Characterization of steady-state fluorescence properties of polystyrene latex spheres using off- and online spectroscopic methods
Abstract. Fluorescent dyed polystyrene latex spheres (PSLs) are commonly used for
characterization and calibration of instruments detecting fluorescence
signals from particles suspended in the air and other fluids. Instruments
like the Ultraviolet Aerodynamic Particle Sizer (UV-APS) and the Waveband
Integrated Bioaerosol Sensor (WIBS) are widely used for bioaerosol research,
but these instruments present significant technical and physical challenges
requiring careful characterization with standard particles. Many other
research communities use flow cytometry and other instruments that
interrogate fluorescence from individual particles, and these also frequently
rely on fluorescent PSLs as standards. Nevertheless, information about
physical properties of commercially available PSLs provided by each
manufacturer is generally proprietary and rarely available, making their use
in fluorescence validation and calibration very difficult. This technical note presents an overview of steady-state fluorescence
properties of fluorescent and non-fluorescent PSLs, as well as of
polystyrene-divinylbenzene (PS-DVB) particles, by using on- and offline
spectroscopic techniques. We show that the “fluorescence landscape” of
PSLs is more complex than the information typically provided by
manufacturers may imply, especially revealing multimodal emission patterns.
Furthermore, non-fluorescent PSLs also exhibit defined patterns of
fluorescent emission originating from a mixture of polystyrene and
detergents, which becomes a crucial point for fluorescence threshold
calibrations and qualitative comparison between instruments. By comparing
PSLs of different sizes, but doped with the same dye, changes in emission
spectra from bulk solutions are not immediately obvious. On a
single-particle scale, however, fluorescence intensity values increase with
increasing particle size. No significant effect in the fluorescence
signatures was detectable by comparing PSLs in dry vs. wet states,
indicating that solvent water may only play a minor role as a fluorescence
quencher. Because information provided by manufacturers of commercially available PSLs
is generally very limited, we provide the steady-state excitation–emission
matrices (EEMs) of PSLs as open-access data within the Supplement. Detergent
and solvent effects are also discussed in order to provide information not
available elsewhere to researchers in the bioaerosol and other research
communities. These data are not meant to serve as a fundamental library of
PSL properties because of the variability of fluorescent properties between
batches and as a function of particle aging and agglomeration. The data
presented, however, provide a summary of spectral features which are
consistent across these widely used fluorescent standards. Using these
concepts, further checks will likely be required by individual researchers
using specific lots of standards.