Lecture

Super Resolution Microscopy: STED Method

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In course

MICRO-562: Experimental biomicroscopy

By combining hands-on and theoretical training, this course introduces commonly used optical microscopy techniques. The lab training takes place in BIOP Core Facility and provides access to use microscopes and image biological samples. Theoretical part cov ...

Description

This lecture covers the principles and applications of Super Resolution Microscopy, focusing on the Stimulated Emission Depletion (STED) method. It explains the key parameters and optimization techniques for achieving high-resolution imaging, including the role of STED laser intensity and saturation intensity of the fluorophore. The instructor discusses the non-linear dependence of the depleted population of excited electrons on the STED laser beam intensity, leading to super-resolution imaging. Examples of suitable dyes and lasers for STED imaging are provided, along with the resolution factors and limitations of STED microscopy.

Instructors (2)

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)

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

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Related concepts (32)

Microscopy

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy. Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image.

Confocal microscopy

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object.

Fluorescent tag

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.

Green fluorescent protein

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.

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