A two-wavelength laser-induced fluorescence (LIF) instrument has been developed and used to characterize individual biological aerosol particles, including biological warfare (BW) agent surrogates. Fluorescence in discrete spectral bands from widely different species, and also from similar species under different growth conditions were measured and compared. The two-wavelength excitation approach was found to increase discrimination among several biological materials, and especially with respect to diesel exhaust particles, a common interferent for LIF BW detection systems. The spectral characteristics of a variety of biological materials and ambient air components have been studied as a function of aerosol particle size and incident fluence.
©2011 Optical Society of America
This paper describes use of an instrument developed for biological aerosol characterization that is based on laser-induced fluorescence (LIF) spectroscopy and elastic scattering measurements on individual aerosol particles suspended in an inlet air flow. The instrument employs two ultraviolet (UV) excitation wavelengths at 266 and 355 nm that sequentially interrogate each particle as they flow through a common focal volume. Previous fluorescence-based bio-aerosol detectors that utilize a single UV wavelength source to excite fluorescence from aerosol particles [1–7] have demonstrated an ability to distinguish between inorganic aerosol particles and particles that contain conjugated and/or aromatic organic material, but have not been particularly effective in distinguishing among different groups of materials that exhibit a fluorescence signature, such as diesel soot, fungi and bacteria. Approaches employing multiple-wavelength excited fluorescence have been developed to explore the possibility of increased discrimination among these aerosol materials [8–10]. Here we present and discuss experimental data that show using two excitation wavelengths provides greater discrimination among bio-aerosols than either wavelength used alone. The motivation for this work was to evaluate the utility of this approach for improving sensitivity and/or discrimination in biological warfare (BW) detection systems. Measurements were performed using the two-wavelength excitation single-particle fluorescence analyzer (2-SPFA) described below on a set of laboratory-generated aerosol particles of single-component materials each with a range of mean particle sizes and narrow distributions. The particle aerodynamic diameters studied range from slightly below 1 micron to about 8 microns. The list of particle compositions includes examples of bacterial spores, fungal spores, vegetative bacteria, proteins, silicates, as well as ambient outdoor air with, and without, diesel engine exhaust present. Based on these results, we conclude that front-end trigger devices for bioaerosol detection that rely on single wavelength excitation will likely provide higher false alarm rates than ones based on a multi-wavelength excitation approach such as described here. More importantly, we show a number of basic results including the dependence of emission on particle size, absence of photo bleaching and saturation effects, and the effects of the presence of media in particle samples. Optical systems such as this one could also be used as a trigger for macroscopic aerosol sample collection for some form of subsequent analysis or assay. Recent efforts have been made to develop means for selective collection of individual particles based on their spectral differences [11,12]. These selected particle samples could then be subjected to either further optical analysis, or bio-molecular assays with significantly reduced burden of interference and/or dilution by relatively large quantities of non-target particles in the sample. This type of capability will be needed to ultimately explore and understand the naturally occurring biological component of ambient air and their etiology.
2. Experimental design
The 2-SPFA optical design is shown schematically in Fig. 1 . An optical system previously described , was modified to accommodate additional spectral channels and incorporate custom data acquisition circuitry . Aerosol particles are entrained in inlet air flow through an aerodynamic nozzle to form a collimated flow of about 0.8 mm in diameter by using a concentric column of sheath air flow. The inlet nozzle directs the aerosol-laden air flow at a right angle to the optical axis of the elliptical mirror (into the page), and positions the flow through the mirror’s primary focal point inside a sealed aerosol sample chamber. (Fig. 3 in Sivaprakasam et al.  shows a 3-dimensional illustration of the particle flow in the aerosol chamber). A fused silica window is used to define the airtight aerosol chamber and pass the collected light from the elliptical mirror to the rest of the instrument. Interrogating laser beams enter and exit the sample chamber orthogonal to both the aerosol sample flow direction and the reflector optical axis. A small pump provides the differential pressure (nominally 5 kPa) to pull both HEPA filtered sheath air as well as the sample air into the aerosol chamber, and out through an exit tube mounted in the bottom chamber wall. The flow velocity of the particles in the sample stream is approximately 4 m/s, corresponding to a gas flow rate of about 0.3 liter/min. Light from a CW 785 nm diode laser is focused to intercept aerosol particles at the focal position of the elliptical mirror. Elastically scattered light from individual particles passing through this beam is used both to provide relative measure of particle size, and to generate a cueing signal for the pulsed excitation lasers when the scattering intensity exceeds a predetermined threshold value, T (see Timing Chart, inset, Fig. 1). Two externally Q-switched diode pumped Nd:YAG lasers (Coherent Model 501QDII and Coherent Vector 532-1000-20) are used to generate the UV excitation pulses. The first laser is frequency-quadrupled to generate a 266 nm pulse having a 20 ns pulse width, while the second laser is frequency-tripled to generate a 5 ns duration, 355 nm pulse. Fused silica beam splitters are used to pick off a fraction of each pulse to monitor the energy using a pair of photodiodes (PD).
