Spectral detection of ultraviolet laser induced fluorescence from individual bioaerosol particles Per Jonsson*a, Fredrik Kullandera, Claes Vahlberga, Pär Jelgerb, Mikael Tiihonenb, Pär Wästerbyc, Torbjörn Tjärnhagec, and Mikael Lindgrenc, d a
FOI - Swedish Defence Research Agency, Sensor Technology, PO Box 1165, SE-581 11 Linköping, Sweden b KTH - Royal Institute of Technology, Dept. of Applied Physics, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden c FOI - Swedish Defence Research Agency, NBC Defence, Cementv. 20, SE-901 82 Umeå, Sweden d Dept. of Physics, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway ABSTRACT We present results of a measurement system designed for detecting the fluorescence spectrum of individual aerosol particles of biological warfare agents excited with laser pulses at wavelengths around 290 or 340 nm. The biological aerosol is prepared and directed into a narrow air beam. A red laser is focused on the aerosol beam and a trigger photomultiplier tube monitor the presence of individual particles by measuring the scattered light. When a particle is present in the detection volume, a laser pulse is triggered from an ultraviolet laser and the fluorescence spectrum is acquired with a spectrometer based on a diffraction grating and a 32 channels photomultiplier tube array with singlephoton sensitivity. The spectrometer measures the fluorescence spectra in the wavelength region from 300 to 800 nm. In the experiment we used different simulants of biological warfare agents. These bioaerosol particles were excited by a commercial available gas laser (337 nm), or a laser (290 nm) that we have developed based on an optical parametric oscillator with intracavity sum-frequency mixing. In the analysis of the experiments we compare the measured signals (fluorescence spectra, total fluorescence energy and the scattered energy) from the individual bioaerosol particles excited with the two different ultraviolet wavelengths. Keywords: bioaerosol detection, biological warfare agent, ultraviolet laser excitation, fluorescence spectroscopy, point detection
1. INTRODUCTION Detection of contagious levels of biological warfare agents (BWA) is genuinely difficult. Already very low doses of BWA in air can infect humans, so the detection system must be very sensitive. The system also needs to be fast so that precautionary measures can be taken before people have been exposed to the threat. To complicate the task even further, there are many harmless naturally occurring biological particles that are hard to distinguish from the hazardous ones. Consequently, it is not a trivial task to construct protection systems against the BWA threat. No single sensor or technology fulfill all the requirements for sensitivity, speed and specificity (distinguish between harmless and hazardous particles). Many BWA detection systems therefore combine different sensors to achieve the best performance possible. These systems normally have an initial warning or trigger detector that continuously monitor the air to achieve a fast response time. The trigger detectors measure some inherent property of the dry aerosol particles and warn personnel and trigger further analysis with other sensors. Examples of warning detectors are the Aerosol Size and Shape Analyzer (ASAS) that measures the particle shape from laser scattering [1], the Fluorescence Aerodynamic Particle Sizer (FLAPS) system that measure size and presence of ultraviolet (UV) induced fluorescence [2] and the Biological Alarm Monitor (MAB) which measure the elemental decomposition by flame spectrophotometry [3]. These detectors can reach close to real-time warning but have relatively low specificity which sometimes results in a high false alarm rate. *
[email protected], Phone +46-13-37 8578, Fax +46-13-37 8066, www.foi.se Optically Based Biological and Chemical Detection for Defence III, edited by John C. Carrano, Arturas Zukauskas, Proc. of SPIE Vol. 6398, 63980F, (2006) 0277-786X/06/$15 · doi: 10.1117/12.689666 Proc. of SPIE Vol. 6398 63980F-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/25/2014 Terms of Use: http://spiedl.org/terms
We are investigating methods of detection that give an increased specificity, with preserved sensitivity and detection speed, by measuring UV laser induced fluorescence spectra from individual bioaersol particles at dual excitation wavelengths (around 290 nm and 340 nm) [4, 5]. All biological particles fluoresce when excited with UV radiation. Aromatic amino acids, such as tryptophan, tyrosine and phenylalanine absorb light at 280-290 nm and they emit in a band between 300 nm and 400 nm [6]. Biogenic chemicals associated with cell metabolism, such as reduced nicotinamide adenine dinucleotide (NADH) and riboflavin have their dominating electronic absorption at around 340 nm and the resulting fluorescence peaks between 450 nm and 560 nm [6]. Furthermore, dipicolinic acid, present in spores, has its maximum absorption around 340 nm with an associated emission-band that peaks between 400 and 450 nm [7]. Our detection method, with dual wavelength excitation and the spectral detection, has the potential to distinguish between different biological and interference particles on a single particle level. Our current efforts aim to characterize the single particle fluorescence in order to determine the performance of a warning system based on this method. Many of the inherent characteristics of single bioaerosol particles such as their spectral fingerprint, the amplitude of the fluorescence and ultimately the scattering and absorption cross section together with the quantum efficiency of emission need to be better understood. The dependence on shape, particle size, excitation pulse energy and excitation wavelength of these properties requires investigation. Multivariate data analysis of these properties on a single particle level could improve the specificity in the detection of BWA [8]. In this paper we present experiments of simultaneously measured fluorescence spectra and scattering from individual particles excited with single UV laser pulses at two different excitation wavelengths (290 nm or 337 nm) and the preliminary data analysis of the results.
