Fluorescence calibration method for single-particle aerosol fluorescence instruments

Abstract

Abstract. Real-time, single-particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This gap limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need, we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wideband Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm, emission = 310-400 nm) and pure quinine particles to calibrate the other (FL2; excitation = 280 nm, emission = 420-650 nm). The relationship between fluorescence and mass for the mixed tryptophan-ammonium sulfate particles is linear, while that for the pure quinine particles is nonlinear, likely indicating that not all of the quinine mass contributes to the observed fluorescence. Nonetheless, both materials produce a repeatable response between observed fluorescence and particle mass. This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve the repeatability of the instrumental setup, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.

Characterization of steady-state fluorescence properties of polystyrene latex spheres using off- and online spectroscopic methods

“…Additionally, PSLs undergo aging processes, even when stored properly (i.e., at 4 • C). According to Robinson et al (2017), the shelf stability of PSLs is widely considered to be poor. However, to our knowledge there have been no published studies that address qualitative variances due to shelf degradation and so these issues cannot be predicted in detail.…”

“…Ultimately, more work will be required to develop more stable and generally accepted particle fluorescence calibration standards that can be applied reliably across instruments, time, and geography. One such example is the recent work presented by Robinson et al (2017), who used a mixture of tryptophan and ammonium sulfate to calibrate one fluorescence channel and pure quinine to calibrate a second channel. The authors of this paper did not, however, present a strategy to calibrate the third WIBS channel (FL3).…”

“…It is important to note that physical properties of PSLs underlie production processes, for which their quality cannot be considered to be consistent and, therefore, might shift even within the same production batch (Robinson et al, 2017). Additionally, PSLs also undergo aging processes (e.g., via reactions with radical species and destruction of aromaticity), which can result in, for example, decreasing fluorescence intensities or size inaccuracies due to particle agglomeration over time (Pellach et al, 2012).…”

“…The issue of fluorescence intensity calibration within UV-LIF instruments is sufficiently important and problematic that it has been discussed by a number of authors. In particular, Robinson et al (2017) developed a fluorescence calibration strategy for WIBS channels FL1 and FL2. The issue requires continued attention, however, from the UV-LIF community.…”

“…Fluorescent polystyrene latex spheres (PSLs) play a particularly important role in the characterization and calibration of instruments that rely on particle autofluorescence (also called intrinsic fluorescence) detection. For example, PSLs are commonly used for testing and calibration of instruments such as flow cytometers and lightinduced fluorescent (LIF) instruments (e.g., Hasegawa, 2013;Healy et al, 2012;Jung et al, 2012;Kanaani et al, 2008;Robinson et al, 2017;Toprak and Schnaiter, 2013).…”