Gustavo E. Fernandes, Yong-Le Pan, and Richard K. ChangDepartment of Applied Physics and Center for Laser Diagnostics, Yale University,
New Haven, CT 06520-8284, USA
Abstract: We present both a computational and an experimental ap-proach to the problem of biological aerosol characterization, joining theexpertises reached in the field of theoretical optical scattering by complex,arbitrary shaped particles (multipole expansion of the electromagneticfields and Transition Matrix), and a novel experimental technique based ontwo-dimensional angular optical scattering (TAOS). The good agreementbetween experimental and computational results, together with the possi-bility for a laboratory single-particle angle-resolved investigation, opensa new scenario in biological particle modelling, and might have majorimplications for a rapid discrimination of airborne particles.
References and links1. K. P. Gurton, D. Ligon, and R. Kvavilashvili, “Measured infrared spectral extinction for aerosolized Bacillus
subtilis var. niger endospores from 3 to 13 μm,” Appl. Opt. 40, 4443-4448 (2001)2. R. G. Pinnick, S. C. Hill, P. Machman, J. D. Pendleton, G. L. Fernandez, M. W. Mayo, and J. G. Bruno, “Flu-
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3. P. P. Hairstone, J. Ho, and F. R. Quant, “Design of an instrument for real-time detection of bioaerosols usingsimultaneous measurement of particle aerodynamic size and intrinsic fluorescence,” J. Aerosol Sci. 28, 471-482(1997)
4. M. J. Seaver, D. Eversole, J. J. Hardgrove, W. K. Cary, Jr., and D. C. Roselle, “Size and fluorescence measure-ments for field detection of biological aerosols,” Aerosol Sci. Technol. 30, 174-185 (1999)
5. W. D. Dick, P. J. Ziemann, P.-F. Huang, and P. H. McMurray. “Optical shape fraction measurements of submi-crometre laboratory and atmospheric aerosols,” Meas. Sci. Technol. 9, 183-196 (1998)
6. B. Sachweh, H. Barthel, R. Polke, H. Umhauer, and H. Buttner, “Particle shape and structure analysis fromthe spatial intensity pattern of scattered light using different measuring devices,” J. Aerosol Sci. 30, 1257-1270(1999)
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7. P. H. Kaye, J. E. Barton, E. Hirst, and J. M. Clark, “Simultaneous light scattering and intrinsic fluorescencemeasurement for the classification of airborne particles,” Appl. Opt. 39, 3738-3745 (2000)
8. S. Holler, Y. Pan, R. K. Chang, J. R. Bottiger, S. C. Hill, and D. B. Hillis, “Two-dimensional angular opticalscattering for the characterization of airborne microparticles,” Opt. Lett. 23, 1489-1491 (1998)
9. Y. L. Pan, K. B. Aptowicz, R. K. Chang, M. Hart, J. D. Eversole, “Characterizing and nonitoring respiratoryaerosols by light scattering,” Opt. Lett. 28, 589-591 (2003)
10. S. A. Burke, J. D. Wright, M. K. Robinson, B. V. Bronk and R. L. Warren, “Detection of molecular diversity inBacillus Atrophaeus by amplified fragment length polymorphism analysis,” Appl. Env. Microbiology, 70, 2786–2790 (2004)
11. F. Borghese, P. Denti, R. Saija, Scattering by model nonspherical particles (Springer, Heildelberg, 2002)12. C. Li, G. W. Kattawar, and P. Yang, “Identification of aerosols by their backscattered Mueller images,“ Opt.