Beams from both pulsed UV lasers and the CW 785 nm lasers are directed collinearly into the aerosol chamber. The output of the first pulsed UV laser occurs 1.2 μs after the cueing signal and the second laser pulse is generated approximately 0.4 μs after the first (see Timing Chart, inset, Fig. 1). The beams are focused using cylindrical lenses to form focal spots roughly 200 μm thick in the direction of the particle flow, and nearly 1mm wide, in order to intercept 100% of particle flow near the mirror focal point. Nearly 2π steradian of light that is either scattered or emitted from individual particles is collected by the elliptical mirror, and directed to a pair of field lenses to collimate the collected light. The collimated light passes through a series of custom dichroic beam splitters that separate the fluorescence into three broad spectral bands. Photomultiplier tubes (PMT’s) are used to detect the fluorescence signals in each of the three bands, as well as the scattered light. The first beam splitter was custom-designed to reflect light in a 70 nm wide band centered at 350 nm. This reflected light is directed to a 266 nm high reflectance (HR) mirror, which is used to reject 266 nm light from the 350 nm UV emission channel PMT. Subsequently, a Hoya U360 color-glass filter is used to further reduce any scattered 266 nm light (>107 rejection of 266 nm), and this channel also uses a gated PMT Hamamatsu H7680-01 to measure the 350 nm fluorescence signal. The gate is on only during the presence of the 266 nm pulse. Gating the PMT prevents damage due to high intensity scattered light from the subsequent 355 nm laser pulse. Reflected light from the 266 nm HR mirror is used to measure the elastic scatter signal from both the 266 nm and 355 nm lasers. The second dichroic beam splitter in the main optical train reflects light passed by the first one in an 80 nm wide band centered at 450 nm. A pair of high pass and low pass interference filters are used to isolate the light in the 450 nm band. The third and final dichroic beam splitter reflects light in a narrow band centered at 785 nm into a Hamamatsu H6780-20 PMT and monitors elastic scattering from the CW laser beam. Fluorescent emission in a 100 nm wide band centered around 550 nm passes through all of the beam splitters to a final PMT. A condensing lens is also used in each of the channels to focus the collimated light onto the PMT light-sensitive anodes. The PMT’s used for the 450 nm and 550 nm fluorescence bands are Hamamatsu models H5783-03 and H6780-02, respectively.
Custom-built electronics were incorporated for the data acquisition . A special daughter board, TPC-100, was used to process the elastic scatter signal from the 785 nm beam. This board detects the peak of the scattered light pulse above a threshold, T, and outputs a trigger pulse after a set delay from the peak (see Timing Chart, inset, Fig. 1). Triggering from the scattering signal peak, instead of using a fixed threshold level, removes uncertainty that would arise from varying size particles, and enabled more precise timing for firing the two UV beams. The TPC board also recorded both the pulse height and the integral of the charge under the scatter pulse to provide particle size information. A dual pulse processor circuit (DPP-266) was also designed to separately integrate the two sequential signal pulses from single PMT outputs separated in time by 400 ns. These data acquisition circuits demonstrated linearity over 105 dynamic range of the input pulse charge in order to accommodate the large variations in the fluorescence signals arising from the differences in particle size and composition. The DPP-266 board could be triggered at rates up to of 10 kHz. The temporally integrated analog signals from these custom data acquisition boards were sampled with a National Instruments M-series DAQ card. A data acquisition and processing program was written in LabVIEW to process the data from the DAQ card. For each particle, the raw recorded data consisted of: a time stamp (0.00036 second resolution), peak-value and integrated 785 nm elastic scattering, 266 and 355 nm elastic scattering, 350, 450 and 550 nm band fluorescence excited by the 266 nm laser, and 450 and 550 nm band fluorescence excited by the 355 nm laser. Additionally, the 266 and 355 excitation laser pulse energies were recorded from the photodiodes (PD), and the 785 nm laser power was monitored. These measurements constitute a total of 13 values that were recorded for each particle. The digitized signals were corrected for the PMT spectral response and gain based on manufacturer’s specifications, the measured filter spectral transmission and reflection efficiencies, and collection efficiency of the elliptical mirror. The fluorescence and scattering data from the particles were then normalized to the relative UV laser pulse energy and previously measured UV laser fluence. Finally, the resulting fluorescence data and scattering values were converted into numbers of photons emitted from each particle per mJ/cm2 of incident energy density and recorded as a binary file.
