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Measurement of fluorescence spectra from atmospheric single submicron particle using laser-induced fluorescence technique
Journal of Aerosol Science 58 (2013) 1–8
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Journal of Aerosol Science journal homepage: www.elsevier.com/locate/jaerosci
Measurement of fluorescence spectra from atmospheric single submicron particle using laser-induced fluorescence technique Fumikazu Taketani a,n, Yugo Kanaya a, Takayuki Nakamura b, Kazuhiro Koizumi b, Nobuhiro Moteki c, Nobuyuki Takegawa d a
Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, 3173-25 Showamachi, Kanazawa, Yokohama 236-0001, Japan b Fuji Electric Co. Ltd., Fuji-machi 1, Hino-city, Tokyo 191-8502, Japan c Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan d Research Center for Advanced Science and Technology, University of Tokyo, Komaba 4-6-1, Meguro, Tokyo 153-8904, Japan
a r t i c l e i n f o
abstract
Article history: Received 28 August 2012 Received in revised form 6 December 2012 Accepted 12 December 2012 Available online 26 December 2012
This paper describes the development of an instrument for online detection of certain types of organic aerosol particles in the atmosphere using the laser-induced fluorescence (LIF) technique. Aerosol particles, sampled from the atmosphere to an optical chamber, is first detected by scattering of a 635-nm continuous wave (CW) laser beam and then excited by a 266-nm pulsed laser, to induce fluorescence emission. The fluorescence in the 300–600 nm wavelength range is spectrally dispersed by a grating spectrometer and then detected by a 32-anode photomultiplier tube (PMT). The performance of the instrument was tested using laboratory-generated particles with known fluorescence properties. We found that pure tryptophan particle as small as 0.3 mm was detectable with fluorescence. Preliminary results from ambient measurements and cluster analysis are presented and the interpretation of the classification is discussed. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Laser-induced fluorescence technique Single particle Fluorescence spectrum ambient particle measurement
1. Introduction Atmospheric aerosol influences the Earth’s radiative budget by scattering and absorbing the sunlight (Finlayson-Pitts & Pitts, 2000). It is important to understand optical and chemical properties of aerosols to adequately estimate the present and future influence on the radiative budget. Optical techniques are useful tools for detecting ambient aerosol particles in real time. Optical particle counters (OPCs) are widely used to measure the distribution of scattered light intensity from individual particles to derive aerosol particle size distribution. Adding compositional information to the size-determined individual particles would improve our understanding of the aerosol particle behavior in the atmosphere. Single-particle analysis would also provide information on the mixing state of the particle. Such compositional/chemical analyses of single particles have been achieved using mass spectrometry (Gard et al., 1997; Murphy & Thomson, 1995; Narukawa et al., 2007), laser-induced breakdown spectroscopy (Dixon & Hahn, 2005; Hybl et al., 2006; Park et al., 2009), and light- or laser-induced fluorescence (Brosseau et al., 2000; Hairston et al., 1997; Hill et al., 1995, 1999; Kaye et al., 2000, 2005;
n
Corresponding author. Fax: þ 81 45 778 5496. E-mail address: [email protected] (F. Taketani).
0021-8502/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jaerosci.2012.12.002
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Kiselev et al.,2011; Pan et al., 1999, 2001, 2003, 2009, 2010, 2011; Pinnick et al., 1995, 1998, 2004; Sivaprakasam et al., 2004, 2009; Wu et al., 2009). Fluorescence technique is useful for the identification and classification of certain types of ¨ organic/biological particles. Pohlker et al. (2012) recently summarized fluorescence properties of various types of aerosol particle composition, including both biological and non-biological compounds. Several types of instruments have been developed for the detection of fluorescence from a single particle. Kaye et al. (2000, 2005) have developed a fluorescence monitoring instrument, Waveband Integrated Bioaerosol Sensor (WIBS), which is capable of measuring light-induced fluorescence (with two different excitation wavelengths at 280 nm and 370 nm). This instrument has three fluorescence detection channels: emission following the 280-nm excitation is recorded at 310–400 nm and 400–600 nm, and fluorescence excited at 370 nm is detected at 400–600 nm. This instrument has been applied for the detection of primary bioaerosol particles that contain compounds such as tryptophan, nicotinamideadenine dinucleotide (NADH), and riboflavin. A commercially available instrument is the ultraviolet aerodynamic particle sizer (UV-APS, TSI, Inc.) (Brosseau et al., 2000; Hairston et al., 1997). The UV-APS uses a pulsed ultraviolet laser (Nd:YAG) at 355 nm for the excitation and measures fluorescence in the 420–575 nm wavelength range by a photomultiplier tube (PMT) through an optical filter. This instrument is also designed for the detection of primary bioaerosol particles, but focuses only on nucleotides and riboflavin. There is another category of instruments that can record fluorescence spectra in the 250–700 nm range using multi-channel detectors such as an intensified charge coupled device (ICCD) or a multi-anode PMT, combined with excitation by a 263 nm or 266 nm laser (Hill et al., 1995, 1999; Pan et al., 1999, 2001, 2003, 2009, 2010, 2011; Pinnick et al., 1995, 1998, 2004). Recently, Pan et al. reported the development of a particle fluorescence spectrometer employing dual-wavelength excitation at 263 nm and 351 nm, aiming at improved assessment of the particle composition (Pan et al., 2010). The main focus of these instruments has been the detection of supermicron bioaerosol particles. The fluorescence technique could be applied to the detection of submicron non-biological compounds such as polycyclic aromatic hydrocarbons (PAHs) generated from combustion (Kameda et al., 2005; Kavouras & Stephanou, 2002), which emit unique fluorescence when excited by ultraviolet light (Bones et al., 2010; Finlayson-Pitts & Pitts Jr., 2000; Niessner et al., 1991; Panne et al., 2000). We report the development of an instrument utilizing the laser-induced fluorescence technique, mostly focusing on extending its capability for the detection of fluorescence from submicron single-particles.
