Page 1
Fluorescence and Confocal Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Fluorescence and Confocal Microscopy
Lesson Developer: Sunita Yadav
College/Department: Hansraj College/Department of Botany
Page 2
Fluorescence and Confocal Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Fluorescence and Confocal Microscopy
Lesson Developer: Sunita Yadav
College/Department: Hansraj College/Department of Botany
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Microscopy- Fluorescence and Confocal
Introduction to fluorescence microscopy
History of fluorescence
Principles of fluorescence
? Excitation and emission
? Excitation and emission spectra and stokes shift
Fluorescence microscope
Fluorophores
Fading
Introduction to Confocal Microscopy
History of CLSM
Modern Confocal Microscopy
How does a confocal microscope work?
? Pinhole size
? Intensity of incident light
? Fluorophore
? Dynamics of living cells
? Light Path in a CLSM
Advantages over conventional wide field microscope
? Optical sectioning
? Information about the spatial structure of the object
Page 3
Fluorescence and Confocal Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Fluorescence and Confocal Microscopy
Lesson Developer: Sunita Yadav
College/Department: Hansraj College/Department of Botany
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Microscopy- Fluorescence and Confocal
Introduction to fluorescence microscopy
History of fluorescence
Principles of fluorescence
? Excitation and emission
? Excitation and emission spectra and stokes shift
Fluorescence microscope
Fluorophores
Fading
Introduction to Confocal Microscopy
History of CLSM
Modern Confocal Microscopy
How does a confocal microscope work?
? Pinhole size
? Intensity of incident light
? Fluorophore
? Dynamics of living cells
? Light Path in a CLSM
Advantages over conventional wide field microscope
? Optical sectioning
? Information about the spatial structure of the object
Institute of Lifelong Learning, University of Delhi 2
? Visualization of the dynamic changes
Alternative methods to CLSM
? Spinning disk confocal microscope
? Multiple photon microscope
? Deconvolution microscope
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Page 4
Fluorescence and Confocal Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Fluorescence and Confocal Microscopy
Lesson Developer: Sunita Yadav
College/Department: Hansraj College/Department of Botany
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Microscopy- Fluorescence and Confocal
Introduction to fluorescence microscopy
History of fluorescence
Principles of fluorescence
? Excitation and emission
? Excitation and emission spectra and stokes shift
Fluorescence microscope
Fluorophores
Fading
Introduction to Confocal Microscopy
History of CLSM
Modern Confocal Microscopy
How does a confocal microscope work?
? Pinhole size
? Intensity of incident light
? Fluorophore
? Dynamics of living cells
? Light Path in a CLSM
Advantages over conventional wide field microscope
? Optical sectioning
? Information about the spatial structure of the object
Institute of Lifelong Learning, University of Delhi 2
? Visualization of the dynamic changes
Alternative methods to CLSM
? Spinning disk confocal microscope
? Multiple photon microscope
? Deconvolution microscope
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Institute of Lifelong Learning, University of Delhi 3
Introduction to fluorescence microscope
Although many of the remarkable discoveries in the field of life science have been made
without the application of specialized technology, technology plays an important role in
understanding new frontiers in life science. It provides as a means to test and to prove new
ideas. It also provides new information to formulate new hypothesis. One of remarkable
developments is microscopy and imaging techniques. These techniques occupy a center
stage in biological as well in material science; an essential tool in assessing the properties of
organic or inorganic substances. It provides the advantage of being able to observe and
measure form and features to reveal the variability. With the development of more refine
techniques couple with the discovery of an array of fluorophores, it has made possible to
visualize and study the cellular and subcellular components and the diverse physiological
processes like protein interactions, ion transports, nutrient mobility and metabolic processes
taking place inside a living cell.
In light microscopy, the differential reflection, diffraction and absorption properties of
different specimens are used to study the specimens. So improvements in microscopy are
mainly emphasized on increasing the contrast between the specimen and the background.
This contrast between the sample and the background is enhanced certainly by staining the
sample with agents that absorb light in light microscopy. However this contrast has been
greatly enhanced with the development of fluorescence microscopy. It provides better
contrast than other optical microscopy techniques. This has been achieved by staining or
tagging the target sample with fluorescence dye or with fluorescence molecules
(fluorophores) amid the non-fluorescing background. On irradiation with high energy light,
only the fluorescence molecules emit light enabling to visualize only the object of interest in
the dark background. With the development of highly specific labeling probes, coupled with
the ability to imaging of individual components and other macromolecular complexes,
fluorescence microscopy brings a revolution in cell biology. (web link no.1)
History of fluorescence
Fluorescence has been described first by Irish Scientist Sir George G. Stokes in 1852 during
the middle of nineteenth century. While working with a mineral named „fluorspar? at the
Cambridge University, he noticed that the mineral emitted red light when illuminated with
blue light. Although this phenomenon has been encountered in microscopy in the early part
of twentieth century by several scientists, including August Köhler and Carl Reichert, they
Page 5
Fluorescence and Confocal Microscopy
Institute of Lifelong Learning, University of Delhi
Lesson: Fluorescence and Confocal Microscopy
Lesson Developer: Sunita Yadav
College/Department: Hansraj College/Department of Botany
Institute of Lifelong Learning, University of Delhi 1
Table of Contents
Chapter: Microscopy- Fluorescence and Confocal
Introduction to fluorescence microscopy
History of fluorescence
Principles of fluorescence
? Excitation and emission
? Excitation and emission spectra and stokes shift
Fluorescence microscope
Fluorophores
Fading
Introduction to Confocal Microscopy
History of CLSM
Modern Confocal Microscopy
How does a confocal microscope work?