3. Experimental data and results
3.1 Spectral differentiation among biological aerosols
The 2-SPFA has been used to measure and quantify fluorescence from a wide range of biological and non-biological aerosol particles generated in the laboratory, representative of typical ambient air samples. Controlled samples of aerosol particles were generated using different dissemination techniques resulting in wet or dry aerosolization including: Collison nebulizer, Sono-tekTM generator , Micro-dropletTM generator  and Pitt generator. These lab-generated aerosols were entrained in HEPA filtered dry nitrogen, prior to introduction into the 2-SPFA. Laboratory calibration particles that were analyzed included: undoped and dye doped polystyrene latex (PSL) spheres (Thermo Scientific, Inc.) and fused silica spheres of varying sizes in the range of 0.7 to 8.0 microns. Some fluorescence measurements obtained with a similar system, have been presented previously [8,16].
LIF signatures of some of the common organic and inorganic sample materials such as: kaolin, Arizona road dust (ARD) and Sporisorium Cruentum (SC) fungal spores; BW agent simulants including: bacterial spores, Bacillus atrophaeus, ssp. globigii (BG) from Dugway Proving Grounds (DPG), bacterial vegetative cells, Bacillus subtilis (BS) in tryptic soy broth (TSB) media and Brucella neotomae (BN) in Brucella broth; and proteins: albumin and ovalbumin (OV) are shown in Fig. 2 . These aerosol particles were generated using a Collison nebulizer, with sufficient dilution to result in relatively small particles around 1.5 micron aerodynamic diameter. For bacterial spore samples these particles would be primarily composed of a single organism. The 2-SPFA elastic scatter threshold was set to trigger on particles greater than 0.7 μm in diameter based on PSL measurements, and size distributions were monitored simultaneously using TSI Inc, aerodynamic particle spectrometer (APS) model 3321.
After relevant normalization, the corrected aerosol particle data sets still include 8 independent channels so that analysis of discrimination involves multivariate classification methods that are not easily visualized directly. Results of that analysis will be presented separately, but for this article, we show differences that can be seen directly in 2-dimensional data plots using selected channels. In Fig. 2, the emission in the three 266 nm excited fluorescence bands are summed and plotted against the sum of the two 355 nm excited fluorescence bands to provide a general sense of the classification capability that this type of data can provide. Each point on the scatter plot represents an individual aerosol particle, and data from approximately 104 particles was plotted for each sample composition. Two contour lines are plotted for each type of particle, which helps clarify cases where there is strong overlap between samples. The kaolin and ARD samples show very little fluorescence in any channel, and form a cluster in the lower left corner of the plot, kaolin particles exhibiting slightly higher signal levels than ARD. The bacterial spore, BG, and vegetative cells, BS and BN, have higher fluorescence signals for both excitation wavelengths and show separable clustering from inorganic samples as well as each other. Note that some of the bacterial aero-sols show multimode distributions, and this feature will be discussed in Sec. 3.6. The tightness and the proximity of these distributions depend on the homogeneity and the growth conditions of the sample. The fungal spores (SC), represent naturally occurring biological aerosol particles (and potential interferents), and Fig. 2 shows a fluorescence distribution in this 2 dimensional data plot that is separable from the biological simulants as well as inorganic samples. Note that this statement would not be true if either excitation wavelength were used alone. The size distributions of the particles (as reflected in their elastic scattering) have not been taken into account in this representation. The aerosolized fungal spores were significantly larger in size, roughly 6.5 μm in mean diameter as measured with the APS unit, compared to a typical mean of 1.5 μm diameter for the bacterial samples shown in Fig. 2. If particle size information was used, even greater separation of these two sample groups could be achieved. Pure protein samples, ovalbumin (OV) and albumin, represent bio-toxin surrogates, and their signatures are also easily separated from other samples due to the strong presence of tryptophan emission in the 350 nm band from 266 nm excitation. The purer protein samples show very little fluorescence in any of the other wavelength bands or from 355 nm excitation, and hence are easily identifiable from other bio-aerosol samples.
3.2 Spectral distribution of ambient air and combustion aerosols
For sampling ambient aerosols, a 2″ diameter pipe was installed in the exterior lab wall through which outside air could be drawn. Ambient air was sampled by the 2-SPFA for extended periods of time to characterize natural outdoor aerosol backgrounds. In Fig. 3 fluorescence data from ambient air background particles (Bkgrd) measured over a 2-hour period is shown, overlaid with data from some of the samples shown in Fig. 2. Note that the axes values in Fig. 3 are specific fluorescence spectral bands (data channels), rather than the sums of emission intensities from all the bands. Data in Fig. 3 are plotted as a scatter plot to illustrate the observation that small fractions of ambient particles occur in essentially all parts of the fluorescence feature space. Although the concentrations of these naturally occurring fluorescent particles are relatively low, depending on what specific region of feature space is being examined, their presence is significant because they establish a background threshold level that a new population of fluorescent particles would need to exceed in order to be detected by an automated algorithm.