2. Instrument Fig. 1a shows the schematic diagram of the instrument for the measurement of the fluorescence spectra from a single particle. The optical chamber consisted of a hexagon cell equipped with four 15-cm long arms containing optical baffles. The optical baffles were placed to minimize the stray light scattered by the entrance and exit windows. The diameters of the aperture holes of the baffles in the entrance and exit arms were 3–5 mm. The particle stream, surrounded by a coaxial sheath airflow, was introduced from top to the center of the optical chamber at atmospheric pressure using a custom-built injector. The total injected flow rate to the optical chamber was 0.7 L/min, of which 0.6 L/min was the filtered sheath air. The exhaust flow was also controlled to be 0.7 L/min. The flow rates in the instrument were controlled using mass flow controllers (Kofloc: model 3660 series). The particle stream travelled vertically between the injector nozzle and receptor nozzle in the optical chamber, and the distance between the nozzles was 10 mm (Fig. 1b).
Fig. 1. (a) Schematic diagram of the developed instrument and (b) the side view of the injector nozzle and receptor nozzle of particles in the optical chamber. ADC: analog to digital converter; PMT: photo multiplier tube; PC: personal computer.
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A 635-nm continuous wave (CW) laser (Excelsior, 635C-35) with an average power of 35 mW and a 266-nm pulsed UV laser (Spectra Physics: YHP40-106QU and HM266-YU) were employed to detect and excite the particles, respectively. Fourth harmonic of a diode-pumped, Q-switched YAG laser was used to generate 266-nm photons. The pulse width and energy were 50 ns and 20 mJ/pulse, respectively. These two laser beams crossed at the center of the optical chamber, which was about 4 mm below the exit of the particle injector nozzle. The particle stream passed vertically through the crossing point of the two laser beams. The particles introduced into the optical chamber were first detected by scattering of the 635-nm CW laser beam, which was focused by a cylindrical plano-convex lens before entering the cell. The scattered light from the individual particles at 635 nm was detected by a PMT (Hamamatsu, R5926) through two lenses and a band-pass optical filter (Semrock, FF01-640/14-25). The scattering signal was amplified and recorded as a time profile (20 ms duration with a time step of 0.5 ms) for each particle after digitized by an analog–digital converter (Contec, AI-1204Z-PCI). The UV laser beam was weakly focused by a lens (with a focal length of 45 cm) before entering the fluorescence detection cell. The fluorescence emitted from the particle was collected using a series of four lenses and a long-pass optical filter (Semrock, FF01-300/LP-50) that allowed the transmission of fluorescence at wavelengths longer than 300 nm but blocked the resonant and Raman scatterings of the UV laser. The collected fluorescence with a wavelength range of about 300–600 nm was spectrally dispersed by a grating spectrometer (Andor, SR163 and SR1-GRT-0300-0300) and then detected by a 32-channel PMT (Hamamatsu, H7260-01). The wavelength range was covered by 16 channels of the PMT. The wavelength was calibrated using the wavelengths of Hg atomic lines from a Pen-Ray lamp. Data acquisition for the each fluorescence intensity was performed using a custom-built charge integrating analog–digital converter. The timings to trigger the pulsed UV laser and to initiate data acquisition were controlled by a delay/pulse generator (Stanford Research Systems, DG645), driven by the detection of a scattering signal whose intensity exceeded a set threshold. The irradiation of the pulsed 266-nm laser was delayed by 1.2 ms after the generation of the trigger pulse. During the 1.2 ms period, the particle travels vertically and therefore, the vertical position of the UV laser beam was precisely optimized. The delay/pulse generator also sent another trigger pulse to the analog–digital converter for the acquisition of the scattering light as well as a gate pulse (250 ns) defining the time period for the charge integrating 32-channel analog–digital converter to integrate each fluorescence signal. A custom software written in Visual Basic acquired all data to a PC, consisting of time stamp of the particle detection, a temporal profile of the scattering signal, and the time-integrated fluorescence signals for 32-channels per each particle. 3. Results and discussion 3.1. Calibration of particle size The particle-size calibration was made using dry polystyrene latex (PSL) particles (Duke Scientific). Mono-dispersed PSL particles generated by an atomizer were passed through a diffusion dryer (TSI, Model 3062) to be dried before entering the optical chamber. The time profiles of the scattering light intensities at 635 nm were measured for PSL particles of known sizes (0.3, 0.5, 0.7, 1.0, and 2.0 mm). The frequency distribution of the peak intensities of the scattering light at 635 nm was recorded. The most frequent signal intensities of the scattering light are shown in Fig. 2 as a function of the diameter (mm) of the PSL particles. They are well separated for different sizes. The intensities of the scattering light were utilized for the calibration of the particle sizes. Although it may be more suitable to use a fit based on Mie theory, we employed a secondorder polynomial fit in this study for simplicity: 2
y ¼ 0:208d þ 0:478d
ð1Þ
Fig. 2. The most frequently observed signal intensities of the scattered light as a function of each PSL particle diameter (0.3, 0.5, 0.7, 1.0, and 2.0 mm). The error bars indicate one-standard deviation (s) for most frequently observed signal intensities of the scattered light.
