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Confocal Microscopy
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"Multifocal Multiphoton Microscopy: New Detection Methods and Biological Applications",
Jörg Martini, Faculty of Physics, Bielefeld University, November 2006
In this work multifocal
multiphoton microscopy (MMM) has, on the one hand, been applied as a technique for
the investigation of certain biological samples. On the other hand, new detection techniques
have been developed and tested in order to advance the capabilities in MMM.
This progress in technical possibilities has been driven by the particular difficulties in
imaging the samples of interest, i.e. cartilage, tissue engineering products for cartilage
implants and tobacco protoplasts transfected with a Arabidopsis thaliana transcription
factor. Some of these techniques became a standard measurement protocol for successful
imaging of the particular sample of interest, while others were found to be better suited
for types of samples that they were not intended for. As this work documents a
contribution to the collaboration between partners both in the field of biology, who are
interested gaining knowledge on their samples, as well as in the field of microscopy, who
are interested in advancing the general techniques, the results serve both groups.
"Calibration and Validation of Confocal Spectral Imaging Systems",
Jeremy M. Lerner, Robert M. Zucker, U.S. Environmental Protection Agency, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Cytometry Part A 62A:8–34 (2004)
Copyright ©2004 Wiley-Liss, Inc.
Confocal spectral imaging (CSI) microscopic
systems currently on the market delineate multiple fluorescent
proteins, labels, or dyes within biological specimens by
performing spectral characterizations. However, some CSI
systems have been found to present inconsistent spectral
profiles of reference spectra within a particular system and
between related and unrelated instruments. This variability
confirms that there is a need for a standardized, objective
calibration and validation protocol. Our protocol uses an inexpensive multi-ion
discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic
fluorophores that emit distinct, stable, spectral features
in place of a sample. We derived reference spectra
from the MIDL data to accurately predict the spectral
resolution, ratio of wavelength to wavelength, contrast,
and aliasing parameters of any CSI system. We were also
able to predict and confirm the influence of pinhole diameter
on spectral profiles.
"Scattering Suppression and Confocal Detection in Multifocal Multiphoton Microscopy",
Jörg Martini, Volker Andresen, Dario Anselmetti, Journal of Biomedical Optics 12(3), 034010 (May/June 2007)
Copyright ©2007 Society of Photo-Optical Instrumentation Engineers
We have developed a new descanned parallel (32-fold)
pinhole and photomultiplier detection array for multifocal multiphoton
microscopy that effectively reduces the blurring effect originating
from scattered fluorescence photons in strongly scattering biological
media. With this method, we achieve a fourfold improvement in photon
statistics for detecting ballistic photons and an increase in spatial
resolution by 21% in the lateral and 35% in the axial direction compared
to single-beam non-descanned multiphoton microscopy. The
new detection concept has been applied to plant leaves and pollen
grains to verify the improvements in imaging quality.
"Laser Scanning Confocal Microscopy",
Nathan S. Claxton, Thomas J. Fellers, Michael W. Davidson, Department of Optical Microscopy and Digital Imaging, National High Magnetic Field Laboratory,
The Florida State University, Tallahassee, Florida
Laser scanning confocal microscopy has become
an invaluable tool for a wide range of investigations
in the biological and medical sciences for imaging
thin optical sections in living and fixed specimens
ranging in thickness up to 100 micrometers. Modern
instruments are equipped with 3-5 laser systems
controlled by high-speed acousto-optic tunable filters
(AOTFs), which allow very precise regulation of
wavelength and excitation intensity. Coupled with
photomultipliers that have high quantum efficiency
in the near-ultraviolet, visible and near-infrared
spectral regions, these microscopes are capable of
examining fluorescence emission ranging from 400
to 750 nanometers. Instruments equipped with
spectral imaging detection systems further refine
the technique by enabling the examination and
resolution of fluorophores with overlapping spectra
as well as providing the ability to compensate for
autofluorescence. Recent advances in fluorophore
design have led to improved synthetic and naturally
occurring molecular probes, including fluorescent
proteins and quantum dots, which exhibit a high level
of photostability and target specificity.
"Spectral Imaging Fluorescence Microscopy",
Tokuko Haraguchi, Takeshi Shimi, Takako Koujin, Noriyo Hashiguchi, Yasushi Hiraoka, Genes to Cells (2002)
Copyright ©2002 Blackwell Science Limited
The spectral resolution of fluorescence microscope images in living cells is achieved by using a
confocal laser scanning microscope equipped with grating optics. This capability of temporal
and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for
example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral
imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent
dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate
(FITC) and green fluorescent protein (GFP).
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