Bio-aerosol detection using intrinsic fluorescence and
elastic light scattering analysis
W R Stanley1, P H Kaye1, V E Foot2, K L Baxter2 and S J Barrington2
1. Science and Technology Research Institute, University of Hertfordshire,
Hatfield, Herts. AL10 9AB. UK.
2. Defence Science and Technology Laboratory, Porton Down, Salisbury,
Wilts. SP4 0JQ. UK.
Abstract. Laser diodes and light-emitting diodes capable of continuous sub-300 nm
radiation emission will ultimately represent optimal excitation sources for compact and
fieldable bio-aerosol monitors. However, until such devices are routinely available and
whilst solid-state UV lasers remain relatively expensive, other low-cost sources of UV
can offer advantages, especially in monitor networks where several tens of discrete
point-sampling detectors may be required. This paper describes one such prototype
that employs compact xenon discharge UV sources to excite intrinsic fluorescence
from individual particles within an ambient aerosol sample. Results from aerosols of
E.coli, BG spores, and a variety of non-biological materials are presented. A
development to add a particle shape assessment capability based on measurement of
azimuthal variation in particle elastic scattering is also described.
1. WIBS2 Wide Issue Bio-aerosol Sensor
The WIBS2 sensor is a development based on previous particle intrinsic fluorescence
research presented elsewhere [1]. The sensor employs a central optical chamber, a model of
which is shown in figure 1. Around this are arranged a continuous-wave 660 nm diode laser
used to detect and size particles, two miniature xenon flash tubes emitting in different
wavebands (set by filters), and two photo-multiplier tube (PMT) based fluorescence detection
channels, FL1 and FL2.
Aerosol in
Xe1 (280nm)
Diode Laser 660nm
FL2
(420-600nm)
FL1
(310-600nm)
Scatter
Beam dump
Xe2 (370nm)
Figure 1. WIBS2 design.
The prototype monitor samples ambient air at a rate of ~4.5 l/min through a bifurcated
delivery system that filters 3.9 l/min of the air and re-introduces this as a sheath around the
remaining 0.6 l/min flow. Photographs of the optical chamber and air delivery system are
shown in figure 2. Particles within this sample flow column are thus rendered in single file as
they intersect the beam from diode laser; each individual particle producing a scattered light
signal from which an estimate of particle size (down to ~1 µm) may be derived.
Xenon 2
power
monitor
Xenon 1
(280nm)
Beam dump
Laser
Xenon 1
power
monitor
Sensing
volume
Xenon 2
(370nm)
Sheathflow
annulus
Figure 2. WIBS2 system configuration.
The same scattered light signal also initiates the sequential firing (~5 µs apart) of two
xenon UV sources. These fire UV pulses that irradiate the particle as it passes through the
sensing region. The xenon sources are used as an alternative to laser diodes and lightemitting diodes capable of continuous sub-300 nm radiation emission, which are also used in
this field [2]. The diodes will ultimately represent optimal excitation sources for compact and
fieldable bio-aerosol monitors. However, until such devices are routinely available and whilst
solid-state UV lasers remain relatively expensive, the low-cost xenon UV sources can offer
advantages.
mm
2
0 uJ/cm2/sr
50
100
150
200
250
300
350
Particle
trajectory
1
Sensing
volume
0
0
1
mm
2
Figure 3. X-Y profile of UV pulse energy
focused onto sensing volume.
A profile of the pulse energy from a xenon flash with respect to the sensing volume can be
seen in figure 3. Wherever the particle is within the sensing volume it should receive an
equal amount of irradiance during a xenon flash. WIBS2 can illuminate the sensing volume
with an essentially uniform fluence (to within ~±6 %) in excess of ~300 µJ/cm2. The xenon
UV pulses are centered (using filters) upon ~280 nm and ~370 nm wavelengths, optimal for
excitation of bio-fluorophores tryptophan and NADH respectively. For each excitation
wavelength, fluorescence is detected across two bands embracing the peak emissions of the
same bio-fluorophores. Thus, for each particle, a 2-dimensional fluorescence excitationemission matrix is recorded together with an estimate of particle size. To achieve the large
solid angle of fluorescent light collection necessary, a pair of concave spherical mirrors (one
for each channel) is used to reflect the light onto the PMT photo-cathodes. This optical
configuration allows a large solid angle (~3 sr) of fluoresced light to be captured.
2. Preliminary results
The plots below are a method of displaying all the recorded data at once in a way that is both
easy to observe and effective in showing the observer how well WIBS2 can resolve different
particle types. On the vertical axis, the two fluorescence measurements FL1 and FL2 for the
Xenon 1 flash are divided to give a value that is independent of particle size.