Diesel soot is known to have UV fluorescence properties that may act as an interferent for UV LIF aerosol detection systems. Consequently, the ambient outdoor air sampling port was also used to obtain combustion engine exhaust data from diesel vehicles. Ambient air background was sampled immediately before and after operating a diesel engine vehicle in the vicinity of the inlet pipe to provide a comparison population of background data of the air with diesel combustion particles present. Figure 4 illustrates data contrasting ambient air particles with and without diesel exhaust particles present. In Fig. 4(a) the same outdoor particle sample data shown in Fig. 3, just prior to starting the diesel engine vehicle, is now presented as contour plots to show the population distribution within the fluorescence feature space. Figure 4(b) shows typical sample data immediately following, when the diesel engine was running. In Fig. 4(b) a bimodal distribution is seen in the ambient air data with a population of relatively highly fluorescent particles appearing in addition to the non-fluorescent particle population observed before (and after) the diesel exhaust was present. The new population was shown to result from the diesel engine exhaust by repeated trials with, and without, the engine running.
The clear separation of these two populations as shown in Fig. 4(b) permits the obvious identification of the second, highly fluorescent particle group as diesel combustion products. As in Fig. 3, data from some of the lab-generated aerosol samples has been overlaid on Fig. 4 (also as contours). In Fig. 4(a) the kaolin sample can be seen to represent a significant subpopulation of ambient outdoor aerosol particles that are essentially non-fluorescent. The ambient aerosol sample also includes populations of particles showing significant 450 nm band fluorescence that extends their contour sufficiently to overlap the fungal spore (SC) sample region. However, in Fig. 4(b) the emission from the diesel exhaust particle population clearly overlaps with the signature profiles of the bacterial spore sample, BG DPG, and bacterial vegetative cell particles, BN. Fluorescence signatures from aerosolized NIST diesel particulate matter (DPM) (black powder) were also measured, but data from these particles was quite different compared to diesel particles measured directly from vehicle exhaust, with the NIST DPM typically having very low UV LIF values (e.g. similar to kaolin).
In Figs. 3 and 4 the data channels (plot axes) are from two emission band intensities excited by 266 nm wavelength light. Figure 5 illustrates the additional discrimination provided by data from 355 nm wavelength excitation. Figure 5(a) shows outdoor aerosol data with diesel particles present using data from the 350 nm and 450 nm emission bands from 266 nm excitation as shown in Fig. 4(b) but with different lab-generated samples overlaid. In Fig. 5(b) the same particle data is presented using 450 nm emission from 355 nm excitation as the vertical axis. Visual comparison of Figs. 5(a) and 5(b) shows the diesel particle population is significantly separated from the bacterial samples by including the 355 nm excited 450 nm band feature space. The fluorescence in the 450 band is observed to be greater for BG spores for the 355 nm excitation compared to the 266 nm excitation and vice-versa for diesel, thus comparing the ratio of the 450 nm band for the two excitations result in better discrimination of diesel engine particles from bacterial sample particles. This qualitatively demonstrates one of the primary advantages of using both 355 nm and 266 nm excitation wavelengths. Other differences are also evident in the fluorescence characteristics of some of the simulant samples in comparing Figs. 5(a) and 5(b). Vegetative cells BN and BS show greater separation for 266 nm excitation (Fig. 5(a)), compared to 355 nm excitation (Fig. 5(b)) while signatures for OV and albumin show more separation in Fig. 5(b) compared to Fig. 5(a).
