Fluorescence Spectra of

Individual Flowing Airborne

Biological Particles Measured in

Real Time

ARL-TR-1961 February 2001

Ronald G. Pinnick, Steven C. Hill, Stanley Niles, Yong-Le Pan, Stephen

Holler, Richard K. Chang, Jerold R. Bottiger, Burt V. Bronk, Dominick

C. Rozelle, Jay D. Eversole, and Mark Seaver

Approved for public release; distribution unlimited.
The findings in this report are not to be construed as an

official Department of the Army position unless so

designated by other authorized documents.

Citation of manufacturer’s or trade names does not

constitute an official endorsement or approval of the use

thereof.

Destroy this report when it is no longer needed. Do not

return it to the originator.
ARL-TR-1961 February 2001

Army Research Laboratory

Adelphi, MD 20783-1197

Fluorescence Spectra of

Individual Flowing Airborne

Biological Particles Measured in

Real Time

Ronald G. Pinnick, Steven C. Hill, Stanley Niles

Computational and Information Sciences Directorate, ARL

Yong-Le Pan

New Mexico State Univ.

Stephen Holler, Richard K. Chang

Yale Univ.

Jerold R. Bottiger

U.S. Army Edgewood Chemical Biological Center

Burt V. Bronk

U.S. Air Force Research Laboratory

Dominick C. Rozelle, Jay D. Eversole, and Mark Seaver

Naval Research Laboratory

Approved for public release; distribution unlimited.
The UV-excited fluorescence spectra of individual flowing biological

aerosol particles as small as 2 μm in diameter have been measured in real

time (rates up to 10 particles per second). The particles are illuminated

with a single shot from a Q-switched 266- or 351-nm laser. The

signal-to-noise ratio and resolution of the spectra are sufficient for

observing small line-shape differences among various types of

bioaerosols (e.g., bacteria versus pollens) and between the same types of

bioaerosol prepared under different conditions (e.g., the unstarved and

four-month-starved E. coli strain DH5). Multiple-wavelength excitation

provides additional information for distinguishing bioaerosols based on

their fluorescence spectra.

Abstract

ii
ARL-TR-1961 February 2001

Army Research Laboratory

Adelphi, MD 20783-1197

Approved for public release; distribution unlimited.
iii

Contents

1. Introduction 1

2. Experimental Setup 3

2.1 Biological Samples ...................................................... 5

2.2 Aerosol Generation ...................................................... 6

3. Results and Discussion 7

3.1 Spectra From Uniform Particles ....................................... 7

3.2 Spectra From Bacterial Particle Aggregates of Different Sizes 8

3.3 Spectra of Similar Bacteria Prepared Under Different

Conditions .................................................................. 9

3.4 Spectra of Pollens and Bacteria ....................................... 11

3.5 Spectra for Multiwavelength Excitation ........................... 12

3.6 Advantages and Limitations of This Technique .................. 13

4. Conclusion 15

Acknowledgments 16

References 17

Distribution 21

Report Documentation Page 25

Figures

1. Schematic of the experimental setup used for detecting single-shot

laser-induced-fluorescence spectra from individual micrometer-sized

aerosol particles .................................................................. 4

2. Frequency-of-occurrence histogram distributions of peak intensity for

1000 consecutive fluorescence spectra of individual tryptophan

particles ........................................................................... 7
iv

3. Single-particle laser-excited fluorescence spectra from clusters of

airborne B. subtilis vegetative cells with nominal diameter

5 μm, 2.3 μm, and 1.8 μm ...................................................... 9

4. Single-particle, 266-nm laser excited fluorescence spectra from

nominal 5-μm-diam clusters of B. subtilis var. niger spores, and B.

subtilis var. niger vegetative cells ............................................. 10

5. Fluorescence spectra of starved and unstarved E. coli strain DH5 ... 10

6. Single-shot, single-particle fluorescence spectra from nominal

5-μm-diam clusters of paper mulberry pollen, meadow oat pollen

allergen, and E. coli, and their corresponding 100-particle

accumulation spectra............................................................ 11

7. Fluorescence spectra of B. subtilis var. niger vegetative cells and

A. versicolor fungal spores ...................................................... 12
1

1. Introduction

Bioaerosols [1–5] have been studied in and around workplaces, homes [5],

hospitals [6,7], buildings with defective air-handling systems, factories where

metal-working fluids are used [8], pig farms [9], dairies [10] and other animal houses [4], agricultural operations, sites of sludge application [11], recycling and composting plants [12], and sewage treatment plants. Unlike

most common atmospheric aerosols, some airborne microorganisms can

cause infectious diseases. Like other biological aerosols (e.g., dust mite allergens) and nonbiological aerosols (e.g., diesel exhaust particles [13]), they

can also cause allergies and other respiratory problems.