2. EXPERIMENTAL SETUP 2.1 Aerosol beam generation The particles used in these experiments were class 1 organisms, often used as simulants for biological warfare agents. Bacillus atrophaeus (also known as Bacillus subtilis var. niger and Bacillus globigii, red strain) and Turex (a non-pure technical preparation of Bacillus thuringiensis) are being used as simulants for pathogenic bacterial spores such as Anthrax. Ovalbumin from chicken egg (Sigma, A-5253, Grade II) is commonly used as a simulant for toxins. We are using the common abbreviations BG, BT and OA, respectively, for the simulants. The micron sized spores of BG and BT and the molecules of OA tend to cluster into larger particles when aerosolized. The typical diameter of the particles investigated was between 1 and 10 µm. The setup for aerosol generation is shown in Fig. 1. The aerosol was generated from a thin layer of dry simulant powder put on the bottom of a beaker. The emission rate of the aerosol was controlled by agitating the beaker by hand and/or by a magnetic stirrer with a 30mm long steel-wire with a diameter of 1mm placed in the beaker. The emission rate was regulated by observing the PMT trigger rate in combination with the aerodynamic particle counter readout and agitating accordingly. The aerosol flow in the system was sustained by the aerodynamic particle counter (TSI 3321 APS) evacuating air at a flow rate of 5 l/min from the enclosed chamber by the collector pipe. In this way the aerosol was driven from the aerosol generator through the coaxial central nozzle of the particle injector along with an enclosing sheet flow supplied with filtered air. The aerosol beam was approximately 1 mm in diameter with a resulting particle speed of approximately 2.5 m/s. In between the measurement sessions the aerosol generator was replaced with an air filter. The equipment was then run until no PMT triggers were obtained and the aerodynamic particle counter readout levels were back to those obtained with a clean system. Occasionally the background level was not reached and the tubes from the aerosol generator had to be replaced. The particle counter logged the number of particles and their size with each value being integrated over 20 s. The counter recordings are anticipated to slightly underestimate the numbers of particles in the aerosol beam since some particle get stuck in the 1 m long tube from the collector pipe to the counter.
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Airtight chamber
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Figure 1. Schematics of the aerosol beam generation.
2.2 Optical setup The schematics of the optical setup are shown in Fig. 2. The setup is similar to the one used in our previous experiments [4, 5]. The largest difference in this new setup is that the cuvette that surrounded the aerosol beam is removed. In the previous experiments particles deposited on its surfaces gave rise to a large fluorescence background. In order to achieve a stable and controlled aerosol beam without the cuvette, the optics and the particle injector was put in a large airtight chamber as described above.
Spectrograph PMT array Readout module 32 channels
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Figure 2. Schematics of the optical setup.