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where y is the scattering intensity and d is the particle diameter in mm. We used Eq. (1) to estimate the spherical PSL-equivalent size of the particles.
3.2. Fluorescence from a single particle In order to test the performance of the instrument, we measured the fluorescence spectra of size-known PSL particles and several primary fluorophores (tyrosine, tryptophan, NADH, and riboflavin), whose fluorescence properties have been reported for 5-mm particles when excited by a 266-nm laser (Hill et al., 1999). These compounds were dissolved in Milli-Q water and their particles (approximate size range 0.3–3.5 mm) were generated using an atomizer. They were then introduced into the optical chamber in the same manner as the PSL particles were introduced. The average background signal in each wavelength channel /SiS was measured in the absence of the particles while operating the UV laser in the internal trigger mode and was subtracted from the total signal. First, we tested the hitting percentage of the UV laser to the particles, taking advantage of the fact that PSL particles emit the fluorescence in the wavelength range of 300–400 nm. The hitting percentage, calculated as the ratio of the frequency of the detection of fluorescence in the 300–400 nm from PSL particles to that of the triggered pulses generated by the scattered light of 635 nm, was 70–80% and independent of the particle size (0.3–2.0 mm) in this system. Fig. 3 shows 100-shot-averaged spectra measured without selecting particle size of tyrosine, tryptophan, NADH, and riboflavin. Here the signal intensities were normalized to the peak intensity of each compound. Our results are in good agreement with the previous study (Hill et al., 1999), where tyrosine and tryptophan have peak emissions at around 310 nm and 340 nm, respectively, NADH has a broad emission at around 450 nm, and the riboflavin emission peak is present at around 560 nm. Therefore, our instrument can effectively differentiate the fluorescence occurring in different spectral regions, which enables basic classification of compounds on the basis of their fluorescence spectral signatures. The detectability of fluorescence from submicron particles was tested. Fig. 4 shows the fluorescence spectra from mono-dispersed particles of tryptophan with a size of 0.3 mm. Scattering light intensities of a 0.3-mm PSL particle and of a
Fig. 3. The averaged dispersed spectra for single particles of tyrosine, tryptophan, NADH, and riboflavin. The signal intensities were normalized to the peak intensity of each compound.
Fig. 4. Fluorescence spectra from mono-dispersed tryptophan particles with a size of 0.3 mm. Gray lines show observed fluorescence spectra. Broken line shows the averaged spectrum.
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mono-dispersed tryptophan (centered on 0.3 mm) selected by the DMA were almost same. Broken line shows the averaged spectrum of them. The particles were selected using a differential mobility analyzer (TSI, DMA3080) and then introduced into the optical chamber. The noise level at each wavelength was estimated from temporal fluctuation of the background signal, and the fluorescent particles were defined as those producing fluorescence signals exceeding /SiSþ 3si, where si represents a standard deviation. Considering the signal intensities of the tryptophan particles in this study in the 320–390-nm wavelength range were generally above 20 and si was in the range of 4–6, our instrument could detect the fluorescence signal from a pure tryptophan particle with a size of 0.3 mm at a sufficient sensitivity. In the previous study (Pan et al., 2010), the detectable size of fluorescence from tryptophan particle was larger than 1 mm. The reasons for our superiority are not very clear. The numerical aperture (NA) for fluorescence collection is similar (0.42 for our case, in comparison to 0.40). Although the same 32-channel PMT was used, gain settings or noise characteristics might be different. Another reason might be that in their instrument the particle detection and size determination by scattering are not optimized to submicron particles, and therefore the fluorescence characterization has not been enabled for submicron particles. Pan et al. (2010) utilize scattering caused by a pulsed UV laser and the scattering light histogram for 1-mm PSL particle exhibits a large uncertainty. Our nozzle might have better characteristics in sending particles to right position to produce scattering and fluorescence. A larger detection cell size (10 cm cube) in comparison to 5 cm cube (Pan et al., 2010) might have helped reducing the background signal levels for scattering and fluorescence. The detectivity of ambient submicron particle should depend on the fluorescence quantum yield and the volume fraction of the fluorophores; therefore it is concluded here that our instrument can detect fluorescence whose intensity is equivalent to that from a 0.3-mm pure tryptophan particle. 3.3. Ambient measurement To demonstrate the instrumental performance, in situ online measurements of ambient aerosol particles were carried out over about 6 h on the evening on April 20, 2011 at our campus in Yokosuka city facing Tokyo Bay (35.321N, 139.651E). Ambient air outside of our laboratory was introduced into the fluorescence monitoring cell at a flow rate of 0.7 L/min through a diffusion dryer by using a conductive tube (12.7 mm inner diameter and 2 m length), aided by supporting bypass flow (6 L/min) to minimize deposition of the particles. Over the course of this experiment, about 20,000 particles (approximate size range 40.7 mm) were detected. Out of the detected particles, about 26% (5171 particles) were fluorescent at one or more wavelength channels. As mentioned above, the threshold of /SiSþ3si was used to select the fluorescent particles for each wavelength channel. We adapted the cluster analysis technique to objectively group the obtained large number of fluorescence spectra into several types. In this study, we tentatively limited data used for the analysis to those having significant fluorescence signal levels (S/N 43) in six or more channels. As a result, the number of fluorescence spectra included in the analysis was reduced to 334. Furthermore, we used