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Here, we summerize our efforts concerning new design concepts and examples for fluorescence standards that can provide traceability to radiometric units and present a first step towards a toolbox of fluorescence standards, currently consisting of:
i) A first set of liquid fluorescence standards enables the determination of a broad variety of fluorescence parameters was developed and certified by BAM and is distributed by Sigma-Aldrich.
ii) Ready-to-use, glass-based fluorescence standards for instrument performance validation (IPV) and determination instrument-to-instrument variations can also be used as wavelength standard for fluorescence instruments with low requirements on spectral resolution and allow monitoring of temporal changes of the wavelength-dependent spectral responsivity.
iii) Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.
iv) We currently develop reference materials, which can be used as reliable quantum yield standards for relative methods for the determination of QY and can be valuable in the evaluation of the performance and sources of uncertainty of absolute, standard-free methods using e.g. integrating spheres.
There is an increasing interest in bridging the gap between the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) commonly assessed in ensemble studies and the PL features of single QDs for life sciences applications such as bioimaging studies or use in microfluidic assays. The fluorescence quantum yield (ΦF) is a key performance parameter for all molecular and nanoscale emitters, increasingly employed in nanoscience, nanotechnology, and medical diagnostics.
ΦF determines not only the signal size together with the reporter´s molar extinction coefficient, yet it is particularly relevant for nanocrystals like QDs with coordinatively bound surface ligands and size- and surface chemistry-dependent PL characteristics.
The importance of ΦF measurements at ultralow concentration encouraged us to explore the potential of fluorescence correlation spectroscopy (FCS) for the relative determination of ΦF of ligand-stabilized CdTe nanocrystals in comparison to molecular dyes with closely matching spectral properties and known ΦF.
We describe a FCS-based method for the relative determination of ΦF of dispersed QDs at ultralow concentrations, and procedures to overcome QD-inherent challenges like complex and power-dependent blinking behavior as well as ligand- and QD-specific aggregation. We could demonstrate the potential of this approach by comparison with steady state ensemble measurements.