Improved methods for measuring biological and other aerosols are needed.

In this report “real time” indicates the capability of sampling a particle within

0.1 s. Presently, methods that measure aerosol size distributions in real time

provide almost no information about particle types* and cannot specifically

identify particular microorganisms. On the other hand, methods that specifically identify microorganisms and/or proteins in a sample are generally

time-consuming and require aerosols to be collected [14–19]. Furthermore,

for most allergens, toxins, and microorganisms, specific identification requires culturing the sample or using specific protein or nucleic acid recognition molecules.†

Instruments that run continuously and classify particles into broad classes

of biological and other aerosols (and also determine size distributions of

the particles in each class) would have several advantages:

(1) In monitoring harmful bioaerosols, a rapid response may be necessary in situations where it would be impractical to continuously run

an identifier (impractical because of requirements for reagents and

for the collection of particles). For example, a continuously running

monitor/classifier could indicate when to sample for and identify

specific harmful bioaerosols. Also, in searching for or studying of intermittent sources of bioaerosols, a rapid response is necessary.

(2) Recognition molecules are not always available for all particle types

of interest.

(3) Improved studies of bioaerosol dynamics and reactions (agglomeration, nucleation [20], evaporation, growth, mixing, etc) may require a

real-time monitoring capability.

*Single particle mass spectrometry and the intrinsic fluorescence-based methods discussed

here are exceptions. Single-particle x-ray dispersive analysis methods require sample

collection. †Some pollens or other particles that can be identified microscopically are exceptions.
2

Fluorescence particle counters that continuously measure the laser-induced

intrinsic fluorescence (total or spectrally dispersed) from individual

bioaerosol particles have been shown to be able to partially discriminate

between biological and nonbiological aerosols [21–27]. Because the biological particles of interest may occur as very minor or rare species entrained

within a main population of nonbiological particles, the fluorescence must

be measured for each particle so that signals are uncontaminated and undiluted by those of the main population.

Classification with the use of only intrinsic fluorescence spectra is

challenging:

(1) There are so many possible types of biological aerosol particles.

(2) Naturally occurring biological aerosols may be mixtures of several

types of particles and compounds.

(3) Differences between the emission spectra of highly purified bacterial

or protein samples may be small (spectra may be dominated by a

small number of primary fluorophors).

(4) Differences in spectra may depend upon growth conditions, the atmospheric environment (e.g., temperature, humidity, and sunlight),

and other factors.

We report a method for measuring high-quality, reproducible, single-shot,

ultraviolet- (UV)-excited fluorescence spectra of individual airborne biological particles. Particles are interrogated by a pulsed UV laser [22,23] as

they are carried by a stream of air through a small sample region defined by

the intersection of two focused continuous-wave (cw) diode-laser beams

[27]. This report extends the findings reported in our recent letter [27] and

shows that (a) single-shot spectra of particles (which are aggregates of bacteria) as small as 1.8 μm in diameter can be measured, (b) particles within

certain size ranges can be selected for measurement, (c) differences between

spectra of bacteria of a single species grown under different conditions can

be seen in single-particle spectra, and (d) biological aerosols that have almost identical single-shot fluorescence spectra when excited at 266 nm can

have 351-nm-excited spectra that differ significantly from each other. Our

results suggest that single-shot spectra from individual bioaerosols can be

measured and may be useful for continuous detection and partial characterization of biological aerosols.
3

2. Experimental Setup

Our goal is to demonstrate a method for the real-time measurement of singleparticle fluorescence spectra for characterization of biological particles.

However, we need to be able to measure single-particle fluorescence spectra with good signal to noise before we can prove the utility of spectra. This

is technically challenging for several reasons. First, fluorescence signals are

very weak: we are trying to detect fluorescence from single particles containing only a few picograms of material, and only a small fraction of the

mass of biological particles consists of fluorophors. Second, because particles are mixed randomly in the air, particles moving rapidly through an

optical sample volume occur at random times, and so we must be able to

measure spectra of particles as they appear at random times; further, particles of interest may exist as a small concentration in a far higher concentration of nonbiological aerosols. Third, the fluorescence spectra from various biological and nonbiological particles extend over a wide wavelength

range, from the UV to the visible and near-infrared (IR) regions (280 to

750 nm). We want to excite particles in the UV wavelength and where most

biological particles (and biological molecules) have a high absorption and

fluoresce efficiently.