fluorescence spectra after normalized to its maximum, in order to make classification based on the spectral patterns, without being affected by the intensity which can be variable with the abundance of the composition. The results of the clustering by are shown in Fig. 5. We obtained nine clusters. The fraction of the particles assigned to each cluster, and the associated information of the averaged size and fluorescence intensity are listed in Table 1. Although it is difficult to identify the specific organic molecular compounds contributing to each fluorescence spectrum considering the general complexity of the organic composition of the particles in the atmosphere, the types of fluorophores could be inferred for the clusters. The fluorescence spectra in clusters 1, 2, and 3 have emission peaks at around 350, 370, and 400 nm, respectively. The fluorescence spectra in cluster 4 also have the emission peak in the 300–400 nm range. These particles could contain either single or double ring aromatics or heterocyclic compounds (Pinnick et al., 2004). The fluorescence spectra in cluster 1 might be caused by tryptophan as it also has a similar fluorescence pattern. We compared the signal intensity for pure tryptophan particles at 351 nm in Fig. 4 with those of cluster 1. In some cases, the signal intensities in cluster 1 were lower than those for pure tryptophan particles of the same size; this suggests either that the particles of cluster 1 include tryptophan as a partial constituent and mixed with other components, or that the fluorophores are other materials that have a lower quantum efficiency of fluorescence emission than that of tryptophan. The spectral patterns of clusters 2 and 3 were similar to those of cluster 1, but the peak positions are shifted to longer wavelengths, suggesting that the aromatics in the particles have some additional groups (Pinnick et al., 2004). The fluorescence spectra in cluster 4 had an emission peak in range of 300–400 nm, and some significant signals were observed at wavelengths greater than 500 nm, suggesting that these particles contain the compounds similar to those in the clusters 1–3, but mixed with other compounds emitting fluorescence with wavelengths of around 500–600 nm. These ¨ could include biological compounds such as flavins, lipofuscin, and terpenoids (Pohlker et al., 2012). The spectra for cluster 5 exhibited an arrow peak around 400 nm and its fluorescence intensity was strongest among all clusters. The peak could have been caused by aromatic and polycyclic aromatic compounds (Pan et al., 2001). The fluorescence spectra in cluster 6 were broad and had relatively low signal intensities at wavelengths of around 300–400 nm, suggesting that either fulvic or humic acids or humic-like substances might be responsible because of the similar fluorescence spectra of these compounds (Chen et al., 2003; Pan et al., 2001). The fluorescence spectra in the clusters 7–9 consisted of signals with large variabilities against wavelength. For cluster 7, it was difficult to suggest the candidates due to their discrete signals. The normal position of the peak in the clusters 8 and 9 was at wavelengths greater than 500 nm, suggesting that some biological compounds such as flavins, lipofuscin, and terpenoids might be contributing to the spectra. The fluorescent ambient particles size in this measurement ranged 0.7–1.3 mm, while those size in the previous study employing ICCD
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Fig. 5. Results of cluster analysis for the normalized fluorescence spectra from single particles as measured at Yokosuka city between 5:20 p.m. on April 20, 2011 and 01:00 a.m. on April 21, 2011. Gray lines show normalized fluorescence spectra. Broken lines show the averaged spectrum. Of the 5171 fluorescent particles detected, 334 showed fluorescence at six or more wavelength channels and their spectra were used in the analysis (see text).
Table 1 Details of each cluster of ambient aerosol particles classified by the fluorescence spectral pattern.
Cluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster
1 2 3 4 5 6 7 8 9
Number of particles
Averaged size (mm)
Averaged intensity (arb. unit)
19 30 33 22 13 33 44 83 57
0.97 0.75 0.88 1.03 0.89 1.00 0.99 1.05 1.13
400 313 578 315 1608 666 104 223 609
(6%) (9%) (10%) (7%) (4%) (10%) (12%) (24%) (17%)
detector were more than 3 mm (Pinnick et al., 2004), suggesting the improved capability of our instrument. In this analysis, the fluorescence patterns of clusters 1, 5 and 6 were similar to the some clusters (clusters 4, 6, and 8) analyzed in the previous report (Pinnick et al., 2004, Fig. 7), suggesting some fluorescent compounds would be ubiquitous in the ambient particles and present from submicron to supermicron particles. Using cluster analysis, we tentatively classified the fluorescence spectra into the nine clusters and some of the compounds potentially responsible for the spectra were inferred. More laboratory and field studies are necessary to characterize fluorescence spectra from various aerosol compounds in order to improve our systematic understanding. This study demonstrates that our instrument can be used for this purpose with the capability of detection down to submicron particles.
4. Conclusion In this study, we reported the development of an instrument for the measurement of fluorescence from single particles using the LIF technique. The performance of the instrument was tested using tyrosine, tryptophan, NADH, and riboflavin as reference compounds, which have distinct fluorescence properties. The major advantage of our instrument compared to
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previously reported ones is the minimum detectable diameter with regard to a single particle fluorescence spectrum (0.3 mm for pure tryptophan particles). During the test measurement of ambient aerosol particles in our campus, 26% of the total number of particles exhibited detectable levels of fluorescence. They were classified into several types by the cluster analysis, and the compounds potentially responsible for the observed fluorescence were inferred for each category. Further laboratory and field studies will be made toward systematic and improved understandings of the behaviors of atmospheric fluorescent aerosol particles.