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Sci. 29, Suppl.1 965-966 (1998)14. P. S. Tuminello, E. T. Arakawa, B. N. Khare, J. M. Wrobel, M. R. Querry, and M. E. Milham, “Optical properties
of Bacillus subtilis spores from 0.2 to 2.5 μm,” Appl. Opt. 36, 2818-2824 (1997)15. A. Katz, A. Alimova, M. Xu, P. Gottlieb, E. Rudolph, J. C. Steiner, and R. R. Alfano, “In situ determination of
refractive index and size of Bacillus spores by light transmission,” Opt. Lett. 30, 589-591 (2005)16. M. I. Mishchenko, L. D. Travis, and A. A. Lacis, in Scattering, Absorption, and Emission of Light by Small
Particles (Cambridge University Press, Cambridge, 2002)17. M. I. Mishchenko, D.W. Mackowski, “Electromagnetic scattering by randomly oriented bispheres: Comparison
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1. Introduction
In the last years there has been a growing interest in methods for the analysis and characteriza-tion of biological aerosols. The main aim is to achieve differentiation between various types ofairborne particles so that the presence of biowarfare agents can be rapidly detected from a safedistance. Although important advances have been made in determining the properties of bio-logical particles, a definitive characterization is still elusive. Both the infrared extinction spectra[1] and the fluorescence spectra [2] of aerosolized biological spores are relatively smooth anddevoid of any sharp identifiable line structure. The most characteristic features associated withthe presence of certain spore proteins occur in the range between 5.6 and 6.6 μm wavelength.Unfortunately, this highly characteristic region lies in the middle of the most opaque portionof the atmosphere and is probably of little value when we are considering possible remotedetection techniques [1].
Methods involving optical or electron microscopy can change the shape and internal struc-ture of the particles, and are also incapable of doing real-time particle-by-particle diagnosis.Methods and instruments have been developed using particle and fluorescence counters [2],and incorporating simultaneous measurement of aerodynamic size and intrinsic fluorescence[3, 4]. However, these monitoring systems are susceptible to the occurrence of false positives,due to the fact that non-biological particles may be present with similar size and fluorescencesignature to biological particles. This implies the need for other methodologies, that are able toprobe additional particle properties. To this aim, elastic light scattering appears to be a suitabletool, since it is dependent on the particle size, shape, surface roughness and complex index ofrefraction [1, 5, 6, 7].
Bioaerosols are typically inhomogeneous and often nonspherical, which makes even accuratepredictive calculations extremely difficult. Holler et al. analyzed clusters of Bacillus subtilis varniger (BG) spores (a simulant for anthrax) through a two-dimensional angular optical scattering(TAOS) technique [8]. Angle-resolved elastic light scattering opens a new window for single-particle investigation and may be developed into a new technology for a fast, highly sensitive,and reliable discriminatory instrument.
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Two research groups, headed by P. H. Kaye and R. K. Chang respectively, have been work-ing intensely over the past few years to develop and refine elastic scattering technologies foraerosol characterization, particularly as an optical diagnostic tool for rapid discrimination ofbiowarfare agents. In this paper we describe a system that can simultaneously obtain TAOSpatterns of aerosol particles (1-10 μm in diameter) in both the forward and the backward hemi-spheres in real time, and with a single laser pulse. Scattering in the forward and backwardhemispheres contain information about particle size, morphology, surface roughness and com-plex index of refraction. TAOS patterns of ambient aerosols and laboratory generated simulantsallowed particle discrimination between B. subtilis spores and aerosol particles found in theambient atmosphere [9]. We notice that a recent paper [10] has pointed out that B. Subtilisvar. niger should actually be reclassified as Bacillus Atrophaeus. Nevertheless, in this paper westick to the denomination B. Subtilis that is perhaps more known to non specialists.
In the next section, we will describe a system to simultaneously acquire TAOS patterns in theforward and backward hemispheres of single particles on-the-fly. The complete angle-resolvedscattering information and data are ready for comparison with theoretical computations.
A theoretical approach that has proven to be suitable for comparison with measured TAOSpatterns is the Multipole Expansion of the electromagnetic fields in the framework of the Tran-sition Matrix (METM) method. Such approach allows us to calculate the scattering propertiesof particles with arbitrary size and to take proper account of asymmetries in the scatterer shape.The Transition Matrix method was found to be very useful and flexible in describing asymmet-ric particles in terms of aggregates of spheres [Borghese, Denti and Saija [11] and referencestherein]. The METM method can also be useful in modelling biological spores, as we will showin Section 3. An alternative computational approach, through the finite-difference time-domain,has been very recently used by Li et al. that model the spore as an ellipsoid with a centered coreand one layer coat [12].