The trigger laser (Flexpoint-76/3TAE) was a laser diode with a wavelength of 670 nm. This laser could be modulated up to 100 kHz with a maximal output power of 3 mW. The focus of the beam was adjusted with the built-in lens set and focused on the aerosol beam resulting in a focus diameter smaller than 0.5 mm in diameter. The purpose of this laser was
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to generate scattered light when particles passed the focus. The radiation from the UV lasers was focused on the same spot via a fibre and focusing probe. The beam diameter at the focus was about 1 mm. Two different UV laser sources were used in the experiments at the wavelengths of 290 nm and 337 nm, respectively, and details about these lasers are found in the next section. When a particle is in the focus of the lasers it will give rise to scattering and, in some cases, fluorescence. Portions of this radiation were collected by the collection probes. One of the probes was connected via a fibre bundle to the trigger PMT (Hamamatsu H6780-4), monitoring the scattering. The diameter of the focus was about 2.0 mm and the collection solid angle was about 1.9% of the whole solid angle sphere. The other collection probe was connected with another fibre bundle to the spectrometer to monitor the fluorescence. The diameter of the collection focus was about 1.6 mm in this case and the collection efficiency of the fluorescence was about 1.2%. The spectrometer consists of a spectrograph (Oriel 77442) and a 32 channel linear PMT array (Hamamatsu H7260-4). The spectrometer covers a wavelength band from approximately 280 nm to 800 nm. An optical filter that blocked the UV scattered radiation was mounted in front of the spectrometer since the scattered radiation is much stronger than the fluorescence and would saturate the PMT array. In some experiments, an optical filter was also mounted in front of the trigger PMT to assure that both the UV scattered radiation and the red scattered light were within the dynamical range of the trigger PMT. Further details of the optics components can be found elsewhere [4, 5]. 2.3 UV lasers Two different UV lasers were used throughout the experiments, one commercial nitrogen gas laser (Oriel79111) and one solid-state laser constructed at KTH – Royal Institute of Technology, emitting at the wavelengths 337 nm and 290 nm, respectively. The nitrogen gas laser has a maximum pulse repetition rate of the 50 Hz. The maximum pulse energy and the pulse length, which depend of the repetition rate, are approximately 100 µJ and 5 ns, respectively. The time delay from trigger to UV pulse output is less than 0.5 µs for this laser. The laser constructed at KTH is based on a pulsed master oscillator power amplifier (MOPA) laser emitting at 1064 nm, which is used as a pump source in a frequency-conversion scheme going from the infrared (IR) to the UV. The frequency-conversion unit has been reported on elsewhere [9, 10], and we will only briefly go through it here, whereas the MOPA system is new and will be described in more detail below. The MOPA consists of two parts: (i), the master oscillator (MO), which is a diode pumped Nd:YAG laser with a Cr:YAG stage as a passive Q-switcher, and (ii), a diode pumped Nd:YVO4 crystal acting as the power amplifier (PA). The setup is shown in Fig. 3. MO pump diode
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The Nd:YAG crystal has the dimension 3×3×4 mm3 (x×y×z) with a doping concentration of 1% atm. and is contacted with indium-foil to a copper block that is cooled thermoelectrically. Both end surfaces of the Nd:YAG crystal are coated for high transmission (HT) at 808 nm radiation, while at 1064 nm the left side is high reflective (HR) and the right side is anti-reflective (AR) coated. This coating arrangement enables us to construct a very compact laser cavity since only one external cavity mirror is needed. The cavity mirror (M1) is essentially a flat-flat mirror (radius-of-curvature: -10 m) with a reflectivity of 70% at 1064 nm. The Q-switching is facilitated by the Cr:YAG which has an initial transmission of 70% at 1064 nm and the dimension 3×3×2 mm3 (x×y×z). The MO is pumped by a fibre-coupled 808 nm (Axcel Photonics) diode, which has a fibre diameter of 200 µm and a maximum continuous wave (CW) output power of 4 W. The output from the fibre is imaged in a one-to-one lens system, consisting of two aspherical lenses (f1) with the focal length of 4.5 mm. The characterization of the master oscillator was first done in the CW regime. The CW cavity showed a slope efficiency as high as 51%, which indicates perfect mode-overlap between the pump (808 nm) and the oscillating 1064 nm wave. When inserting the saturable absorber (Cr:YAG) inside the cavity, pulse energies of 33 µJ in 3.2 ns long pulses could be achieved. In order to be able to synchronize the MO pulses with the power amplifier, we operate the fibre-coupled pump diode in a pulsed mode, with a duty-cycle of 23% at a pulse repetition rate of 1 kHz. The build-up time for the cavity is around 230 µs, with a measured pulse-to-pulse jitter of ± 5 µs. The transversal beam parameter, M2, was measured to 1.1 and the output from the MO is collimated to a beam radius of approximately 300 µm using a lens with a focal length of f2 = 150 mm.
