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@PHDTHESIS{Ledesch:851775,
author = {Ledesch, Ralph},
title = {{S}imultaneous dual-color imaging on single-molecule level
on a {W}idefield microscope and applications},
volume = {178},
school = {RWTH Aachen},
type = {Dr.},
address = {Jülich},
publisher = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
reportid = {FZJ-2018-05290},
isbn = {978-3-95806-348-8},
series = {Schriften des Forschungszentrums Jülich. Reihe
Schlüsseltechnologien / Key Technologies},
pages = {IX, 119 S.},
year = {2018},
note = {RWTH Aachen, Diss., 2018},
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 [...]},
cin = {ICS-5},
cid = {I:(DE-Juel1)ICS-5-20110106},
pnm = {899 - ohne Topic (POF3-899)},
pid = {G:(DE-HGF)POF3-899},
typ = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
urn = {urn:nbn:de:0001-2018091935},
url = {https://juser.fz-juelich.de/record/851775},
}