? Pinhole size
? Intensity of incident light
? Fluorophore
? Dynamics of living cells
? Light Path in a CLSM
Advantages over conventional wide field microscope
? Optical sectioning
? Information about the spatial structure of the object
Institute of Lifelong Learning, University of Delhi 2
? Visualization of the dynamic changes
Alternative methods to CLSM
? Spinning disk confocal microscope
? Multiple photon microscope
? Deconvolution microscope
Summary
Exercise/ Practice
Glossary
References/ Bibliography/ Further Reading
Institute of Lifelong Learning, University of Delhi 3
Introduction to fluorescence microscope
Although many of the remarkable discoveries in the field of life science have been made
without the application of specialized technology, technology plays an important role in
understanding new frontiers in life science. It provides as a means to test and to prove new
ideas. It also provides new information to formulate new hypothesis. One of remarkable
developments is microscopy and imaging techniques. These techniques occupy a center
stage in biological as well in material science; an essential tool in assessing the properties of
organic or inorganic substances. It provides the advantage of being able to observe and
measure form and features to reveal the variability. With the development of more refine
techniques couple with the discovery of an array of fluorophores, it has made possible to
visualize and study the cellular and subcellular components and the diverse physiological
processes like protein interactions, ion transports, nutrient mobility and metabolic processes
taking place inside a living cell.
In light microscopy, the differential reflection, diffraction and absorption properties of
different specimens are used to study the specimens. So improvements in microscopy are
mainly emphasized on increasing the contrast between the specimen and the background.
This contrast between the sample and the background is enhanced certainly by staining the
sample with agents that absorb light in light microscopy. However this contrast has been
greatly enhanced with the development of fluorescence microscopy. It provides better
contrast than other optical microscopy techniques. This has been achieved by staining or
tagging the target sample with fluorescence dye or with fluorescence molecules
(fluorophores) amid the non-fluorescing background. On irradiation with high energy light,
only the fluorescence molecules emit light enabling to visualize only the object of interest in
the dark background. With the development of highly specific labeling probes, coupled with
the ability to imaging of individual components and other macromolecular complexes,
fluorescence microscopy brings a revolution in cell biology. (web link no.1)
History of fluorescence
Fluorescence has been described first by Irish Scientist Sir George G. Stokes in 1852 during
the middle of nineteenth century. While working with a mineral named „fluorspar? at the
Cambridge University, he noticed that the mineral emitted red light when illuminated with
blue light. Although this phenomenon has been encountered in microscopy in the early part
of twentieth century by several scientists, including August Köhler and Carl Reichert, they
Institute of Lifelong Learning, University of Delhi 4
failed to recognised the fluorescence and instead reported it as a background noise in
ultraviolet microscopy. The first fluorescence microscopes were developed by German
physicists Otto Heimstädt and Heinrich Lehmann between 1911 and 1913 as variant from
the ultraviolet microscopy. These microscopes have used to observe autofluorescence in
bacteria, animal, and plant tissues. A new era of fluorescence microscopy aroused
thereafter the development of a technique for labelling antibodies with fluorescent dyes by
Albert Coons during the early 1940s. (web link no. 2)
Principles of Flourescence
Excitation and Emission
Every molecule can absorb light of certain wavelength. When these molecules are subjected
to radiant energy, they absorb the energy and become excited to a higher energy state. The
inherent property of every molecule to stay at the lowest energy state made much of the
trapped energy to be released in the form of heat or light. However some atoms or
molecules after absorbing light, it reradiates back the energy in the form of light within
nanoseconds after absorption. This physical phenomenon is known as fluorescence and is
first describe d by Sir George G. Stokes. He coined the term “fluorescence” named after the
mineral „fluorspar? he is working with. He also pointed out that, the light emitted by an
excited molecule have a wavelength longer than the wavelength of light originally absorbed.
Like, upon absorption of blue light, green light is emitted soon afterwards. Green is changed
to yellow, yellow to reddish orange and invisible UV light to visible light. The time delay
between the absorption and emission of the light in fluorescence is less than a microsecond.
However, the phenomenon phosphorescence occurs when the emission persists even after
the excitation light has been discontinued. (web link no.1)
A useful explanation of the various energy levels involved in excitation and emission process
by a fluorophore is illustrated by the Jablonski energy diagram named after the polish
physicist Alexander Jablonski.
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