Output power of MOPA (mW)
The power amplifier (PA) consists of a Nd:YVO4, a fast-axis collimated quasi-CW diode bar (Jenoptik) with the dimension 1 µm×10 mm, three routing mirrors (M4, M5, M6) that are HR-coated for an angle of incidence of 45 degrees at 1064 nm, and a cylindrical lens with a focal length of fcyl = 13 mm. The 3×3×4 mm3 (x×y×z) laser crystal had a doping concentration of 1 % atm. and is also contacted with indium-foil to a copper block, which is cooled thermoelectrically. The fcyl lens focuses the diode’s slow-axis (i.e. in the y-direction), so that an elliptical cross section (~1×2 mm2 x-,ydirection) within the crystal is excited. The routing mirrors M4, M5, and M6 are placed approximately 60 mm from the right surface of the Nd:YVO4 crystal. In order to extract as much gain as possible, the collimated beam from the MO is passed through the Nd:YVO4 crystal four times by using the routing mirrors. At a duty-cycle of 9.5% (1 kHz repetition rate), the pump diode delivers a peak power of 100 W in 95 µs long pulses, which are synchronized with the MO. The resulting gain after four passes is 15, delivering output pulse energies of close to 500 µJ. However, to decrease the pulseto-pulse power fluctuations the output pulse energy was limited to 400 µJ for 1 kHz repetition rate. The main reason for the instability was parasitic lasing between the amplifier and the output coupler of the MO. In Fig. 4 the output power from the power amplifier as a function of incident power from the MO is depicted. Also seen in the figure is the difference between two and four passes through the PA. In the amplification process the pulse length became slightly longer and was measured to be 3.5 ns. 550 500 450 400 350 300 250 200 150
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The frequency converting unit developed consists of a periodically poled KTiOPO4 (PPKTP) crystal [11], deployed in an optical parametric oscillator (OPO) with an intracavity sum-frequency mixing scheme using a BBO crystal. The frequency converting processes can be described in a step wise manner: (i), the 1064 nm output from the MOPA laser is frequency double to 532 nm by a 3 mm long PPKTP crystal. (ii), the 532 nm is then used to pump the PPKTP OPO, which generates a signal. Depending on the grating period of the PPKTP, signals between 610 nm to 940 nm can be generated. (iii), the signal and the undepleted 532 nm pump is then sum-frequency mixed (SFM) in a BBO crystal in order to generate ultraviolet radiation between 290 nm and 345 nm. The frequency converting unit is shown in Fig. 5. The step-wise tunable parametric device is described in detail elsewhere [9, 10]. In this particular setup, we use a PPKTP crystal with a grating period of Λ = 13.5 µm yielding a signal wavelength of 640 nm. As a result the sum-frequency mixing in the BBO crystal, between the signal and the green pump, becomes 290 nm. The UV output pulse energy is 2 µJ delivered in 3 ns long pulses (full width at half maximum). The lower pulse energies compared to those reported on in Ref. [9], is first of all due to lower pump energies emitted at 1064 nm. Secondly, due to the robustness and compactness of the constructed mechanical mount holding the PPKTP and BBO crystals (seen in Ref. [10]), there was limited room for fine tuning (optimization) of the crystals’ positions. Although the lower UV output, the frequency converting unit generates sufficient UV radiation in order to conduct the spectroscopic measurements.
U V-output 532 nrn
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OPO signal Figure 5. The frequency converting unit. The OPO consist of two cavity mirrors coated for signal resonance, a dichroic mirror that couples out the UV from the cavity, a PPKTP crystal with a period of 13.5 µm, and BBO crystal for the UV generation.