3.3 Linearity of fluorescent emission with incident energy
At the outset of this investigation of UV excitation of fluorescence from individual aerosol particles, there were concerns over the issues of photobleaching and excitation saturation. Photobleaching of fluorescence from biological materials is a well-known effect in epifluorescent microscopy that results from photo-degradation of the fluorophore molecules. Saturation will occur at some fluence level, when all of the fluorphores in a micron-sized particle are simultaneously excited, depleting the lower state, so that further increase in incident radiation will not result in greater (linear) increase in emission. Especially with regard to sequential excitation by two UV beams, there was concern that the signal from the second excitation might be altered or degraded by the first due to either of these effects. Previous studies on biological aerosols have reported possible photobleaching effects with increasing incident fluence [17,18]. In order to evaluate possible effects of two-excitation beam measurement on individual lab-generated aerosol particles, an experiment was performed in which aerosol particles were sequentially interrogated by two 266 nm laser nanosecond pulses separated by 200 ns. Fluorescent emission was recorded in all of the 3 spectral bands independently for both excitation sources. In measurements taken on a given aerosol sample material, data was obtained for a large number of particles, and then the first laser’s energy was increased. This process was repeated as the initial laser pulse energy was incrementally increased from 0.1 μJ to 16 μJ per pulse, corresponding to fluences ranging from about 0.05mJ/cm2 to 8 mJ/cm2; while the second laser was operated at a constant fluence of about 0.5 mJ/cm2. The fluorescent intensity measured in the spectral band centered at 350 nm for both excitation sources is plotted in Fig. 6 for a selected set of samples. Fluorescence values were measured for samples ranging from low-yield particles such as silica spheres, to relatively bright fluorescent particles such as bacterial vegetative cells. In Fig. 6, the top set of slanted curves (solid lines) corresponds to the fluorescence measured for the first excitation source (plotted with the left vertical scale). The lower set of curves (dotted lines) represents fluorescence measured from the second excitation (plotted with the right-hand scale) which was held at constant energy. The fluorescence response from the first excitation appears to be linearly proportional to excitation energy (for a log-log plot the slope of the curve is approximately unity). The lower set of curves showing the emission from the second excitation pulse are essentially flat, indicating constant fluorescence values for the second excitation held at constant energy which implies that no photobleaching effects are present for this range of fluences for the initial excitation pulse. As the first laser’s fluence increases past about 4-5 mJ/cm2 (8-10 µJ), the fluorescence response appears to slightly drop below the linear projection for many of the samples, with the possible exception of BG from Air Force Institute of Pathology (AFIP) spore samples. While the value of the emission intensity at the highest intensity point is still within the standard deviation of the linear projection for each sample set of points, the observation that nearly all the samples are consistently lowered is suggestive that this intensity level may be the beginning of an excitation saturation effect for these samples.
To explore the possibility of saturation effects, a power-law analysis was performed for the data presented in Fig. 6, and the results are shown in Table 1 . A least squares fit to a straight line was performed on the logarithm of the emission intensity values first, for all the data points for each sample (Column 2), and then repeated for a subset of the data excluding values of the incident fluence greater than 1 mJ/cm2 (Column 3). The resulting values for the fitted slopes of the log emission vs. the log excitation are shown in columns 2 and 3 below. For all samples, the exponent values in column 3 are closer to unity than their corresponding value in column 2. These results suggest that potential saturation for most of the biological material samples could be beginning to occur at higher fluences. However, additional study at fluences higher than we were able to achieve would be necessary to confirm that possibility.
Reference PSL particles appear to be an exception to the linear dependence model, showing not only potential saturation effects at higher fluences but a power-law dependence that was sub-linear (exponent less than one) even at lower incident fluences. The measured deviation is greater than our apparent experimental uncertainty. While the source of this behavior is not understood at present, it may be connected to the high absorptivity of PSL material at these wavelengths. The estimated absorption depth for the PSL material is 1.6 μm at 266 nm. The silica particles had minimal fluorescence and thus yielded signals slightly higher than stray light signal at each of the fluence settings. The exponent value obtained for silica essentially characterizes the stray light noise response of the system as linearly dependent on the incident fluence. The exponent values obtained for the fluorescence measured in the 450 nm band showed very similar characteristics to that shown in Table 1 for the 350 nm fluorescence band. A similar computation was carried out for power-law exponent values for the second excitation pulse that was held constant at 0.5 mJ/cm2. The results showed that essentially all the samples emission from the second excitation remained constant within experimental error, indicating that no measureable photobleaching or other photochemical degradation effects were seen at these fluences.
These results imply that limiting the incident fluence to less than 5 mJ/cm2 should ensure that the fluorescence from these types of particles does not show any saturation effects, and that the emission characteristics of the aerosol particles under investigation remain unaltered by sequential interrogation excitation laser pulses. This conclusion is consistent with previous work  reporting non-linearity for incident fluence higher than 10 mJ/cm2 in both aqueous and aerosol samples.