Our strategy to overcome these technical difficulties [22,23,25,27] has several key elements: First, we use a focused pulsed UV probe laser, which is

triggerable on demand and has a sufficiently high intensity to excite measurable fluorescence in microparticles. Second, to increase our fluorescence

collection ability, we use a large numerical aperture (NA) reflecting objective that can collect fluorescence from the emitting particle over a large solid

angle and focus it onto the slit of a spectrograph without chromatic aberration. Third, to sense the fluorescence at the exit port of the spectrograph, we

use an intensified charge-coupled device (CCD) detector (ICCD), which is

gated on only during the laser pulse so that it records the fluorescence of

the targeted particles alone, undiluted and largely uncontaminated by background. Finally, the focused probe laser and large NA collection objective

demand a tiny sample volume. To define a small sample volume through

which particles must traverse to be sampled, we require that particles pass

through crossed cw diode-laser beams that are focused immediately upstream from the UV probe laser.

The key equipment is as follows. The Q-switched UV laser output is either

the 266-nm fourth harmonic of an Nd:YAG laser, 30- or 70-ns pulse duration (Spectra Physics, model X-30 or Y-70 with the 532-nm output converted

to 266 nm by a beta-barium borate doubling crystal from Superoptronics),

or the 351-nm third harmonic of an Nd:YLF laser, 120-ns pulse duration

(Quantronix). The Q-switched laser fires within ~3 μs of the trigger pulse.

The Schwarzchild reflecting objective (Ealing) has a 0.5 NA. The
4

spectrograph (Acton model SP-150, NA = 0.125) has a 300-groove/mm grating blazed at 500 nm. The input slit width is 1 mm (resolution is about

4 nm). The ICCD camera (Princeton Instruments, ITE/CCD-1024MLDS-E

with PG200 pulse generator) is a fiber-optic-coupled, thermoelectrically

cooled ICCD gatable to 50 ns with 1024 × 256 pixels. The ICCD’s image

intensifier acts as a fast shutter, opening when the targeted particle is illuminated by the UV laser. A long-pass filter is placed in front of the spectrograph to block elastically scattered light of the UV laser and to pass the

fluorescence. A liquid cell containing N, N-dimethyl formamide is used to

absorb the 266-nm light.

Figure 1 illustrates schematically our experimental setup. Particles entrained

within a stream of air are directed downward toward the sample volume.

In the experiments reported here, there is no air-tight chamber, and so the

air stream is blown into the system toward the sample volume.

The requirement for a precisely defined sample volume is met by the use of

two nearly orthogonal, different-wavelength diode-laser beams (diode lasers (LaserMax) 1 and 2 emit at 635 and 670 nm). These lasers define a

15-μm-diam volume centered in the focal plane of the reflecting objective

(the beams are crossed so that the focal volume is approximately spherical).

As an aerosol particle flows through the focal volume of the crossed beams,

the near-forward elastic scattering is measured with photomultipliers PMT 1

and PMT 2 (with narrow-band interference filters at 635 and 670 nm). The

PMT signals are fed into two single-channel analyzers (SCA 1 and SCA 2)

Reflecting

objective lens

Long-pass

filter

Spectrograph with

ICCD camera

PMT 2 with 670-nm

interference filter

Nd:YAG laser

(266 nm or 351 nm)

To trigger

ICCD and Nd:YAG laser

Diode laser 1

(635 nm)

SCA 2

Dried

particles

SCA 1

AND

gate

Diode laser 2

(670 nm)

PMT 1 with 635-nm

interference filter

Ink-jet aerosol

generator

Figure 1. Schematic of

the experimental setup

used for detecting

single-shot laserinduced-fluorescence

spectra from

individual

micrometer-sized

aerosol particles.

Sample volume is

defined by two

intersecting cw diodelaser beams (at 635

and 670 nm). When a

particle traverses the

sample volume, (1) it

scatters the light from

two diode laser beams; (2) this light is detected by photomultipliers PMT 1 and PMT 2;

(3) if signals from each PMT fall within preset voltage windows, each single-channel

analyzer produces an output; (4) if outputs of both SCAs are logical ones, the AND gate

indicates that a spectrum is to be measured for this particle (by sending a triggering signal

to the UV laser and ICCD); (5) Q-switched UV laser fires and excites fluorescence in the

particle; (6) the fluorescence is collected by the reflecting objective and focused onto the

input slit of spectrograph; (7) the spectrograph disperses energy; and (8) the spectrum is

recorded with ICCD, which is gated to be on when the UV laser fires.
5

set to operate as discriminators in a window mode. We ensure that the

particles have sizes within a certain range by requiring the intensity of the

diode-laser light scattered by the particle (assumed to increase with particle size) to fall within a preset interval. The two SCA outputs are fed into a

logical AND gate, which produces an output pulse only when the two SCAs’

output signals overlap for at least 3 ns. The AND gate output then sends a

trigger pulse to the Q-switched laser and the controller of the ICCD; i.e.,

particles must pass through the intersection of both focused-trigger beams

before a logic pulse triggers the UV laser to fire and the ICCD to turn on.