2.4 Data acquisition The data acquisition systems were either triggered by the signal from the trigger PMT or by an external signal with a fixed pulsed repetition rate. A cycle of the data acquisition, shown graphically in Fig. 6, included the following sequential events; at time t1, the cycle is initiated either by the trigger PMT signal reaches the trigger level or by an external trigger operating at fixed repetition rate. at time t2, the trigger laser (670 nm) is turned off. at time t3, the PMT array detector acquisition is triggered. at time t4, the UV laser pulse (290 or 337 nm) is triggered. at time t5, the trigger laser (670 nm) is turned on again, and a new cycle can begin. The trigger laser was turned off during the acquisition of the PMT array data to reduce the background level. The gate time, i.e. the time the trigger laser is turned off (t5- t2), was adjusted to match with the maximum pulse repetition rate of the different laser sources. The allowable rate of the data collection systems was higher than 1 kHz, the highest possible pulse repetition rate of the UV lasers.
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trigger level
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Figure 6. The timing of the data acquisition systems.
Since the readout module of the PMT array has fixed integration time of 1 µs, it was necessary to carefully adjust the timing of all events in order to assure that the PMT array data were collected in coincidence with the UV laser excitation. The internal time-delay of the lasers required that they were triggered in advance of the PMT array acquisition so that the UV pulse occurs within the integration window of the PMT array. The fluorescence lifetime is in the order of nanoseconds, therefore it is important that the PMT array acquisition starts before the UV pulse in order to collect all the fluorescence. The timing was problematic especially when the 290 nm laser was used due to its pulse-to-pulse jitter of about ± 5 µs as will be seen in the results. Two separate acquisition systems were used to record the PMT array data and the trigger PMT signal, respectively. An internal digital hardware signal from the first system, indicating the trigger instances, was used to synchronize the data in the second system. In the second system, operating continuously at 500 kS/s, the analogue trigger PMT signal was bandwidth limited to 200 kHz and two carrier waves at 220 kHz and 235 kHz were added prior to the A/D conversion. The two carrier waves were BPSK (binary phase shift keying) modulated with the trigger indicator signal and a digital time code signal, respectively. The time codes were generated at a 1 Hz rate by a separate system. The unpacking and synchronization of data was carried out by filtering, demodulation and decoding of the trigger indicators and time codes from the carriers in the trigger PMT system data. The results in the form of a trigger marker vector and a timestamp vector were then used to extract the trigger PMT signal in time frames around each trigger instance. Each of these trigger PMT signals were then paired with the corresponding spectrum collected with the other acquisition system.
3. RESULTS AND DISCUSSION Fig 7 shows the 50 first triggered events of a measurement series taken with BG aerosols excited with 337 nm. The left plot shows the signal measured with the trigger PMT and the right plot shows the corresponding signal on the PMT array. The laser pulse energy was measured before the measurement and was dependent on the pulse repetition rate. The energy per pulse was 38, 37 and 30 µJ for the repetition rates 10, 20 and 40 Hz, respectively. The repetition rates varied during the measurements since the UV pulses were triggered by the presence of a particle in the detection volume as described above. The triggered events started at around 6.6 s when the red trigger laser was turned on. In our previous publication [4, 5] we have estimated the total fluorescence energy from the detected signal on the PMT array and corrected the results using the spectral response of the laser blocking filter, the spectrograph and the detector itself. Due to lack of time we have not performed these calculations yet and therefore we will use the measured data directly throughout this paper. However, all the data has been background corrected. The background was measured without any particles present in the air beam.
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Figure 7. The first 50 triggered events of a measurement series taken with BG aerosol excited with 337 nm. The left plot shows the signal on the trigger detector and the right plot shows the corresponding signal on the spectrometer.
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The left plot in Fig. 8 shows three example of the signal on the trigger detector for three different triggered events. Using the notation in Fig. 6 above; the event is triggered at t1 = 0 µs, the trigger laser was turned off at t2 = 200 µs, the data acquisition of the PMT array was triggered at t3 = 221 µs, and the UV pulse was triggered at t4 = 220 µs (before t3 due to the internal delay in the UV laser from its trigger to the output pulse). The trigger laser was turned on again at t5 = 20.7 ms (not shown in the plot) allowing for a new cycle to begin, hence the maximum pulse repetition rate was limited to 48 Hz in this measurement. The ripples around 220 µs and 330 µs are artifacts due to the demodulation described above in Sec. 2.4.
