Book/Dissertation / PhD Thesis

FZJ-2018-05290

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Simultaneous dual-color imaging on single-molecule level on a Widefield microscope and applications

Ledesch, R. (Corresponding author)FZJ*

2018
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-348-8

Please use a persistent id in citations: http://hdl.handle.net/2128/19699  urn:nbn:de:0001-2018091935

Abstract: The epifluorescence microscope, as we know it today, has come a long way. Starting as a spin-off to the UV microscope at the beginning of the twentieth century, its development over the last century has made it a powerful tool, allowing the study of biological processes on single-molecule level in unprecedented detail. The first fluorescence microscope, with UV illumination, was developed at Carl Zeiss by the german physicists Otto Heimstaedt and Heinrich Lehmann. Between 1925 and 1932, Philipp Ellinger and August Hirt from Heidelberg conceived the UV intravital microscope, that allowed them to study the distribution of previously injected fluorescent dyes in living kidney and liver of frogs and mice [5, 6] to study their deposition and transport through the blood vessels. The setup is considered the prototype epifluorescence microscope: Unlike the previously built transmitted light microscopes, where the illumination light source is transmitted from the opposite side of the specimen from the objective, in their setup, the objective itself acted as the illumination condenser. While the emitted red-shifted fluorescence was transmitted and imaged on a diapositive, the reflected UV light was blocked by a yellow filter, placed between the objective and the ocular. In contrast to the transmitted light microscope, the illumination light is not detected, resulting in a higher image contrast, while at the same time alignment problems could be avoided. The evolution to the modern epifluorescence microsope is due to the contributions of the russian scientist Evgenii Brumberg (State Optical Institue of Leningrad) and Johann Sebastiaan Ploem, a microscopist from the University of Amsterdam. They are responsible for the development of the dichroic beamsplitter, a key element in every modern fluorescence microscope. It physically separated the excitation light from the much weaker fluorescence signal, by deflecting the unwanted back-reflected excitation light [7]. Up to the 1950s, fluorescence microscopes used excitation light ranging from the UV to the blue spectra, that was isolated from (mercury or xenon) arc lamps by optical filters. The steadily increasing number of developed fluorophores were not necessarily optimally illuminated within the UV spectra only. In 1962, Ploem collaborated with the Schott glass company in Mainz to extend the spectral range for illumination, by producing dichroic filters (or mirrors), that deflected the blue and green spectra [8]. Furthermore, Ploem collaborated with the Ernst Leitz company, which constructed the first inverted microscope with epi-illumination and combined the optical filter(s) and the dichroic mirror in a unit (the filter cube). The filter cubes were arranged to match the excitation/emission spectra of the employed fluorophore. Mounted in a turret below the objective, they could be interchanged [...]

Note: RWTH Aachen, Diss., 2018

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Contributing Institute(s):

1. Molekulare Biophysik (ICS-5)

Research Program(s):

1. 899 - ohne Topic (POF3-899) (POF3-899)

Appears in the scientific report 2018

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