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Journal of Aerosol Science journal homepage: www.elsevier.com/locate/jaerosci
Measurement of fluorescence spectra from atmospheric single submicron particle using laser-induced fluorescence technique Fumikazu Taketani a,n, Yugo Kanaya a, Takayuki Nakamura b, Kazuhiro Koizumi b, Nobuhiro Moteki c, Nobuyuki Takegawa d a
Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, 3173-25 Showamachi, Kanazawa, Yokohama 236-0001, Japan b Fuji Electric Co. Ltd., Fuji-machi 1, Hino-city, Tokyo 191-8502, Japan c Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan d Research Center for Advanced Science and Technology, University of Tokyo, Komaba 4-6-1, Meguro, Tokyo 153-8904, Japan
a r t i c l e i n f o
abstract
Article history: Received 28 August 2012 Received in revised form 6 December 2012 Accepted 12 December 2012 Available online 26 December 2012
This paper describes the development of an instrument for online detection of certain types of organic aerosol particles in the atmosphere using the laser-induced fluorescence (LIF) technique. Aerosol particles, sampled from the atmosphere to an optical chamber, is first detected by scattering of a 635-nm continuous wave (CW) laser beam and then excited by a 266-nm pulsed laser, to induce fluorescence emission. The fluorescence in the 300–600 nm wavelength range is spectrally dispersed by a grating spectrometer and then detected by a 32-anode photomultiplier tube (PMT). The performance of the instrument was tested using laboratory-generated particles with known fluorescence properties. We found that pure tryptophan particle as small as 0.3 mm was detectable with fluorescence. Preliminary results from ambient measurements and cluster analysis are presented and the interpretation of the classification is discussed. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Laser-induced fluorescence technique Single particle Fluorescence spectrum ambient particle measurement
1. Introduction Atmospheric aerosol influences the Earth’s radiative budget by scattering and absorbing the sunlight (Finlayson-Pitts & Pitts, 2000). It is important to understand optical and chemical properties of aerosols to adequately estimate the present and future influence on the radiative budget. Optical techniques are useful tools for detecting ambient aerosol particles in real time. Optical particle counters (OPCs) are widely used to measure the distribution of scattered light intensity from individual particles to derive aerosol particle size distribution. Adding compositional information to the size-determined individual particles would improve our understanding of the aerosol particle behavior in the atmosphere. Single-particle analysis would also provide information on the mixing state of the particle. Such compositional/chemical analyses of single particles have been achieved using mass spectrometry (Gard et al., 1997; Murphy & Thomson, 1995; Narukawa et al., 2007), laser-induced breakdown spectroscopy (Dixon & Hahn, 2005; Hybl et al., 2006; Park et al., 2009), and light- or laser-induced fluorescence (Brosseau et al., 2000; Hairston et al., 1997; Hill et al., 1995, 1999; Kaye et al., 2000, 2005;
n
Corresponding author. Fax: þ 81 45 778 5496. E-mail address: [email protected] (F. Taketani).
0021-8502/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jaerosci.2012.12.002
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Kiselev et al.,2011; Pan et al., 1999, 2001, 2003, 2009, 2010, 2011; Pinnick et al., 1995, 1998, 2004; Sivaprakasam et al., 2004, 2009; Wu et al., 2009). Fluorescence technique is useful for the identification and classification of certain types of ¨ organic/biological particles. Pohlker et al. (2012) recently summarized fluorescence properties of various types of aerosol particle composition, including both biological and non-biological compounds. Several types of instruments have been developed for the detection of fluorescence from a single particle. Kaye et al. (2000, 2005) have developed a fluorescence monitoring instrument, Waveband Integrated Bioaerosol Sensor (WIBS), which is capable of measuring light-induced fluorescence (with two different excitation wavelengths at 280 nm and 370 nm). This instrument has three fluorescence detection channels: emission following the 280-nm excitation is recorded at 310–400 nm and 400–600 nm, and fluorescence excited at 370 nm is detected at 400–600 nm. This instrument has been applied for the detection of primary bioaerosol particles that contain compounds such as tryptophan, nicotinamideadenine dinucleotide (NADH), and riboflavin. A commercially available instrument is the ultraviolet aerodynamic particle sizer (UV-APS, TSI, Inc.) (Brosseau et al., 2000; Hairston et al., 1997). The UV-APS uses a pulsed ultraviolet laser (Nd:YAG) at 355 nm for the excitation and measures fluorescence in the 420–575 nm wavelength range by a photomultiplier tube (PMT) through an optical filter. This instrument is also designed for the detection of primary bioaerosol particles, but focuses only on nucleotides and riboflavin. There is another category of instruments that can record fluorescence spectra in the 250–700 nm range using multi-channel detectors such as an intensified charge coupled device (ICCD) or a multi-anode PMT, combined with excitation by a 263 nm or 266 nm laser (Hill et al., 1995, 1999; Pan et al., 1999, 2001, 2003, 2009, 2010, 2011; Pinnick et al., 1995, 1998, 2004). Recently, Pan et al. reported the development of a particle fluorescence spectrometer employing dual-wavelength excitation at 263 nm and 351 nm, aiming at improved assessment of the particle composition (Pan et al., 2010). The main focus of these instruments has been the detection of supermicron bioaerosol particles. The fluorescence technique could be applied to the detection of submicron non-biological compounds such as polycyclic aromatic hydrocarbons (PAHs) generated from combustion (Kameda et al., 2005; Kavouras & Stephanou, 2002), which emit unique fluorescence when excited by ultraviolet light (Bones et al., 2010; Finlayson-Pitts & Pitts Jr., 2000; Niessner et al., 1991; Panne et al., 2000). We report the development of an instrument utilizing the laser-induced fluorescence technique, mostly focusing on extending its capability for the detection of fluorescence from submicron single-particles.