Figure 4 shows that in most cases discrimination is readily achieved. Where the
fluorescence and/or size signals are weak and signal-to-noise S/N ratio is low (small or
weakly fluorescing particles), widespread distributions occur in the plot. This can be
observed for the 1 µm PSL (relatively small) and the Kettle Scale and Gypsum (low
fluorescence). The Tonic Water (diluted 1 % in distilled water) and the fluorescently doped
1.7 µm latex spheres produced expected high values of fluorescence, especially in the FL2
channel band.
Figure 5 shows results from aerosols more representative of the type of biological
materials and organisms that a prospective bio-aerosol sensor would need to detect and,
ideally, discriminate. These data recorded at DSTL Porton Down, UK, show results from
aerosols of: washed BG spores (Bacillus atrophaeus, a simulant of B. anthracis spores); dry
non-viable BG spores; washed and unwashed E. coli vegetative cells; 0.1 mmol solutions of
tryptophan and NADH (both in 1 % sucrose solution); a 1 % solution of ovalbumen in water;
and 3 µm latex spheres. The Tonic Water and 1.7 μm fluorescent PSL results from figure 4
have also been included for reference. These data show a promising degree of
discrimination between the various particle types.
FL1 280 / FL2 280
10
10
10
10
Paper mulberry pollen
Kettle scale
Gypsum
Cornflour
5m PSL
3m PSL
1m PSL
1.7m fluorescent PSL
Tonic water (1%)
1
0
-1
-2
-2
10
-1
10
0
10
1
10
2
10
3
10
10
10
2
FL2 370
10
1
0
10
-1
Size (m)
Figure 4. Preliminary WIBS2 data recorded from test
aerosol materials.
FL1 280 / FL2 280
10
10
10
3m PSL
1.7m fluorescent PSL
BG spores (dry)
BG spores (washed)
NADH (0.1mmol)
Tryptophan (0.1mmol)
E. coli veg. (unwashed)
E. coli veg. (washed)
Ovalbumen (1%)
Tonic water (1%)
1
0
-1
-2
10
-2
10
10
-1
10
0
10
10
1
10
10
2
10
FL2 370
3
10
-1
0
1
Size (m)
Figure 5. Preliminary WIBS2 data recorded from a variety
of biological and non-biological aerosols.
3. Conclusions
The dual-channel WIBS2 prototype has demonstrated its potential for the real-time
characterisation of aerosol particles down to ~1 µm in size. With future refinement, it is
hoped the WIBS2 will become the basis of low cost networks of bio-aerosol sensors that
could be used to provide 24/7 monitoring of atmospheres in both military sites (e.g. airfields,
troop encampments, ships, etc.) and civilian locations (e.g. underground systems, shopping
arcades, airport terminals, etc).
4. Further development in progress
The current WIBS2 prototype uses a 660 nm diode laser for triggering and particle size
analysis only. A development is in progress to utilise the elastically scattered laser light for
shape definition of particles. The optical system shown in figure 6 shows how light scattered
from a particle at angles up to 15° will be projected onto three sensors arranged around the
optic axis at different azimuth angles. The azimuthal variation in particle elastic scattering,
chiefly dependent on particle shape, will result in an imbalance in the light intensities
detected by the sensors. This additional data will add significantly to the particle
discrimination performance of the monitor.
Figure 6. Shape definition optics to be added to WIBS2
flow chamber.
5. Acknowledgement
This work has been supported by the Defence Science & technology Laboratory, Porton
Down, U.K.
References
[1] P.H. Kaye, J.E. Barton , E. Hirst and J.M. Clark, “Simultaneous light scattering and
intrinsic fluorescence measurement for the classification of airborne particles”,
Applied Optics 39, 21, 3738-3745 (2000).
P.H. Kaye, E. Hirst, V.E. Foot, J.M. Clark and K. Baxter, “A low-cost multichannel
aerosol fluorescence sensor for networked deployment”, in Optically Based
Biological and Chemical Sensing for Defence. J.C. Carrano and A. Zukauskas, eds.
Proc. SPIE 5617, 388-398 (2004).
V.E. Foot, J.M. Clark, K.L. Baxter and N. Close, “Characterising single airborne
particles by fluorescence emission and spatial analysis of elastic scattered light”, in
Optically Based Biological and Chemical Sensing for Defence. J.C. Carrano and A.
Zukauskas, eds. Proc. SPIE 5617, 292-299 (2004).
Y-L Pan, J. Hartings, R.G. Pinnick, S.C.Hills, J. Halverson and R.K. Chang, “Single
particle fluorescence spectrometer for ambient aerosols”, Aerosol Sci. Tech., 37,
628-639 (2003).
[2] SUVOS – Semiconductor Ultraviolet Optical Sources, J.C. Carrano, director,
http://www.darpa.mil/mto/suvos/ (2002).
T.H. Jeys, L. Desmarais, E.J. Lynch, and J.R. Ochoa, “Development of a UV LED
based biosensor”, in Sensors and Command, Control, and Intelligence Technologies
for Homeland Defence and Law Enforcement. E.M. Carrapezza, ed. SPIE 5071,
234-240 (2003).