with an electron microscope and never in real colour.  
Applications of the microscope in biomedical research may be relatively simple and routine, 
for example, a quick check of the status of a preparation or the health of cells growing in a 
plastic dish in the tissue culture. The application may be more involved, for example, 
measuring the concentration of calcium in a living embryo over a millisecond timescale 
through a more advanced light microscope (often called an imaging system). Some 
microscopes are more suited to specific applications than others but there may be 
constraints imposed by the specimen. Images may be required from specimens of vastly 
different sizes and magnifications; for example, for imaging whole animals (metres), 
through tissues and embryos (micrometres) and down to cells, proteins and DNA (nm). The 
study of living cells may also require time resolution from days (like when imaging neuronal 
development or disease processes) to milliseconds (like when imaging cell signalling 
events).  
Principles of microscopy 
The principles of microscopy need to be examined through special emphasis on the factors 
that determine how small an object can be observed and analyzed with current 
technologies.  
Illuminating wavelength  
Three elements are always needed to form an image, regardless of the type of microscope 
being used:   
? a source of illumination 
? a specimen to be examined 
? a system of lenses that focuses the illumination on the specimen and forms the 
image.