2. Instrument Fig. 1a shows the schematic diagram of the instrument for the measurement of the fluorescence spectra from a single particle. The optical chamber consisted of a hexagon cell equipped with four 15-cm long arms containing optical baffles. The optical baffles were placed to minimize the stray light scattered by the entrance and exit windows. The diameters of the aperture holes of the baffles in the entrance and exit arms were 3–5 mm. The particle stream, surrounded by a coaxial sheath airflow, was introduced from top to the center of the optical chamber at atmospheric pressure using a custom-built injector. The total injected flow rate to the optical chamber was 0.7 L/min, of which 0.6 L/min was the filtered sheath air. The exhaust flow was also controlled to be 0.7 L/min. The flow rates in the instrument were controlled using mass flow controllers (Kofloc: model 3660 series). The particle stream travelled vertically between the injector nozzle and receptor nozzle in the optical chamber, and the distance between the nozzles was 10 mm (Fig. 1b).
Fig. 1. (a) Schematic diagram of the developed instrument and (b) the side view of the injector nozzle and receptor nozzle of particles in the optical chamber. ADC: analog to digital converter; PMT: photo multiplier tube; PC: personal computer.
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A 635-nm continuous wave (CW) laser (Excelsior, 635C-35) with an average power of 35 mW and a 266-nm pulsed UV laser (Spectra Physics: YHP40-106QU and HM266-YU) were employed to detect and excite the particles, respectively. Fourth harmonic of a diode-pumped, Q-switched YAG laser was used to generate 266-nm photons. The pulse width and energy were 50 ns and 20 mJ/pulse, respectively. These two laser beams crossed at the center of the optical chamber, which was about 4 mm below the exit of the particle injector nozzle. The particle stream passed vertically through the crossing point of the two laser beams. The particles introduced into the optical chamber were first detected by scattering of the 635-nm CW laser beam, which was focused by a cylindrical plano-convex lens before entering the cell. The scattered light from the individual particles at 635 nm was detected by a PMT (Hamamatsu, R5926) through two lenses and a band-pass optical filter (Semrock, FF01-640/14-25). The scattering signal was amplified and recorded as a time profile (20 ms duration with a time step of 0.5 ms) for each particle after digitized by an analog–digital converter (Contec, AI-1204Z-PCI). The UV laser beam was weakly focused by a lens (with a focal length of 45 cm) before entering the fluorescence detection cell. The fluorescence emitted from the particle was collected using a series of four lenses and a long-pass optical filter (Semrock, FF01-300/LP-50) that allowed the transmission of fluorescence at wavelengths longer than 300 nm but blocked the resonant and Raman scatterings of the UV laser. The collected fluorescence with a wavelength range of about 300–600 nm was spectrally dispersed by a grating spectrometer (Andor, SR163 and SR1-GRT-0300-0300) and then detected by a 32-channel PMT (Hamamatsu, H7260-01). The wavelength range was covered by 16 channels of the PMT. The wavelength was calibrated using the wavelengths of Hg atomic lines from a Pen-Ray lamp. Data acquisition for the each fluorescence intensity was performed using a custom-built charge integrating analog–digital converter. The timings to trigger the pulsed UV laser and to initiate data acquisition were controlled by a delay/pulse generator (Stanford Research Systems, DG645), driven by the detection of a scattering signal whose intensity exceeded a set threshold. The irradiation of the pulsed 266-nm laser was delayed by 1.2 ms after the generation of the trigger pulse. During the 1.2 ms period, the particle travels vertically and therefore, the vertical position of the UV laser beam was precisely optimized. The delay/pulse generator also sent another trigger pulse to the analog–digital converter for the acquisition of the scattering light as well as a gate pulse (250 ns) defining the time period for the charge integrating 32-channel analog–digital converter to integrate each fluorescence signal. A custom software written in Visual Basic acquired all data to a PC, consisting of time stamp of the particle detection, a temporal profile of the scattering signal, and the time-integrated fluorescence signals for 32-channels per each particle. 3. Results and discussion 3.1. Calibration of particle size The particle-size calibration was made using dry polystyrene latex (PSL) particles (Duke Scientific). Mono-dispersed PSL particles generated by an atomizer were passed through a diffusion dryer (TSI, Model 3062) to be dried before entering the optical chamber. The time profiles of the scattering light intensities at 635 nm were measured for PSL particles of known sizes (0.3, 0.5, 0.7, 1.0, and 2.0 mm). The frequency distribution of the peak intensities of the scattering light at 635 nm was recorded. The most frequent signal intensities of the scattering light are shown in Fig. 2 as a function of the diameter (mm) of the PSL particles. They are well separated for different sizes. The intensities of the scattering light were utilized for the calibration of the particle sizes. Although it may be more suitable to use a fit based on Mie theory, we employed a secondorder polynomial fit in this study for simplicity: 2
y ¼ 0:208d þ 0:478d
ð1Þ
Fig. 2. The most frequently observed signal intensities of the scattered light as a function of each PSL particle diameter (0.3, 0.5, 0.7, 1.0, and 2.0 mm). The error bars indicate one-standard deviation (s) for most frequently observed signal intensities of the scattered light.
