Analytische Chemie
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Optical spectroscopy – Techniques, instrumentation, and typical molecular and nanoscale reporters
(2018)
Different types of optical spectroscopies are introduced with special emphasis on method-inherent limitations and reliable instrument calibration and performance validation. In addition, different classes of molecular and nanocrystalline emitters are presented and the underlying photophysical processes are briefly described.
In all fluorescence-based techniques, the measured signals contain not only sample-related but also instrument-specific contributions, which limit the direct comparison of fluorescence data obtained e.g. on different devices or at different times and often hamper quantification. To rule out instrumentation as major source of variability of emission data, accepted fluorescence standards and procedures for the control of instrument specifications and long-term performance are required. For flow cytometry (FCM), a broad variety of fluorophore-stained polymer beads differing in emission wavelength and intensity is available for the testing of the alignment, sensitivity, and other parameters of FCM. These calibration tools are intended to facilitate the assessment of instrument performance to ensure reliable measurements and to improve the comparability of FCM experiments.
As a step towards an improved comparability of fluorescence data, with special emphasis on spectroscopic methods measuring nano- and micrometer-sized fluorescent objects, we are currently developing a set of fluorescent polystyrene (PS) beads loaded with luminophores from the certified BAM-Kit “Spectral fluorescent standards”, initially developed for the calibration of fluorescence spectrometers. Here, we present first results from studies of these fluorophore-loaded polymer beads. Moreover, new beads are made to supplement this kit by encapsulating near-infrared (NIR)-emissive luminophores in PS beads to cover the UV/VIS, and NIR wavelength range.
These beads are designed for calibration of flow cytometers and other fluorescence imaging systems to meet the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas like e.g. medical diagnostics.
Different types of optical spectroscopies are introduced with special emphasis on method-inherent limitations and reliable instrument calibration and performance validation. In addition, procedures for the determination of spectroscopic key parameters like the photoluminescence quantum yield are presented including required instrument calibrations and material-specific effects related to certain emitters.