Emission wavelength dependence of fluorescencelifetimes of bacteriological spores and pollens

Ann Thomas, David Sands, Dave Baum, Leleng To, and Glenn O. Rubel

Concern about biological terrorism has greatly increased in the 21st century, and correspondingly, so hasthe need for accurate detection and identification of biological hazards, such as Bacillus anthracis. Opticaltechniques have been shown to be useful for this purpose. Use of fluorescence lifetimes as a function ofemission wavelength for different materials using point-detection methods appears to be an additionalviable option. Although the lifetimes range only between 2 and 6 ns, most biological materials tested in thisstudy were distinguishable. A preliminary database has been compiled for use in a possible futuredetection system. © 2006 Optical Society of America

OCIS codes: 170.0170, 170.6280, 300.2530, 300.6500.

1. Introduction

A number of techniques are currently used to identifyand distinguish biological spores, especially for possi-ble bioterrorism detection. The current methods ap-proved by the U.S. Centers for Disease Control andPrevention for identifying Bacillus anthracis, for ex-ample, involve collecting samples and growing them inlaboratory conditions.1 Recent work has been done todevelop methods of direct examination of the sus-pect materials for identification. In particular, Pinnicket al.2 examined the emission wavelength dependenceof the time-integrated fluorescence of spores, pol-lens, and other materials with a similar appearance.Méjean et al.3 demonstrated that the fluorescent sub-stances in such materials can be detected remotely.Recent reports4,5 of work done at the U.S. Army Re-search Laboratory at Aberdeen Proving Ground indi-cate that some species of bacteria used as surrogatesfor B. anthracis can be identified by vaporizing thematerial and analyzing the characteristic emissionlines generated during recombination.

The current study is, to our knowledge, a first at-tempt to make use of time-resolved fluorescence life-time measurements directly from bulk materials forthe purpose of generating a database of such lifetimesfor future comparison. The emissions of a number ofbacteriological materials, as well as of other similarlyappearing materials such as pollens, are investi-gated. The three goals of the study: (i) determine iffluorescence lifetimes of these biological materialsare readily measurable; (ii) construct a database ofmaterials for 337 nm excitation; (iii) determine ifthere are sufficient differences to distinguish materi-als. Results indicate that the lifetime spectra gener-ated could be useful in identifying and distinguishingsuch materials, and the use of fluorescence lifetimesas a function of emission wavelength for differentmaterials appears to be an additional viable option asa point-detection method.

2. Procedure and Equipment

A schematic of the experimental apparatus is pre-sented in Fig. 1. The excitation source for this studywas a Laser Science VSI-337 nitrogen laser that gen-erated 337 nm, 4 ns, 100 �J pulses at rates of up to20 Hz. The laser light passed through a microscopecoverslip used as a beam splitter; thus a small per-centage of the light was deflected through a set ofneutral density filters and was incident on a fastdiode detector (Thorlabs DET 110) used as the timingtrigger [Fig. 1(A)]. Initially, quartz tubes and “non-fluorescing” powder cells were used in a traditional90° orientation, but they each fluoresced about asstrongly as did the samples. Instead, the powderedsamples rested in a horizontal, nonfluorescing tanta-

A. Thomas, D. Sands, D. Baum (rgbaum@verizon.net), and L. Toare with Goucher College, 1021 Dulaney Valley Road, Baltimore,Maryland 21204. A. Thomas, D. Sands, and D. Baum are with theDepartment of Physics. L. To is with the Department of BiologicalSciences. G. O. Rubel is with the U.S. Army Edgewood Chemicaland Biological Center, Aberdeen Proving Ground, Maryland21010.

Received 16 September 2005; revised 23 March 2006; accepted13 April 2006; posted 14 April 2006 (Doc. ID 64828).

