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.
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