Thus, the system ignores particles not flowing through this intersecting region (which may not be illuminated by the central portion of the UV laser

beam and may not be in the focal region of the reflecting objective).

2.1 Biological Samples

Several of the samples were used as purchased: meadow oat pollen allergens (Greer Laboratory), paper mulberry pollen (Duke Scientific), Bacillus

subtilis (lyophilized cell that are probably cells, American Type Culture Collection (ATCC) 6633, Sigma Chem.), B. subtilis var. niger spores (Dugway

Proving Ground, UT), Erwinia herbicola (ATCC 33243), and fungal spores

(Aspergillus versicolor, ATCC 9577).

B. subtilis var. niger (lyophilized cells) and E. herbicola were grown by streaking onto tryptic soy agar plates. E. coli strain DH5 was maintained on glucose M9 minimal media agar (Sigma Chemical Co., St. Louis, MO) enriched

with thiamine (Fisher Scientific, Pittsburgh, PA) [28]. For sample preparation, 1-ml aliquots from overnight cultures were transferred to sterile 1.5-ml

polypropylene microcentrifuge tubes. Cells were gently pelleted, the supernatant fluids were removed, and the pellets were gently suspended in

1 ml of sterile 0.9% NaCl. The washing process was performed three times

to ensure removal of all nutrient media [29]. Viable but not culturable bacteria were grown and measured as described in Roselle et al [30] (see next

paragraph), except that 50-ml cultures of bacteria in minimal media were

grown to a stationary phase and then stored in the dark at room temperature in the original flasks without further manipulation for four months.

To characterize the aged cells, we began by centrifuging 1-ml aliquots for

5 min at 4000 rpm to remove larger (viable and culturable) bacteria. The

supernatant fluid was isolated, the pellet was resuspended in 0.9% NaCl,

and the supernatant fluid was centrifuged for 15 min at 14,000 rpm to pellet

the smaller bacteria. Again the supernatant fluid, which contained small

unpelleted bacteria (<0.5 μm), was isolated, and the remaining pellet was

resuspended in 0.9% NaCl. A portion of this last supernatant fluid was further processed by a Centricon 100 concentrator that elutes soluble components (less than 100,000 MW) through a membrane into a lower collection

chamber while retaining the small bacteria in the upper chamber. The separation procedures just described yield four samples (a pellet of larger bacteria, a pellet of smaller bacteria, a supernatant fluid with smallest bacteria,
6

and the effluent from the Centricon concentrator). The number of colonyforming units from each E. coli sample was determined by serial dilution

titers and plating onto LB agar. Direct counts were done with acridine orange

stain and epifluorescence microscopy as described elsewhere [30,31]. Direct viable counts were also done, as previously described, by incubation of

bacterial samples in 0.25 g/l YT media and 20 g/ml naladixic acid (which

inhibits cell division but not cell growth) for 24 hr, followed by acridine

orange staining [30,32]. The samples were visually examined for growth by

epifluorescence microscopy, and elongated cells were counted.

2.2 Aerosol Generation

An ink-jet aerosol generator (IJAG) [33] was used to generate test aerosols.

It produces dry aerosol particles that are somewhat monodispersed (the

typical standard deviation in size distribution is 15 percent). The material

to be aerosolized in the IJAG is suspended in distilled, deionized, and filtered water and then inserted into an ink-jet cartridge (HP 51612A, purchased empty) having 12 nozzles. Each cartridge nozzle can be activated

through its resistive heater to generate a droplet approximately 50 μm in

diameter, plus one or two smaller satellite droplets. Droplets are generated

sequentially in the 12 nozzles at a frequency selected to be between 1 Hz

and 2 kHz, or singly on demand. These droplets are fired downward, carried by a filtered stream of air, and passed through a drying chamber heated

to about 104 °C. As these droplets traverse the drying chamber, their water

component evaporates, leaving the residual particles, which dry into roughly

spherical aggregates. Smaller aggregates produced from the satellite droplets are swept out of the drying chamber by a purge flow system. Because

the size of the primary ink-jet droplet is fixed, the size of the dried particle

depends only on the initial sample concentration in the water suspension.

For this series of measurements, we have in most cases prepared

0.1-percent suspensions by weight, resulting in 5-μm-diam dried particles.

A dedicated controller operates the ink-jet cartridge and drying chamber.

At the exit port of the generator, the dried particles are carried in an air

stream through a tapered 1-mm-diam exit nozzle.

