In the presented study we characterized the suitability of 15 conventional fluorescence dyes for GSDIM microscopy. For
all dyes involved in the screening labeled secondary antibodies for immunohistochemistry are commercially available.
The dye performance was tested after staining to fixed mammalian cells. Chemical environments were chosen to be
compatible with the applicative and spectroscopic demands. Investigated watery environments are suitable for TIRF
based applications. To the best of our knowledge, we present for the first time systematic screening for configurations of
dyes embedded in solid polymer. The polymer mounting matches well to the refractive index of oil immersion optics.
This is crucial for applications at high penetration depth into the sample and suitable for long-term sample storage.
We rated the final super-resolution image quality additional to quantitative characterization of important spectroscopic
parameters. Therefore, this dye screening is optimized for various biological imaging applications. Control of the single molecule blinking rate by 405nm light exposure is quantified, as well. It is shown that this important effect is applicable to numerous fluorescent dyes. Thus, the controlled application of low intensities of 405nm light allows to maximize recording speed. As this option is already included in commercial GSDIM microscopes the results of our study allow optimized super-resolution imaging down to ~20nm with multiple dyes and multi-color staining.
STED microscopy enables confocal imaging of biological samples with a resolution that is not limited by diffraction. It
provides new insights in various fields of biology, such as membrane biology, neurobiology and physiology. Its three
dimensional sectioning ability allows the acquisition of high resolution image stacks. Furthermore, STED microscopy
enables the recording of dynamic processes and live cell images. We present two-color imaging in confocal STED
microscopy with a single STED wavelength. Pulsed and continuous wave lasers in the visible and near infra-red
wavelengths range are used for stimulated emission. The resolution enhancement is demonstrated in comparison to
confocal imaging with biological specimens.
STED microscopy has gained recognition as a method to break the diffraction limit of conventional light microscopy.
Despite being a new technique, STED is already successfully implemented in life science research. The resolution
enhancement is achieved by depleting fluorescent markers via stimulated emission. The performance is significantly
dependent on the laser source and the fluorescence markers. Therefore the use of novel fluorescent markers in
conjunction with the right laser system was the main focus of our research. We present new developments and
applications of STED microscopy, unraveling structural details on scales below 90nm and give an overview of required
specifications for the solid state laser systems.
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