In Section 4 we will present our computational results, studying the effect of the refractiveindices on the scattering behaviour of some spore simulants. A comparison will be done be-tween experimental TAOS and theoretical patterns, computed through the METM approach.Finally, in Section 5 we will draw our main conclusions.
2. The Forward-Backward TAOS experimental set-up
The experimental setup used for simultaneously collecting TAOS patterns in the forward andbackward hemispheres is illustrated in Fig. 1. The particle travels downward along the y-axisand is irradiated by a 50 ns pulse of a 532 nm Nd:YAG laser (Spectra Physics, X-30), propagat-ing along the z-axis. The symmetry axis of the truncated ellipsoidal reflector is oriented alongthe x-axis and thus is perpendicular to the direction of laser propagation. The scattering eventoccurs at the first focal point of the truncated ellipsoid (F1). A large portion (63% of 4 sr) ofthe light scattered by the particle is intercepted by the reflector, and projected onto the ICCDdetector (1024x1024 pixels, Andor iStar). The ICCD detector is positioned on the x-axis, afterthe second focus (F2) of the reflector. Half of the ICCD detector detects the forward-scatteringpattern, and the other half detects the backward-scattering pattern.
TAOS is measured for the scattering angles in the range 15◦ < θ < 165◦ and for azimuthalangles covering as much as 360◦ in the near-forward and near-backward scattering. The fullrange of azimuthal angles (0◦ < φ < 360◦) is collected for all θ except where the reflector hasparts removed by truncation of the ellipsoid and five holes drilled through the reflector. Thetruncated hole will restrict 0◦ < φ < 270◦ at θ = 90◦. The five drilled holes also caused a smallamount of loss of φ . The reason and location for each of the five holes are explained below.The particles enter through the top hole at θ = 90◦, φ = 270◦, and exit through the bottom holeat θ = 90◦, φ = 90◦. The laser beam enters through a side hole centered at θ = 180 ◦ and exits
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Fig. 1. Spherical coordinate system as defined for the backward/forward TAOS experiment.
through a side hole at θ = 0◦. Lastly, there is the fifth hole in the back (θ = 90◦, φ = 180◦) usedfor the passage of the trigger laser diode beam. The particles are generated by putting acqueoussolutions of polystyrene (PSL) microspheres or BG spores in an Ink-Jet Aerosol Generator [13].
It is quite instructive to transform the recorded patterns into the traditional spherical coordi-nate system. The resulting TAOS patterns were divided into forward and backward halves (Fig.2) for easier comparison with the theoretical calculations. The dark shadows at the center, theleft side, and the right side in both patterns are caused by the loss of the reflection from the openspace and holes of the reflector. This loss of reflection restricts the range of angles φ collectedfor each θ .
Fig. 2. Simultaneously recorded forward and backward scattering patterns after processingfor a single polystyrene microsphere (diameter: 1.44 μm), illuminated by a single shot ofthe second harmonic of a Nd:YAG laser.
3. A model for spore simulants
The choice of a suitable model for the spore simulants has been suggested by the electron mi-croscope images. These images give quite accurate indication about size and shape of biologicalparticles. From SEM microscopy BG spores appear to have a cylindrical-like shape. The modelthat we adopt in our computations for spore simulants consists of a cluster of two identical,mutually contacting, spheres. At the laser wavelenght used in the TAOS set-up, such model is
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appropriate. Indeed, as expected from the general scattering theory, the difference between thepattern from such a cluster and a cylindrical particle is not appreciable. This suggests that thebinary cluster model is a reasonable choice. Our choice is further supported by the comparisonbetween the measured TAOS patterns of a cluster of two PSL microspheres and the pattern ofa single BG spore (Figs. 5 and 7). In the calculations, the radius of the spherical monomers ischosen so to reproduce, as best as possible, the actual size of the BG spore.