An aerodynamic particle sizer (TSI, Inc., model 3310A) was used to provide

an aerodynamic size distribution of the particles in real time. In most cases,

the sizes were also measured through scanning electronic microscope (SEM)

images of particles collected on filters.
7

3. Results and Discussion

3.1 Spectra From Uniform Particles

We initially tested the apparatus with tryptophan aerosols. Tryptophan is

highly absorbing at wavelengths below 295 nm, has a relatively large fluorescence quantum efficiency (0.1 to 0.2), and makes up 3 to 5 percent of the

dry weight of typical bacteria [34] (e.g., for B. subtilis, the percentage is

3 percent for vegetative cells and 5 percent for spores). Measured absolute

fluorescence cross sections of B. subtilis spores are approximately proportional to the tryptophan content [35]. The peak emission of tryptophan is in

the 320- to 360-nm range but depends upon its environment. Particles were

generated with the IJAG with aerodynamic diameters of ~5 μm.

Figure 2 shows frequency-of-occurrence histograms of peak intensities of

1000 consecutive single-shot fluorescence spectra of individual tryptophan

particles excited by a 266-nm laser. The insets in both (a) and (b) indicate

the first 20 consecutive single-shot fluorescence spectra. In figure 2(a), the

0 1 2 3 4 5 6 7 8 9 10

0

100

200

300

400

(b) (a)

0 1 2 3 4 5 6 7 8 9 10

0

20

40

60

80

100

120

140

Relative peak intensity (a.u.) Relative peak intensity (a.u.)

LL = 0.25 V

UL = 10 V

LL = 4.0 V

UL = 6.0 V

Counts

4

3

2

1

20

15

10

5

Flu. intensity (a.u.)

Laser firing

200 300 400 500 600

Wavelength (nm) 4

3

2

1

20

15

10

5

Flu. intensity (a.u.)

Laser firing

200 300 400 500 600

Wavelength (nm)

Figure 2. Frequency-of-occurrence histogram distributions of peak intensity for 1000 consecutive

fluorescence spectra of individual tryptophan particles. Particles nominally 5 μm in diameter are

excited with a pulsed 266-nm-wavelength laser (0.1 mJ/pulse, 70-ns duration, focused to 40 μm in

diameter). In (a), discriminator levels for intensities of scattered light from diode-laser beams are

wide (UL = 10 V, LL = 0.25 V), defining a sample volume that is larger than the UV probe laser beam.

Consequently, a few strong fluorescence signals (corresponding to particles illuminated by the central

portion of the UV beam), but mostly weak fluorescence signals (corresponding to particles in the

wings of the UV beam), are measured. The resulting histogram is an inaccurate portrayal of the

particle fluorescence. In (b), discriminator levels define a relatively narrow window (UL = 6 V, LL =

4 V), and all particles measured are illuminated by the central portion of the UV beam. Insets in both

(a) and (b) indicate the corresponding spectra of the first 20 consecutive single-shot fluorescence

spectra.
8

discriminator levels (for the PMT outputs proportional to the intensities of

scattered light from the two diode-laser beams) are set to be wide: the preset upper (UL) and lower (LL) voltage levels of the two SCAs are set to be

UL = 10 V and LL = 0.25 V. The sample volume corresponding to this wide

voltage window is large because particles that are several beam diameters

from the center of the beams can give signals as large as 0.25 V. The resulting sample volume is larger than either the UV probe laser beam or the

focal region of the reflective objective. Consequently, figure 2(a) shows a

few spectra with strong fluorescence (corresponding to particles well illuminated in the central portion of the UV beam), but mostly spectra with

weak fluorescence (corresponding to particles not well illuminated because

not centered in the UV beam). The resulting histogram is an inaccurate portrayal of the particle fluorescence.

On the other hand, in figure 2(b), the discriminator levels are set to be relatively narrow (UL = 6 V, LL = 4 V). The resulting sample volume is smaller

and lies more within the central portion of the UV beam and the focal region

of the reflective objective. The distribution of peak intensities is approximately Gaussian, with the ratio of standard deviation to average intensity

about 0.6. If the particle size distribution has a ratio of standard deviation to

average radius of 0.15 (a typical value for the ink-jet generator), if the peak

fluorescence is proportional to particle volume (not a bad assumption in

this size range), and if the fluorescence is not saturated, then the ratio of the

standard deviation of fluorescence to the average fluorescence should be

about 3 × 0.15 = 0.45, if only the particle-size dispersion contributes to the

deviation. That result would suggest that the instrument response contribution to the distribution is relatively small (ratio of standard deviation of

intensity to intensity is about 0.6 – 0.45 = 0.15). (The overall standard deviation is the sum of the standard deviations of the particle-size distribution

and the instrument response width (caused by laser shot-to-shot intensity

variations, etc).)