3.4 Fluorescence and elastic scatter dependence on particle size
For BW aerosol detection, particles in the “respirable range” from 1 to 10 μm have been of primary interest. For a given aerosol material, fluorescence and elastic scatter intensities from individual particles are obviously strongly dependent on particle size. Although an extremely large compilation of prior computational and theoretical model work exists for elastic scattering, relatively little prior investigation has been focused on fluorescent emission dependence on particle size or shape. As part of the study in this paper, fluorescence and elastic scattering intensities were measured for aerosol particles of a constant composition over a range of particle sizes. Aerosols with narrow size distributions were generated in a range of 1 to 7 μm in aerodynamic diameter using a MicrodropletTM  generator. These particles were interrogated simultaneously by the 2-SPFA, and an APS. Particles generated using this technique had a size distribution FWHM of typically less than 14% of the mean diameter for particles of homogeneous composition such as ovalbumin, while for particles of heterogeneous composition such as bacterial cells in media, the variation in size increased to as much as 22%. As examples of fluorescence dependence on particle size, Fig. 7 plots the measured values for the 266 nm excited 350 nm band emission versus the 355 nm excited 450 nm band emission band for discrete particle sizes composed of ovalbumin (Sigma-Aldrich grade VI) and vegetative bacteria sample Pantoea agglomerans (PA) grown in TSB media. The scatter plots are shown along with 2 contour lines for each sample and the modes of the various size distributions are labeled on the plot. The fluorescence signals for the individual size distributions are sufficiently tightly clustered to clearly differentiate the size increments shown. As the particle size increases from 1 μm to 7 μm, the emission signal increases, spanning 2-3 orders of magnitude in dynamic range.
In order to quantify the size response, the mean fluorescence signal measured for ≈104 particles for the various types of particles were studied as a function of particle size. Figure 8 shows graph of the 266 nm excited 350 nm fluorescence band intensity as a function of the APS aerodynamic particle size. A power-law fit was performed on each sample, and the exponent value is labeled for each sample type. This analysis was also performed on all emission bands for both the 266 nm and 355 nm excitations, and results are shown in Table 2 . For homogeneous isotropic particles, in which the absorption coefficient is low enough to permit roughly uniform excitation throughout the particle, the fluorescence emission is expected to be proportional to the particle volume (i.e. total number of fluorophores) resulting in a power-law exponent value of 3. As can be seen from Table 2, for homogeneous particles such as ovalbumin and dye doped PSL particles (green dye doped PSL (Thermo Scientific, Inc.)) the observed exponent values range between 2.4 and 3.0 for most of the emission bands. For particles of mixed composition like bacterial cells in media, the fluorophores may not be evenly distributed throughout the particle, and this could lead to compromised particle size dependence. Power-law exponent values as low as 1.6 were observed for such inhomogeneous particles. Other factors that might influence the fluorescence yield as particle size is increased are: non-uniform absorption of the excitation radiation and/or re-absorption of the emitted fluorescence.
The fluorescence emitted in the 450 nm and 550 nm bands for both excitations of the PSL series has repeatedly shown weaker fluorescence yield for the 7 μm particle compared to the 5 μm particle, and this alters the value of fluorescence versus particle size exponent. To illustrate this point, the power-law fit was performed on PSL particles including and excluding 7 μm particles and both sets of values are listed in Table 2. As the fluorescence signal emitted in the 350 nm band doesn’t show much degradation with increasing size particles, the power-law dependence doesn’t vary much in this band as for the two above mentioned cases. The exponent values change significantly for the two longer emission bands for both laser excitation pulses when the 7 μm PSL particles are excluded, illustrating that saturation and/or re-absorption of the emitted light are likely effects for larger PSL particles
For all the measurements made using the 2-SPFA, the elastic scatter data was recorded at 785 nm, 266 nm and 355 nm wavelengths for each particle. To explore the elastic scatter dependence on particle size, a range of particle sizes composed of: silica particles, PSL particles and NIST diesel particulate matter (DPM) were studied. Similar to the fluorescence data discussed previously, a power-law fit was performed on the elastic scatter data as a function of particle size, and the results are summarized in Table 3 . The integrated elastic scatter signal depends on the geometry of the collection optics, as well as the scattering parameter (2πr/λ where r is the equivalent sphere radius) and the refractive index (real and imaginary) of the particles being interrogated. Taking into account all the apertures milled into the elliptical reflector for the laser beams and particle flow access, a calculation was performed for the same set of particles at the 3 wavelengths of interest using a model based on Mie theory (assumed spherical particle shape). The index of refraction data available from literature for silica , thin nm layer of PSL , PSL particle available at 785 nm  and black carbon  was used for these calculations. In the same way as the experimental results, a power-law fit was performed on the computed scatter intensities as a function of particle size, and the resulting slopes are also listed in Table 3. As silica particles have relatively low absorption across the visible region, we measure the highest slope values for these particles (approximately values of 2, meaning that the scattered intensity is proportional to the cross sectional area of the particle). For silica, the experimental values agree reasonably well with the model calculations. As absorption comes into play for PSL particles, we can observe less than quadratic dependence for all excitations with an almost linear relationship at 266 nm. Higher than expected elastic scatter signal has been consistently measured from 1 μm particles (size parameter of 4) for 785 nm excitation (i.e., value for 1 μm size particles falls consistently above the fit). Such an observation was also made for the fit for the theoretical Mie calculations. Perhaps for scattering parameter values of 4, more of the forward lobe is collected compared to elastic scatter from particles with scattering parameter ≥ 8, thus skewing the power-law fit. By excluding the 1 μm data points for the 785 nm excitation the calculated Mie theoretical values and experimental data fit a linear power-law dependence better, and discrepancy between experimental and theoretical values also decreased significantly. Thus computed power dependence values are tabulated in column 6 of Table 3. As an example by excluding the 1 μm silica particles, the percent difference between theory and experiment was reduced to 4% from −17%.