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where y is the scattering intensity and d is the particle diameter in mm. We used Eq. (1) to estimate the spherical PSL-equivalent size of the particles.
3.2. Fluorescence from a single particle In order to test the performance of the instrument, we measured the fluorescence spectra of size-known PSL particles and several primary fluorophores (tyrosine, tryptophan, NADH, and riboflavin), whose fluorescence properties have been reported for 5-mm particles when excited by a 266-nm laser (Hill et al., 1999). These compounds were dissolved in Milli-Q water and their particles (approximate size range 0.3–3.5 mm) were generated using an atomizer. They were then introduced into the optical chamber in the same manner as the PSL particles were introduced. The average background signal in each wavelength channel /SiS was measured in the absence of the particles while operating the UV laser in the internal trigger mode and was subtracted from the total signal. First, we tested the hitting percentage of the UV laser to the particles, taking advantage of the fact that PSL particles emit the fluorescence in the wavelength range of 300–400 nm. The hitting percentage, calculated as the ratio of the frequency of the detection of fluorescence in the 300–400 nm from PSL particles to that of the triggered pulses generated by the scattered light of 635 nm, was 70–80% and independent of the particle size (0.3–2.0 mm) in this system. Fig. 3 shows 100-shot-averaged spectra measured without selecting particle size of tyrosine, tryptophan, NADH, and riboflavin. Here the signal intensities were normalized to the peak intensity of each compound. Our results are in good agreement with the previous study (Hill et al., 1999), where tyrosine and tryptophan have peak emissions at around 310 nm and 340 nm, respectively, NADH has a broad emission at around 450 nm, and the riboflavin emission peak is present at around 560 nm. Therefore, our instrument can effectively differentiate the fluorescence occurring in different spectral regions, which enables basic classification of compounds on the basis of their fluorescence spectral signatures. The detectability of fluorescence from submicron particles was tested. Fig. 4 shows the fluorescence spectra from mono-dispersed particles of tryptophan with a size of 0.3 mm. Scattering light intensities of a 0.3-mm PSL particle and of a
Fig. 3. The averaged dispersed spectra for single particles of tyrosine, tryptophan, NADH, and riboflavin. The signal intensities were normalized to the peak intensity of each compound.
Fig. 4. Fluorescence spectra from mono-dispersed tryptophan particles with a size of 0.3 mm. Gray lines show observed fluorescence spectra. Broken line shows the averaged spectrum.
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mono-dispersed tryptophan (centered on 0.3 mm) selected by the DMA were almost same. Broken line shows the averaged spectrum of them. The particles were selected using a differential mobility analyzer (TSI, DMA3080) and then introduced into the optical chamber. The noise level at each wavelength was estimated from temporal fluctuation of the background signal, and the fluorescent particles were defined as those producing fluorescence signals exceeding /SiSþ 3si, where si represents a standard deviation. Considering the signal intensities of the tryptophan particles in this study in the 320–390-nm wavelength range were generally above 20 and si was in the range of 4–6, our instrument could detect the fluorescence signal from a pure tryptophan particle with a size of 0.3 mm at a sufficient sensitivity. In the previous study (Pan et al., 2010), the detectable size of fluorescence from tryptophan particle was larger than 1 mm. The reasons for our superiority are not very clear. The numerical aperture (NA) for fluorescence collection is similar (0.42 for our case, in comparison to 0.40). Although the same 32-channel PMT was used, gain settings or noise characteristics might be different. Another reason might be that in their instrument the particle detection and size determination by scattering are not optimized to submicron particles, and therefore the fluorescence characterization has not been enabled for submicron particles. Pan et al. (2010) utilize scattering caused by a pulsed UV laser and the scattering light histogram for 1-mm PSL particle exhibits a large uncertainty. Our nozzle might have better characteristics in sending particles to right position to produce scattering and fluorescence. A larger detection cell size (10 cm cube) in comparison to 5 cm cube (Pan et al., 2010) might have helped reducing the background signal levels for scattering and fluorescence. The detectivity of ambient submicron particle should depend on the fluorescence quantum yield and the volume fraction of the fluorophores; therefore it is concluded here that our instrument can detect fluorescence whose intensity is equivalent to that from a 0.3-mm pure tryptophan particle. 3.3. Ambient measurement To demonstrate the instrumental performance, in situ online measurements of ambient aerosol particles were carried out over about 6 h on the evening on April 20, 2011 at our campus in Yokosuka city facing Tokyo Bay (35.321N, 139.651E). Ambient air outside of our laboratory was introduced into the fluorescence monitoring cell at a flow rate of 0.7 L/min through a diffusion dryer by using a conductive tube (12.7 mm inner diameter and 2 m length), aided by supporting bypass flow (6 L/min) to minimize deposition of the particles. Over the course of this experiment, about 20,000 particles (approximate size range 40.7 mm) were detected. Out of the detected particles, about 26% (5171 particles) were fluorescent at one or more wavelength channels. As mentioned above, the threshold of /SiSþ3si was used to select the fluorescent particles for each wavelength channel. We adapted the cluster analysis technique to objectively group the obtained large number of fluorescence spectra into several types. In this study, we tentatively limited data used for the analysis to those having significant fluorescence signal levels (S/N 43) in six or more channels. As a result, the number of fluorescence spectra included in the analysis was reduced to 334. Furthermore, we used fluorescence spectra after