3.2 Spectra From Bacterial Particle Aggregates of Different Sizes

We tested the sensitivity and reliability of the system for bacteria using

B. subtilis. Tryptophan makes up approximately 3 percent of the dry weight

of B. subtilis and is usually the primary fluorophor in bacteria illuminated

with 266-nm light. Therefore, we expect the overall fluorescence quantum

efficiency of B. subtilis excited at 266 nm to be about 3 percent of that of pure

tryptophan. Figure 3 shows 10 consecutive single-shot fluorescence spectra

of B. subtilis vegetative cells (Sigma Chemical) in aggregates of nominal

5.0-, 2.3-, and 1.8-mm diam. For each cluster size, a typical SEM micrograph

is shown on the right. The broad fluorescence peak (at 350 nm) is mainly

from tryptophan, and the tail from 400 to 500 nm may be partially attributed to fluorescence from residues of the nutrient growth material and may

have contributions from reduced nicotinamide compounds. The sharp peaks

at 532 nm are from a leakage of the primary 532-nm beam that generates the

266-nm beam. The sharp peak in some of the 1.8-μm cluster spectra near
9

440 nm may be laser-induced plasma-excited atomic emission (possibly from

nitrogen, calcium, or hydrogen), which only appears at high laser intensity.

These data demonstrate that this system can capture the fluorescence spectra from single bacterial aerosols with sizes as small as 1.8 μm in diameter.

Even for such small particles, the single-shot spectral lineshapes appear to

be quite distinct and consistent.

3.3 Spectra of Similar Bacteria Prepared Under Different Conditions

Figure 4 shows single-particle, single-shot fluorescence spectra of clusters

(aerodynamic diameter ~5 μm) of B. subtilis var. niger spores (Dugway) and

B. subtilis var. niger cells (Sigma). A corresponding typical SEM micrograph

for each sample is presented at the right of each spectrum. The spores exhibit more fluorescence in the 400- to 500-nm tail band, possibly because of

impurities and differences in the ways the samples were prepared.

Figure 5 shows fluorescence spectra of E. coli strain DH5 (unstarved and

four-month-starved samples). Aggregates of unstarved cells are about 4 μm

in diameter, while aggregates of starved cells are about 2.5 μm in diameter.

Starved sample data in figure 5 were obtained from the second pellet,

Figure 3. Singleparticle laser-excited

fluorescence spectra

from clusters of

airborne B. subtilis

vegetative cells

(Sigma) with nominal

diameter (a) 5 μm,

(b) 2.3 μm, and

(c) 1.8 μm. Particles are

illuminated with a

single shot from a Qswitched 266-nm laser

(frequency quadrupled

Nd:YAG), with 0.1 mJ/

pulse and 70-ns

duration, focused to a

40-μm spot size.

Electron micrographs

of typical particles are

shown for each size of

cluster.

5000

2000

2000

1000

10

8

6

4

2

10

8

6

4

2

10

8

6

4

2

200 300 400 500

200 300 400 500

200 300 400 500

Wavelength (nm)

Wavelength (nm)

Wavelength (nm)

Laser shot

Laser shot

Laser shot

Flu. intensity (a.u.) Flu. intensity (a.u.) Flu. intensity (a.u.)

(a) 

(b) 

(c) 
10

(a)

(b)

200 300 400 500 600 700

Wavelength (nm)

Fluorescence intensity (a.u.)

B. subtilis var. niger

B. subtilis var. niger

Figure 4. Singleparticle, 266-nm laser

(0.1 mJ/pulse, 70-ns

duration, focused to

40-mm diam) excited

fluorescence spectra

from nominal 5-μmdiam clusters of B.

subtilis var. niger

spores (Dugway), and

B. subtilis var. niger

vegetative cells,

(Sigma Chem).

Electron micrographs

of typical particles are

shown for each

sample.

Figure 5. Fluorescence

spectra of starved and

unstarved E. coli strain

DH5. Twenty

consecutive singleshot, single-particle

spectral results of

(a) unstarved (4-μm

diam) and (b) fourmonth-starved (2.5-μm

diam) E. coli strain

DH5, and

(c) corresponding 100-

shot (100 particles)

averaged fluorescence

spectra of E. coli strain

DH5. Excitation is at

266 nm (0.085 mJ/

pulse, 30-ns duration,

focused to 40-mm

diam).

3000

1000

10

8

6

4

2

200

400

600

Wavelength (nm)

Flu. intensity (a.u.)

Laser firing

1200

400

10

8

6

4

2

200

400

600

Wavelength (nm)

Flu. intensity (a.u.)

Laser firing

3

2

1

0

200 300 400 500 600 700

DH5

4 K, starved

100-shot average, 266-nm

E. coli

Fluorescence intensity (a.u.)

Wavelength (nm)

(a) (b)

(c)

centrifuged at 14 krpm for 15 min, that includes mostly smaller bacteria.