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Figure 8. The left plot shows three examples of signal on the trigger detector. The right plot shows the integrated signal (energy) for the red scattered light (diamond, red), for the UV scattered radiation (square, green), and for the fluorescence (circle, blue) of the first 50 triggered events. The amplitude of the red scattered energy was divided with a factor of 10 to fit the same plot.
The right plot shows the integrated signal (energy) for the red scattered light, the scattered UV radiation and the fluorescence, respectively. The red scattered light was integrated between 0 µs and 200 µs. In the plot the amplitude of the red scattered energy was divided with a factor of 10 for clarity. The scattered UV radiation was integrated between 220 µs and 300 µs. The long signal from the UV pulse (5 ns long or less) is due to RC constant of the trigger PMT readout circuit. The fluorescence signal was integrated between channel 6 and 23 on the PMT array, which is
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approximately corresponding to the wavelength interval 360-650 nm. From this plot in it is evident that correlation between the scattered UV radiation and the fluorescence energy is good, while it is difficult to see any correlation between the scattered red light and the fluorescence. We will discuss the lack of correlation in the latter case below. However, due to this lack of correlation, the fluorescence is compared only with the scattered UV radiation in the rest of the paper. The left plot in Fig. 9 shows the integrated fluorescence signal versus the integrated UV scattered radiation for the whole measurement series using the same intervals as mentioned above. The red line is a linear least mean square fit of the data for UV scattered energy below 3 V. Above this value the fluorescence energy shows saturation behavior. The slope of the fit was 2.0. The inset plot is an enlargement of the origin region to show that most of the triggered events had fluorescence and scattering close to zero indicating that no particles were present in the detection volume during those measurements. This is surprising since the measurements were triggered by the presence of red scattered light from particles. One explanation could be a poor overlap between the focus of the red trigger laser and the collection optics. However, since the red laser beam was carefully centered in the detection volume, this is not the most probable explanation. Another possible reason is that the particles are moving faster than estimated and are out of the detection volume before the UV pulse is fired. A final contributing effect is that we probably see scattering from smaller particles with the red light than possible with the UV radiation due to the smaller laser focus of the trigger laser and the wavelength dependent filter in front of the trigger PMT. We cannot distinguish the three explanations from each other at this point. The right plot in Fig. 9 shows the results from a measurement series taken with BT aerosol. All the experimental settings and data analysis parameters were the same as in the BG measurement. The slope of the fit for the BT data is slightly higher, 2.6, than for BG, indicating that the fluorescence cross section is higher for BT at this wavelength. BG excited with 337 nm
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Figure 9. Scatter plots of the fluorescence energy versus the UV scattered energy for BG and BT, respectively. The red lines are linear least mean square fit of the data for UV scattered energy below 3.0 V.
The scattering energy is proportional to the particle size in the first approximation. Fig. 10 shows the mean value of the fluorescence spectra for different intervals of the corresponding UV scattered energy. To indicate the variance between the spectra within an interval, the standard deviation is plotted for one of the curves. As expected from Fig. 9, both the amplitude and its variance are higher for BT than for BG. There is no notable change of the spectral shapes between the different intervals. The triggering rate for the different UV scattered energy intervals is plotted together with the data from the particle counter in Fig. 11 in order to relate the UV scattered energy intervals to different particle sizes. This estimation is marred by a large uncertainty. Some of the uncertainty comes from the fact that particles get stuck in the tube between the aerosol beam collector and the particle counter. An even larger source of error is that the maximum pulse repetition rate of the UV laser was set to 48 Hz. Therefore many particles may pass the detection volume without getting detected,
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resulting in a lower count rate. In addition it is possible that a considerable fraction of the particles passes by the side of the focus of the trigger laser, leading to a reduced count rate. In Fig 11 it is evident especially for the BT measurement, after about 400 s that the measured rates underestimate the particle rate, since the pulse repetition rate of the UV laser is constantly close to the allowed maximal frequency of 48 Hz. Taking this saturation into account, the curves indicate that particles as small as 3 µm are detected. A signal strength above 1 V and 3 V seem to correspond to particles sizes larger than 4 µm and 5 µm, respectively. We need to analyze our data further to establish this correspondence. BG excited with 337 nm
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The same analysis was performed on OA aerosol, but with 2.5 times lower amplification on the trigger PMT. The result is presented in Fig. 12. The linear least mean square fit is more uncertain than for BG and BT since most of the measurement point cannot be distinguished from the noise in the origin region. The slope of the fit is 1.80 after compensation for the lower amplification on the trigger PMT, i.e. slightly less than both BG and BT. The right plot in Fig. 12 shows the mean fluorescence spectra of BT, BG and OA with UV scattering energy between 1 V and 3 V (0.4 V-1.2 V for OA), estimated to correspond to particles between 4 and 5 µm. There is no large difference in the spectral fingerprint between the different particles. However, the average fluorescence energies differ for the same scattering energy interval of the three species which is one potential parameter to use in distinguishing them.