The elastic scatter signals for NIST DPM, which is a highly absorbing (black powder) particle shows no measurable signal dependence on particle size for all three wavelengths studied. The experimental values show no correlation to the calculated values obtained using carbon black refractive index data. Prior study measuring the angular scatter signal from DPM concluded that Mie theory cannot adequately account for the measured angular scatter signal from such particles, and a different kind of particle scattering model such as a fractal surface description would need to be considered to obtain reasonable agreements .
These measurements show that elastic scatter alone cannot be reliably used to provide size measurement of ambient particles. If an independent measure of the particle size was to be made, then variations in the elastic scatter could be used to obtain additional absorption information about the particles. Moreover, as the functional form of the particle size dependence for fluorescence and the elastic scatter vary, (depending on the composition of the particle) normalizing the fluorescence to the elastic scatter signal to de-convolve the particle size is only possible for well characterized materials.
3.5 Fluorescence dependence on aerosol generation technique
In order to study the effect of moisture on the fluorescence signature, aerosols of three of the simulants: PA, BG and OV, were generated from a dry powder using a dissemination technique known as a Pitt generator. This method produces a wide particle size distribution, as shown by the data graphed on Fig. 9(a) , measured using an APS. For comparison, these same materials were suspended/dissolved in water, and a Collison nebulizer was used for droplet generation. As discussed earlier, concentrations of these materials were adjusted so that the resulting dried aerosol from the nebulizer has a mean diameter of about 1 micron. In Fig. 9(b) the fluorescence values measured in the 350 nm band for 266 nm excitation are plotted versus those from the 450 nm band. The fluorescence distributions for the dry disseminated particles form long, straight strands with unit slope on the logarithmically scaled fluorescence plot. The length of these narrow strands is due to the wide size distribution of these aerosols. The unit slope results from the fact that emission in these two spectral bands has the same proportion regardless of the particle size. The fluorescence distributions for the Collison-generated particles of the same sample materials from solution droplets are also shown on the same plot. These nebulizer-generated aerosols are more tightly clustered around a central mean size. The fluorescence from ovalbumin generated using the droplet nebulizer overlaps and extends the powder disseminated distribution to lower values as would be expected from smaller particles. The two distributions from the ovalbumin aerosols show that the characteristic emission properties of the ovalbumin particles appear to be independent of the particle generation method. However, for the other two sample materials, PA and BG, the fluorescence distributions do not align or overlap in a smooth way. The fluorescence in the 450 nm emission band for PA and BG shows 2 to 4 times lower fluorescence for the wet disseminated particles compared to the dry particles. Similar differences were evident when comparing the 355 nm excited 450 nm fluorescent band. The results reported here compare dry aerosols generated using either a dry or wet generation technique. In an earlier study (Faris et al. ), a wet aerosol generation technique was used, and particles were measured either prior to or after drying. They reported a factor of 4 lower fluorescence yields from dry B. Subtilis aerosol compared to wet aerosols. A plausible explanation has not been offered for either set of observations.