Figures 5(a) and (b) show single-shot spectra while figure 5(c) shows the

average spectrum from 100 consecutive shots. All E. coli single-shot fluorescence spectra show good spectral quality. These fluorescence results are

qualitatively similar to those measured by a spectrofluorometer on a bulk

liquid suspension of bacteria [30]. This similarity suggests that the method

described here acquires accurate single-shot fluorescence spectra.
11

The spectral features of starved and unstarved cells are different: First, the

absolute fluorescence peak intensity from the region of tryptophan emission (about 330 nm) is about five times larger in the unstarved samples.

This ratio is comparable to the ratio of the volumes of the particles, i.e.,

(4.0/2.5)3 = 4.0. Second, in the 400- to 500-nm range, the fluorescence of the

starved bacteria is within 10 to 50 percent of the fluorescence of the unstarved

bacteria. It appears that as the cells are starved, the tryptophan fluorescence

per volume does not change markedly (it may decrease), but the fluorescence in the 400- to 500-nm range increases significantly, until the emission

near 420 is nearly as large as the tryptophan peak. The compound(s) responsible for the increased emission in the 400- to 500-nm region have not

yet been identified.

3.4 Spectra of Pollens and Bacteria

Figure 6 presents the single-shot, single-particle spectra of paper mulberry

pollen, meadow oat pollen allergen, and E. coli (solid lines), excited by a

266-nm laser, as well as their corresponding 100-shot averaged results (dotted lines). The high quality of the single-shot spectral results shows that

there are no substantive differences between the single-shot spectra and

their corresponding 100-shot accumulation spectra. This confirms that the

single-shot spectra of the individual bioaerosols represent the real spectral

lineshape of the measured bioaerosols.

The spectra of paper mulberry pollen, meadow oat pollen allergen, and E.

coli show clear differences. The highest intensity peaks in the pollen and

pollen allergen spectra are located around 400 to 550 nm, but the E. coli

emission is dominated by tryptophan (330 to 350 nm). The meadow oat

Figure 6. Single-shot,

single-particle

fluorescence spectra

(solid lines) from

nominal 5-μm-diam

clusters of paper

mulberry pollen,

meadow oat pollen

allergen, and E. coli

(solid lines), and their

corresponding 100-

particle accumulation

spectra (dotted lines).

No difference in shape

exists between singleshot spectra and their

corresponding

100-shot accumulation

spectra. Excitation is at

266 nm (0.085 mJ/

pulse, 30-ns duration,

focused to 40-μm diam).

200 300 400 500 600 700

Wavelength (nm)

Fluorescence intensity (a.u.)

Meadow oat

Paper mulberry

E. coli
12

sample has significant emission in the tryptophan range, but the emission

in this range from paper mulberry pollen emission is very low. These results, including those shown in figures 2 to 5 for bacteria, suggest that the

relative intensities (tryptophan versus different bands in the longer wavelength emission) in single-shot fluorescence spectra could be useful for realtime discrimination between bacteria (in which the tryptophan peak is at

least as large as any other peak in all of our measurements) and certain

pollens and allergens (which have smaller tryptophan emission compared

to that in the 400- to 500-nm band).

3.5 Spectra for Multiwavelength Excitation

Figure 7 shows fluorescence spectra of B. subtilis var. niger vegetative cells

and fungal spores (Aspergillus versicolor), both 5 μm in diameter. The spectra in figure 7(a) are excited by 266-nm light and are similar for the two

samples. Both spectra are dominated by the emission from the region of

tryptophan emission.

In figure 7(b) the samples are excited with 351-nm light and the spectra are

far less similar: The fluorescence peaks are at 450 nm for B. subtilis and 420 nm

for the fungal spores. The photon energy of 351-nm light is too low to excite

fluorescence from the aromatic amino acids (tryptophan and tyrosine are

very poorly excited at wavelengths longer than about 305 nm), so these do

Figure 7. Fluorescence

spectra of B. subtilis

var. niger vegetative

cells and A. versicolor

fungal spores (both

with nominal 5-μm

diam). Excitation in (a)

is with a 266-nm laser

(0.085 mJ/pulse, 30-ns

duration, focused to

40-μm diam), and in

(b) is with a 351-nm

laser (1.65 mJ/pulse,

120-ns duration,

focused to 40-μm

diam).

200 300 400 500 600 700

300 400 500 600 700

3

2

1

0

2

1

0

(b) 100-shot average

351-nm excitation

B. subtilis var. niger

vegetative cells

Fungal spores

(a) 100-shot average

266-nm excitation

Fungal spores

B. subtilis var. niger

vegetative cells

Wavelength (nm)

Wavelength (nm)

Fluorescence intensity (a.u.)
13

not contribute to the spectra in 7(b). The fluorescence is dominated by other

compounds, which we conjecture may be flavins, nicotinamide nucleotides,

secondary metabolites, and components of culture media (these may have

more variability from sample to sample than do the amino acids). The fact

that the spectra of the two samples are nearly the same at 266-nm excitation, but differ appreciably at 351-nm excitation, suggests that fluorescence

spectra excited at different wavelengths may provide more discrimination

among bioaerosols than would be possible with single-wavelength

excitation.

