Lecture 11 - Fluorescence and Confocal Microscopy | Cell Biology- Botany PDF Download

 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 
 
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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.