normalized to its maximum, in order to make classification based on the spectral patterns, without being affected by the intensity which can be variable with the abundance of the composition. The results of the clustering by are shown in Fig. 5. We obtained nine clusters. The fraction of the particles assigned to each cluster, and the associated information of the averaged size and fluorescence intensity are listed in Table 1. Although it is difficult to identify the specific organic molecular compounds contributing to each fluorescence spectrum considering the general complexity of the organic composition of the particles in the atmosphere, the types of fluorophores could be inferred for the clusters. The fluorescence spectra in clusters 1, 2, and 3 have emission peaks at around 350, 370, and 400 nm, respectively. The fluorescence spectra in cluster 4 also have the emission peak in the 300–400 nm range. These particles could contain either single or double ring aromatics or heterocyclic compounds (Pinnick et al., 2004). The fluorescence spectra in cluster 1 might be caused by tryptophan as it also has a similar fluorescence pattern. We compared the signal intensity for pure tryptophan particles at 351 nm in Fig. 4 with those of cluster 1. In some cases, the signal intensities in cluster 1 were lower than those for pure tryptophan particles of the same size; this suggests either that the particles of cluster 1 include tryptophan as a partial constituent and mixed with other components, or that the fluorophores are other materials that have a lower quantum efficiency of fluorescence emission than that of tryptophan. The spectral patterns of clusters 2 and 3 were similar to those of cluster 1, but the peak positions are shifted to longer wavelengths, suggesting that the aromatics in the particles have some additional groups (Pinnick et al., 2004). The fluorescence spectra in cluster 4 had an emission peak in range of 300–400 nm, and some significant signals were observed at wavelengths greater than 500 nm, suggesting that these particles contain the compounds similar to those in the clusters 1–3, but mixed with other compounds emitting fluorescence with wavelengths of around 500–600 nm. These ¨ could include biological compounds such as flavins, lipofuscin, and terpenoids (Pohlker et al., 2012). The spectra for cluster 5 exhibited an arrow peak around 400 nm and its fluorescence intensity was strongest among all clusters. The peak could have been caused by aromatic and polycyclic aromatic compounds (Pan et al., 2001). The fluorescence spectra in cluster 6 were broad and had relatively low signal intensities at wavelengths of around 300–400 nm, suggesting that either fulvic or humic acids or humic-like substances might be responsible because of the similar fluorescence spectra of these compounds (Chen et al., 2003; Pan et al., 2001). The fluorescence spectra in the clusters 7–9 consisted of signals with large variabilities against wavelength. For cluster 7, it was difficult to suggest the candidates due to their discrete signals. The normal position of the peak in the clusters 8 and 9 was at wavelengths greater than 500 nm, suggesting that some biological compounds such as flavins, lipofuscin, and terpenoids might be contributing to the spectra. The fluorescent ambient particles size in this measurement ranged 0.7–1.3 mm, while those size in the previous study employing ICCD
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Fig. 5. Results of cluster analysis for the normalized fluorescence spectra from single particles as measured at Yokosuka city between 5:20 p.m. on April 20, 2011 and 01:00 a.m. on April 21, 2011. Gray lines show normalized fluorescence spectra. Broken lines show the averaged spectrum. Of the 5171 fluorescent particles detected, 334 showed fluorescence at six or more wavelength channels and their spectra were used in the analysis (see text).
Table 1 Details of each cluster of ambient aerosol particles classified by the fluorescence spectral pattern.
Cluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster
1 2 3 4 5 6 7 8 9
Number of particles
Averaged size (mm)
Averaged intensity (arb. unit)
19 30 33 22 13 33 44 83 57
0.97 0.75 0.88 1.03 0.89 1.00 0.99 1.05 1.13
400 313 578 315 1608 666 104 223 609
(6%) (9%) (10%) (7%) (4%) (10%) (12%) (24%) (17%)
detector were more than 3 mm (Pinnick et al., 2004), suggesting the improved capability of our instrument. In this analysis, the fluorescence patterns of clusters 1, 5 and 6 were similar to the some clusters (clusters 4, 6, and 8) analyzed in the previous report (Pinnick et al., 2004, Fig. 7), suggesting some fluorescent compounds would be ubiquitous in the ambient particles and present from submicron to supermicron particles. Using cluster analysis, we tentatively classified the fluorescence spectra into the nine clusters and some of the compounds potentially responsible for the spectra were inferred. More laboratory and field studies are necessary to characterize fluorescence spectra from various aerosol compounds in order to improve our systematic understanding. This study demonstrates that our instrument can be used for this purpose with the capability of detection down to submicron particles.
4. Conclusion In this study, we reported the development of an instrument for the measurement of fluorescence from single particles using the LIF technique. The performance of the instrument was tested using tyrosine, tryptophan, NADH, and riboflavin as reference compounds, which have distinct fluorescence properties. The major advantage of our instrument compared to
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previously reported ones is the minimum detectable diameter with regard to a single particle fluorescence spectrum (0.3 mm for pure tryptophan particles). During the test measurement of ambient aerosol particles in our campus, 26% of the total number of particles exhibited detectable levels of fluorescence. They were classified into several types by the cluster analysis, and the compounds potentially responsible for the observed fluorescence were inferred for each category. Further laboratory and field studies will be made toward systematic and improved understandings of the behaviors of atmospheric fluorescent aerosol particles.
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