3.6 Advantages and Limitations of This Technique

Our single-particle fluorescence-based method for detecting and partially

classifying bioaerosols has several advantages. It should be capable of—

(1) Detecting almost all bioaerosols at rates up to 50 to 1000 particles per

second, if the particle diameters are on the order of 2 μm or larger

(and possibly as small as 1 mm in diameter with less spectral

resolution).

(2) Determining the presence of bioaerosols of any origin without the

use of reagents or prior knowledge of the types of aerosols that may

be present.

(3) When combined with pattern classification techniques, specifying

bioaerosols into some as-yet-unspecified set of classes based on their

sizes and spectra. These classes may discriminate between (for example) pollen allergens and bacteria, or bacteria generated under different conditions.

(4) Detecting a very small concentration of biological particles mixed with

a far higher concentration of nonbiological particles that either fluoresce very little or have a different spectral emission.

The approach also has some disadvantages:

(1) Because our technique requires such a small sample volume (because

we need to collect a large fraction of the emission and measure its

spectrum), the rate at which we are able to sample the air is low. If

the diameter of the sample region is about 20 μm and if the air traverses

this region at 10 m/s (these are numbers comparable to what we use

now), the sample rate is only 0.2 ml/min. Even if the particles are

preconcentrated in air at a ratio of 1000:1 (e.g., if we use multiple

virtual impactors), the equivalent sample rate is only 0.2 l/min, which

is not high enough if the particles of interest have a concentration of

1/l or less. For such single-particle sampling, one would need greater

preconcentration to keep the sampling time short (say, a few minutes). The flow rate can be increased in proportion to the sampleregion cross section but at the cost of lower spectral resolution. We

cannot significantly increase the excitation intensity, because nonlinear effects begin to appear [27].
14

(2) We do not expect UV-excited fluorescence spectra by themselves to

be used to specifically identify airborne microorganisms, pollens, or

allergens: there are too many possible types, possible mixtures, and

possible growth conditions. This is a disadvantage, but it is the other

side of the coin mentioned as advantage (2) above: The method should

be capable of detecting all bioaerosols above 1 μm or so and do this

without prior generation of specific molecules to detect specific proteins, nucleic acids, or polysaccharides.
15

4. Conclusion

We have obtained single-shot UV-excited intrinsic fluorescence spectra of

individual flowing biological aerosol particles as small as 2 μm in diameter.

The spectra measured from single bioaerosols have high signal-to-noise ratios and good spectral resolution and reveal small differences that may depend on the type of primary material in the bacteria and the method of

preparation. With multiple-wavelength excitation (e.g., one wavelength

within the absorption band for tryptophan and the other outside the band)

and with pattern-recognition algorithms, this single-particle fluorescencespectral technique may be useful for rapid detection and partial characterization of natural and anthropogenic indoor and outdoor aerosols.
16

Acknowledgments

New Mexico State University acknowledges financial support from the U.S.

Army Research Laboratory (ARL, DAAL01-98-C0056). Yale University acknowledges the partial financial support from the U.S. Army Research Office (DAAG55-97-1-0349), the U.S. Air Force Research Laboratory and ARL

(DAAL01-97-2-0128), and an Augmentation Award for Science and Engineering Research Training (AASERT) Fellowship (DAAG-97-1-0199) for

S. Holler.
17

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298-102

Fluorescence Spectra of Individual Flowing Airborne

Biological Particles Measured in Real Time

February 2001 Progress 7/1/1998 to 3/1/1999

The UV-excited fluorescence spectra of individual flowing biological aerosol particles as small as

2 μm in diameter have been measured in real time (rates up to 10 particles per second). The

particles are illuminated with a single shot from a Q-switched 266- or 351-nm laser. The

signal-to-noise ratio and resolution of the spectra are sufficient for observing small line-shape

differences among various types of bioaerosols (e.g., bacteria versus pollens) and between the same

types of bioaerosol prepared under different conditions (e.g., the unstarved and four-month-starved

E. coli strain DH5). Multiple-wavelength excitation provides additional information for

distinguishing bioaerosols based on their fluorescence spectra.

bioaerosols, aerosol detection, aerosol fluorescence, biological

aerosols, fluorescence spectroscopy, single-particle spectroscopy

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R. G. Pinnick, S. C. Hill, S. Niles (ARL), Y.-L. Pan (New Mexico State

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D. C. Rozelle, J. D. Eversole, and Mark Seaver (Naval Research Laboratory)

29

25