One of the problems with the characterization of bacteriological agents lies in determiningtheir exact refractive index. This is a very relevant parameter for the correct reproduction of thefeatures of the optical spectra. Thus, the refrative index plays an important role in the discrim-ination of biological spores. B. subtilis is generally considered the best simulant of the sporesof anthrax. The refractive index of the B. subtilis spore has been published by Tuminello et al.[14], who reported four kinds of refractive indices corresponding to spores that were exposed(used) or not exposed (as-received) to water in preceding experiments. Values were reported forthe refractive index measured both in water and in glycerol, in order to discriminate the effectof the chemico-physical nature of the environment on the measured spectra. The various formsof the refractive index reported for the B. subtilis spores differed rather little from each other. Inour computations we use the values of the refractive index for the as-received B. subtilis sporesin water. All the theoretical results presented in the following section are obtained through theMETM method.
4. Results and discussion. A comparison between computational and experimental data
The sensitivity of the optical properties to the particle refractive index is shown in Fig. 3, wherewe present the scattering and extinction cross sections computed for a dispersion of spores in arandom orientation. Results obtained using the refractive indices for B. subtilis and B. cereus,another spore belonging to the same family of anthrax, are shown. We model a single spore asa cluster of two spheres, each with a radius of 0.35 μm. This choice for the spore size followsthe suggestion given by Katz et al. [15] relative to the radius of dry homogeneous spores. Theresults for random orientation are independent of the state of polarization.
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Fig. 3. Extinction σe and scattering σs cross sections for a dispersion of cluster of spores ina random orientation. Each spore is modelled as a cluster of two spheres, with radius 0.35μm. The solid line is referred to a B. cereus spore; the dotted one to a B. subtilis spore.
No relevant differences appear in the reported extinction cross sections. More interesting
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Fig. 4. Z11(θ ) in the forward (left part in figure) and backward (right part) regions forclusters of two spheres with different choices of the refractive indexes and fixed monomerradius (0.35 μm).
is the comparison between the scattering cross sections: differences appear in the ultraviolet(between 0.2 and 0.4 μm) and near infrared regions (between 0.7 and 0.9 μm). These are thewavelength ranges in which the refractive indices of B. subtilis and B. cereus show the largestdifferences. The results in Fig. 3 suggest that a multi-wavelength analysis could be useful indiscriminating between the B. subtilis and B. cereus spores. We are currently working to reachthis goal with our instrumental set-up.
Figure 4 shows the the element Z11 of the modified Stokes phase matrix for φ = 90◦ andas a function of θ [16]. We present the results both in the forward and backward regions,for B. subtilis and B. cereus spores, and PSL particles. We model both the spores and thePSL particles as clusters of two spheres, keeping the monomer radius fixed at 0.35 μm. Norelevant difference is observed in the forward scattering pattern. A difference only occurs inthe backward region. The results in Fig. 4 confirm that the scattering pattern in the backwardregion may be more sensitive to the refractive index of the particle than the scattering patternin the forward region. The symmetry axis of the cluster is along the z axis in Fig. 1. Here andhereafter we are considering the same polarization for incident and scattered field, that has beenassumed to be linear and parallel to the y axis (see Fig. 1).
Figure 5 shows the TAOS patterns for a cluster of two PSL spheres, illuminated by a singlepulse of the second harmonic of a Nd:YAG laser, at 532 nm. The primary particle size is 1.44μm in diameter. The relatively noisy appearance of the backward hemisphere portion of the pat-tern relative to the forward portion might be related to two factors: first, the scattered intensityin the near forward hemisphere is roughly one hundred times larger than the scattered intensityin the near backward hemisphere. Thus the two hemispheres are measured at two distinct re-gions of the ICCD’s efficiency curve. Second, the more intricate appearance of the backwardhemisphere, as compared to the forward hemisphere, might follow from the considerations thatscattering in the backward hemisphere is more sensitive to the particle refractive index than thatin the forward hemisphere.
Figure 6 shows the computed forward and backward hemisphere scattering pattern, producedby a cluster of two PSL spheres with the same diameter as the ones used in the measurementshown in Fig. 5 (i.e. 1.44 μm). Here and hereafter, the computed scattering patterns representthe element Z11 of the modified Stokes phase matrix. The computations were performed byslightly varying the orientation of the cluster around the z axis, aiming to best reproduce theasymmetry of the experimental patterns. In fact, the experimental setup does not guarantee the
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Forward hemi. Backward hemi.
