ABSTRACT
Olfactometry is globally acknowledged as a technique to determine odor concentrations, which are used to characterize odors for regulatory purposes, e.g., to protect the general public against harmful effects of air pollution. Although the determination procedure for odor concentrations is standardized in some countries, continued research is required to understand uncertainties of odor monitoring and prediction. In this respect, the present paper strives to provide answers of paramount importance in olfactometry. To do so, a wealth of measurement data originating from six large-scale olfactometric stack emission proficiency tests conducted from 2015 to 2017 was retrospectively analyzed. The tests were hosted at a unique emission simulation apparatus-a replica of an industry chimney with 23 m in height-so that for the first time, conventional proficiency testing (no sampling) with real measurements (no reference concentrations) was combined. Surprisingly, highly variable recovery rates of the odorants were observed-no matter, which of the very different odorants was analyzed. Extended measurement uncertainties with roughly 30-300% up to 20-520% around a single olfactometric measurement value were calculated, which are way beyond the 95% confidence interval given by the widely used standard EN 13725 (45-220%) for assessment and control of odor emissions. Also, no evidence has been found that mixtures of odorants could be determined more precisely than single-component odorants. This is an important argument in the intensely discussed topic, whether n-butanol as current reference substance in olfactometry should be replaced by multi-component odorants. However, based on our data, resorting to an alternative reference substance will not solve the inherent problem of high uncertainty levels in dynamic olfactometry. Finally, robust statistics allowed to calculate reliable odor thresholds, which are an important prerequisite to convert mass concentrations to odor concentrations and vice versa.
Subject(s)
Air Pollutants/analysis , Environmental Monitoring/methods , Odorants/analysis , Environmental Monitoring/standards , Humans , Olfactometry , Pheromones , Retrospective StudiesABSTRACT
Exploring the maximum spatial resolution achievable in far-field optical imaging, we show that applying solid immersion lenses (SIL) in stimulated emission depletion (STED) microscopy addresses single spins with a resolution down to 2.4 ± 0.3 nm and with a localization precision of 0.09 nm.
Subject(s)
Image Enhancement/instrumentation , Lenses , Microscopy, Fluorescence/instrumentation , Nanotechnology/instrumentation , Equipment Design , Equipment Failure Analysis , SolutionsABSTRACT
We show stimulated emission depletion microscopy to break the diffraction limit in the all-far-field-optical detection of magnetic fields and resonances. Electron spin resonances from single nitrogen-vacancy centers in diamond located at subdiffraction proximities are fully discerned. Since diffraction is overcome by disallowing the signaling state through an optical transition such as stimulated emission, the spin state remains unaffected and amenable to microwave manipulation. Stimulated emission depletion presents a universal scheme for superresolving spin resonances detectable by fluorescence.
Subject(s)
Electron Spin Resonance Spectroscopy/methods , Optical Phenomena , Magnetic Fields , Microscopy , Nitrogen/chemistry , Spin LabelsABSTRACT
We describe a STED microscope optimized for colocalization experiments with up to three colors. Two fluorescence labels are separated by their fluorescence lifetime whereas a third channel is discriminated by the wavelength of fluorescence emission. Since it does not require a second STED beam, separating by lifetime is insensitive to drift and thus optimally suited for colocalization analyses. Furthermore, we propose a setup having a second STED beam for long duration multicolor recording.
Subject(s)
Imaging, Three-Dimensional/methods , Microscopy, Fluorescence/methods , Animals , Cell Line , Color , Lamins/metabolism , Time Factors , Tubulin/metabolismABSTRACT
STED microscopes are commonly built using separate optical paths for the excitation and the STED beam. As a result, the beams must be co-aligned and can be subject to mechanical drift. Here, we present a single-path STED microscope whose beams are aligned by design and hence is insensitive to mechanical drift. The design of a phase plate is described which selectively modulates the STED beam but leaves the excitation beam unaffected. The performance of the single-beam setup is on par with previous dual-beam designs.
Subject(s)
Image Enhancement/instrumentation , Microscopy, Fluorescence/instrumentation , Spectrum Analysis, Raman/instrumentation , Calibration , Computer-Aided Design , Equipment Design , Equipment Failure Analysis , Germany , Microscopy, Fluorescence/standards , Reproducibility of Results , Sensitivity and Specificity , Spectrum Analysis, Raman/standardsABSTRACT
We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.