3.6 Fluorescence dependence on biological sample protocol
For some of the microbiological materials, fluorescence spectra displayed multi-mode distributions even though the size distribution was single mode. Presumably, these structured emission profiles are indicative of composite fluorescent materials such as media or metabolic products in addition to the organisms themselves. To test this idea, samples were investigated as aliquots in which one remained as prepared in the media, another was separated from the media material by centrifugation and washed in pure water (3X), and a third sample is formed from the leftover media supernatant. BG spores (DPG), and Bacillus subtilis (BS) and Yersenia rohdei vegetative cells in TSB media were chosen for this study. Figure 10 shows 266 nm excited, 350 nm band fluorescence values plotted vs. the 450 nm band as contour plots for (a) BG spores, (b) BS Vegetative cells and (c) Y. rohdei. In all three of the plots the sample in media is shown in dark blue, the spent leftover media in green, the washed sample in red and the pure nutrient broth in light blue. The spectral signature for Y. rohdei in media shows 3 distinct peaks, or maxima, with the signature for the spent media overlapping with two of these peaks, while that of the washed sample shows a strong correlation with the third peak. Similar results has been observed consistently with multiple Y. rohdei samples, and the growth media typically shows higher fluorescence intensities in the longer wavelength emission bands compared to the organism. However, the fluorescence signatures of the BG spores and BS vegetative cells do not show similar patterns. From the plots for the BG spores,the spores in the media show two distributions with the left over media overlapping with one of the distributions. The signature of the washed sample doesn’t overlap with the second distribution as one would expect, but instead shows a disjoint distribution from the spectrum of the sample in media showing that the fluorescence of the particle is spectrally modified in the presence of surrounding media. The washed sample shows higher fluorescence in the 350 nm band compared to the sample in media and lower fluorescence in the 450 nm band. One possible explanation for this behavior is that some of the emitted 350 fluorescence from the microorganisms embedded in the particle is absorbed by the surrounding media, which then re-emits that energy at longer wavelengths. Additionally, or alternatively, the incident intensity interior to the particle could be significantly attenuated by the presence of the media. The fluorescence distributions of the BS vegetative cells show a similar pattern. BS cells in media and the spent media are so overlapped that they are essentially indistinguishable, while the emission distribution for the washed cells are completely disjoint. In this case, the washed cells emission distribution is shifted vertically down with roughly an order of magnitude lower fluorescence in the 450 nm band, but roughly the same intensity in the 350 nm band. Similar spectral pattern is observed from the 450 nm emission band for the 355 nm excitation source as well. In summary, significant and non-intuitive changes are observed in the samples of the same microorganism depending on the sample preparation in terms of whether the growth media is present or not. Further investigation is required to determine the detailed mechanisms involved in each case. For example, for vegetative cell samples the possibility, or degree, of cellular lysing would be an important parameter to be determined. Moreover, a quantitative model of particle fluorescence is needed. Such a model would permit a direct comparison for the spectral mixing of multiple components with their own absorption and emission spectral profiles, in addition to calculating the source function of the incident excitation radiation in the particle interior.
As a final consideration, we wanted to determine the effect of gamma irradiation on at least a couple of samples in terms of the fluorescence spectra. The fluorescence spectra of Yersinia rhodei in TSB media and DPG BG, also in TSB media, were obtained for both non-irradiated and gamma irradiated samples. Figure 11 shows plots of the 266 nm excited, 350 nm band emission vs. the 355 nm excited 450 nm band fluorescence as contours for (a) Y. rohdei and (b) BG. For the Y. rohdei samples, the tri-modal distribution of the cells in media is shown in green, and the result after gamma radiation shows a single emission mode, which is roughly an average of the spent media and washed cell samples. The intensities of the two samples are strongly over-lapped. For BG samples a similar result is obtained. Overall, the main effect of gamma irradiation appears to be a noticeable reduction in the media component of the signature that results in an increased emphasis of the cellular spectral component, but no dramatic effects in the overall emission intensities are observed.
The fluorescence of a number of calibration standard particles and biological simulants has been studied. The employment of two UV excitation wavelengths was shown to increase discrimination among several biological materials, and especially between BW simulants and diesel exhaust. The fluorescence of aerosol particles was studied as a function of particle size, and for some cases where composition homogeneity and smaller absorption coefficients are obtained, the fluorescence of homogeneous samples show close to a cubic dependence on the particle size (proportional to particle volume). However, for inherently heterogeneous particles, this size dependence shows much greater variation. Particle absorption was seen to play a strong role in the dependence of elastic scatter on particle size. For highly absorbing particles, such as NIST diesel particulate matter, no size dependence was observed.
However, the power-law exponent for particles or wavelengths with weaker absorption coefficients could range from values approaching 2 (proportional to the cross sectional area of the particle) down to 1. This suggests the need for a particle size measurement that is not determined soley by elastic scatter. The linearity of the fluorescence yield was studied as a function of incident energy for particles of different known compositions and known sizes. No photo-bleaching effects were observed, and no saturation effects were observed for fluences of 5 mJ/cm2 or less. The presence and absence of growth media plays a strong role on the fluorescence signature. Understanding these signatures will be important in order to refine detection algorithms and defining the threat features of interest.
This work was supported by chemical and biological defense tech-base, administered by the Defense Threat Reduction Agency (DTRA). DTRA was founded in 1998 to integrate and focus the capabilities of the Department of Defense that address the weapons of Mass Destruction (WMD) threat. The mission of the DTRA is to safeguard America and its allies from WMD threat (e.g. chemical, biological, radiological, nuclear and high yield explosives) by providing capabilities to reduce, eliminate and counter the threat and mitigate its effects. Under DTRA, Department of Defense resources, expertise and capabilities are combined to ensure the United States remains ready and able to address the present and future WMD threats. For more information on DTRA, visit _www.dtra.mil/_ <file://www.dtra.mil/>.
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