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Lecture

Fluorescence in Microscopy: Basic Principles and Processes

In course

MICRO-561: Fundamentals of biomicroscopy

Introduction to the basic operational principles of the most commonly used optical microscopes such as widefield and fluorescence.
Introduction to essential microscopy components such as lenses, objectives, filters, cameras and light sources. ...

Description

This lecture introduces the basic principles of fluorescence in microscopy, focusing on the three-stage process that occurs in fluorescent molecules. It covers topics such as photon absorption and emission, energy level diagrams, Stokes shift, additional processes, and the Jablonski diagram.

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Instructors (2)

Hatice Altug

2020-current Full Professor at the Institute of Bioengineering, EPFL, Switzerland2013-2020 Associate Professor (with tenure) at the Institute of Bioengineering, EPFL, Switzerland 2013 Associate Professor (with tenure) at Electrical and Computer Engineering Department of Boston University, USA 2007-2013 Assistant Professor (tenure-track) at Electrical and Computer Engineering Department of Boston University, USA 2007 Post-doctoral Fellow at Center for Engineering in Medicine of Harvard Medical School, USA 2000-2007 PhD. in Applied Physics at Stanford University, USA 1996-2000 B.S. in Physics at Bilkent University, Turkey

Arne Seitz

04/1996-06/2000 Scientific co-worker at Philipps-University of Marburg, Dep. of Physical Chemistry 07/2000-10/2002 Post Doc at Max-Planck Unit for Structural Molecular Biology in Hamburg 11/2002-11/2005 Post Doc at European Molecular Biolohy Laboratory (EMBL), Cell Biology and Cell Biophysics Programme 11/2005-03/2009 Staff Scientist at Advanced Light Microscopy Facility, EMBL 04/2009- Head of Bioimaging and Optics platforme (BIOP), Ecole Polytechnique Fédérale de Lausanne (EPFL)

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Related concepts (13)

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light.

Fluorescence spectroscopy

Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.

Jablonski diagram

In molecular spectroscopy, a Jablonski diagram is a diagram that illustrates the electronic states and often the vibrational levels of a molecule, and also the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity. Nonradiative transitions are indicated by squiggly arrows and radiative transitions by straight arrows. The vibrational ground states of each electronic state are indicated with thick lines, the higher vibrational states with thinner lines.

Energy level

A quantum mechanical system or particle that is bound—that is, confined spatially—can only take on certain discrete values of energy, called energy levels. This contrasts with classical particles, which can have any amount of energy. The term is commonly used for the energy levels of the electrons in atoms, ions, or molecules, which are bound by the electric field of the nucleus, but can also refer to energy levels of nuclei or vibrational or rotational energy levels in molecules.

Fluorescence microscope

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

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