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OA excited with 337 nm
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Figure 12. Scatter plot of OA to the left and comparison of spectra for the different particles to the right.
Since the triggering with the red laser did not work well as describe above, we decided to use a fixed pulse repetition rate of 1 kHz for the measurements with the 290 nm laser. The plot to the left in Fig. 13 shows the signal measured on the trigger PMT. The acquisition cycle is triggered at t1 = 0 µs together with the UV laser (t4 = 0 µs due to the 320 µs the internal delay in the UV laser), the trigger laser was turned off at t2 = 300 µs, the data acquisition of the PMT array was triggered at t3 = 320 µs, the trigger laser is turned on at t5 = 700 µs (not shown in the plot). The ripples around 320 µs and 425 µs are artifacts due to the demodulation as before. We tried to perform the same data analysis as described above for this data. However, we did not obtain the same good results in this case. The reason is the pulse-to-pulse time jitter of the UV laser, causing some of the UV laser pulses to be fired outside of the 1 µs integration window of the PMT array readout module. The jitter seen in Fig. 13 around t = 320 µs is due to this laser jitter (± 5 µs) in combination with a jitter caused by the demodulation and decoding of the triggers. An improved and more sophisticated data analysis, with selection of only the events when the UV pulse and the integration window of the PMT array coincide, is possible but was not yet made.
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The right plot of Fig 13 shows mean spectra from the measurements with BT, BG and OA excited with 290 nm. In contrast to the results with 337 nm excitation, the spectral fingerprints are quite different and could be used to distinguish the particles from each other.
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Figure 13. The left plot shows three examples of signal on the trigger detector with the 290 nm laser. The right plot shows a comparison of fluorescence spectra for the different particles excited with 290 nm.
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4. CONCLUSION AND FURTHER WORK We have previously showed that it is possible to measure fluorescence spectra from single bioaerosol particles and that two different excitation wavelength (290 nm or 337 nm) result in significantly different normalized fluorescence spectra (spectral fingerprints) [4, 5]. In the experiments described in this paper we have improved the experimental setup by simultaneously measuring the fluorescence spectra and scattering from individual particles. We have also removed sources of background noise, which has increased the detection sensitivity. The data analysis of the experiments shows a strong correlation between the fluorescence and the scattering energy that could be used as an extra parameter to discriminate different types of particles from each other on a single particle level. We are confident that further insight into the detailed properties of single particle fluorescence and scattering will result in new means of designing earlywarning detection systems for BWA threats based on single particle fluorescence. Such system will have close to realtime detection capability, be sensitive and have a higher degree of discrimination and classification of different substances than systems available today. We will evaluate the measurement further by including the spectral response in the data analysis to get an estimate of the scattering and absorption cross section together with the quantum efficiency of the emission. The improved data analysis of our results with 290 nm excitation will be done in the near future. We will improve the detection system, especially the triggering method by improving the trigger laser scheme and reducing the time jitter of the UV laser. We will also perform the measurements on known interference particles and use some more excitation wavelength for comparison, e.g. 266 nm and 355 nm, to find the optimal wavelengths for distinguishing different particle species from each other.
ACKNOWLEDGEMENTS This work was funded by the Swedish Defence Material Administration and the Swedish Armed Forces.
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