Fig. 5. Experimental scattering pattern, in both the forward and backward hemispheres, fora cluster of two PSL microspheres (primary particle size is 1.44 μm in diameter) illumi-nated by a 532 nm pulsed laser.
Fig. 6. Computational results for the same PSL spheres of Fig.5 in the forward (see leftpanel) and backward (right panel) regions. Both axes report cosθ according to the experi-mental setup (see Fig. 1)
vertical orientation of the cluster. The computed pattern that we report in Fig. 6 is the one that,in our opinion, achieves the best agreement between experimental and theoretical results.
The encouraging agreement obtained for PSL spheres led us to extend the comparison tothe case of BG spores. The experimental TAOS pattern is presented in Fig. 7. Concerning thecomputational results, some previous considerations are necessary. The scattering computedthrough the METM is strongly dependent on the particle size, so that even a slight variationcan affect the results in a relevant way [17]. This consideration, together with the uncertainty inthe experimental determination of the actual spore size, led us to consider the monomer radiusa free parameter, varying between 0.25 and 0.45 μm. Then we looked for the best fit of theexperimental data. The best fit was obtained for a binary cluster with a monomer radius of0.38 μm. The computed pattern is shown in Fig. 8. To better show the comparison betweenexperimental and computed results, we extracted the intensity profiles from Figs. 7 and 8 alongthe direction indicated by the broken line in the computed patterns. The comparison betweenexperimental (dotted line) and computational (solid line) results, shown in Fig. 9 for both theforward and backward zone, is very satisfactory.
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Forward hemi. Backward hemi.
Fig. 7. Experimental TAOS patterns in the forward and backward hemispheres for a B.subtilis spore, illuminated by a 532 nm pulsed laser.
Fig. 8. Computed patterns in the forward (see left panel) and backward (right panel) regionsfor a B. subtilis spore, modelled as a cluster of two spheres, each with a radius of 0.38 μm,illuminated by a 532 nm pulsed laser. Both axes report cosθ according to the experimentalsetup (see Fig. 1)
5. Conclusions
The motivation of this work comes from the growing interest in the characterization ofaerosolized biological particles. We propose a twofold approach to the problem, joining thetheoretical experience reached in the field of optical scattering by complex particles, and anovel experimental method (TAOS) for aerosol characterization. The METM method, basedon the multipole expansion of the electromagnetic field within the framework of the Transi-tion Matrix approach, proved to be powerful and flexible in describing scattering of light bycomposite, arbitrary shaped particles [11], overcoming most of the rough approximations oftenused in the literature when dealing with non-spherical particles. A reliable model to simulateparticles with complex morphology is the cluster of spheres, that proved to be successful also
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Fig. 9. Intensity profiles as a function of cosθ along an arbitrary direction (indicated bythe broken line in Fig. 8) extracted by the experimental (dotted line) and computational(solid line) scattering patterns for BG spores, both in the forward (left part in figure) andbackward (right part) hemispheres.
when modelling biological spores. Specifically, through a binary cluster with appropriate size,suggested by experimental evidences.
Angle-resolved elastic light scattering offers a great opportunity to the development ofsingle-particle investigation techniques. In this context, the TAOS technique appears a verypromising approach for a fast and highly sensitive aerosol characterization [18]. The systemthat we described to simultaneously obtain the forward and backward scattering patterns al-lows to partially discriminate between biological spores and aerosol particles. Scattering in theforward direction mainly allows to reconstruct information about particle size and morphol-ogy; while the backward scattering gives more detailed information about internal structure,composition, and surface roughness. The joint examination of the forward and backward TAOSpatterns, and the good comparison with the theoretical results opens a new window for a deepanalysis of the optical properties of biological spores and will hopefully enable us to overcomemany of the limits encountered in this field up today (structural changes induced on investigatedparticles, incapability to perform a real-time particle-by-particle analysis, occurrence of falsepositives).
The computational approach that we used enables us to carry out a complete angle-resolvedanalysis in terms of particle refractive index, size, and shape. The results presented also showthe importance of a multi-wavelength analysis of the particle scattering behaviour to achievedifferentiation between various types of airborne particles and/or spore simulants. This is thenext goal that will be hopefully reached with the TAOS instrumental set-up.
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