0003-6935/06/256634-06$15.00/0© 2006 Optical Society of America

6634 APPLIED OPTICS � Vol. 45, No. 25 � 1 September 2006

lum tray [Fig. 1(B)], and the excitation light wasdirected with a front surface mirror onto the sample.Emitted light was collected by a lens above the trayand redirected horizontally with a second plane mir-ror. (Note that the angle between incident and emit-ted light propagation vectors was approximately135°, rather than the more usual 90°.) The secondarylens coupled this light to the entrance slit of a Spex1680B double 1�4 m monochromator through anOriel 59470 385 nm long-pass filter used to minimizethe detection of scattered laser light. The signal wasmeasured by a Hamamatsu R7400U fast photomul-tiplier tube (PMT) operated at �800 V with a transit-time spread (TTS) of 0.25 ns. The signal was recordedat 100 ps intervals on an HP 54121T 20 GHz digitaloscilloscope; this scope was chosen for its high analogbandwidth �20 GHz�, in spite of its low digitizationrate. A 52 � cable was used to match the oscilloscopeinput impedance at this frequency. The usable spec-tral range of the system was limited by the long-passfilter and PMT response to the range 380–600 nm;data from 610 to 640 nm were recorded, but the life-time fits are of low reliability due to the low signal-to-noise ratio and so were discarded. Each lifetime

measurement was fit to eight averaged curves with atotal of 4000 laser pulses. Since fluorescence lifetimesare expected to change slowly with emission wave-length, measurements were taken at 10 nm intervals,and the bandpass widths of individual wavelengthswere adjusted for optimal signal strength rather thanfor uniform spectral resolution. Lifetime values weredetermined using a commercially available softwarepackage, FLUOFIT.6 The system was tested usingCoumarin 540A in methanol, which is known to havea single lifetime of 4.16 ns.7 The system test resultwas 4.24 ns, well within the estimated uncertainty(see the discussion below). In theory, this system wasable to determine lifetimes on the order of 500 ps orlonger by deconvoluting the excitation pulse out ofthe fluorescence signal; however, despite the expec-tation that the decay curves are combinations of life-times from several materials present in the samples,only single lifetime fits were possible with this appa-ratus.8

An analysis of contributions to the uncertainty ofthe lifetime fits was undertaken. The issue of inho-mogeneities in the samples was addressed by per-forming ten lifetime measurements of B. globigii withthe sample returned to its storage container andshaken between each, resulting in a variation of ap-proximately �0.2 ns. Ten repeated measurementswere then made without shaking the sample, alsoresulting in a spread of about �0.2 ns, suggestingthat any sample inhomogeneities do not affect thelifetime results. Slight variations in the initial condi-tions set for the fitting program FLUOFIT resulted invariations of the lifetime fits that ranged from essen-tially zero (common) to roughly 0.2 ns (fairly rare);this was estimated by performing multiple fits onsingle data sets. Since this factor is necessarily in-cluded in the estimates described earlier, it might beconcluded that the largest contribution to the uncer-tainty is due to noise in the signal and possible effectsfrom the PMT time response. Therefore it was as-sumed that spectra for other materials possess ap-proximately the same uncertainty; a representativeerror bar (for B. globigii) is shown in each lifetimedecay figure.

A series of time-integrated (TI) fluorescence emis-sion spectra was also collected. The system was thesame as described above, except that the detectorwas replaced with a slower Hamamatsu R928 PMT,which, with the use of the long-pass filter, resulted ina usable spectral range from 380 to 750 nm; thisupper limit was chosen to avoid second-order outputfrom the monochromator. The output was fed to aPAR 160 boxcar integrator, the gate of which wasadjusted to measure all the light output of each pulse.The analog output of the boxcar was digitized andrecorded by computer as a function of emission wave-length. The PMT sensitivity in this range is fairlyconstant as a function of wavelength, but no correc-tion was made for the grating efficiency, since in thiswork the curves were used for comparison only. Sucha correction was, of course, unnecessary for the life-time measurements.

Fig. 1. (A) Schematic of time-resolved fluorescence apparatus; theoscilloscope was replaced by a boxcar integrator for the TI mea-surements. (B) Details of the excitation and fluorescence-collectionoptics for the sample configuration used in this study.

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3. Samples

The materials tested included vegetative cells of bac-teria, such as B. globigii, B. atrophaeus, B. subtilis,and B. subtilis spizizenii, which were of greater than99% purity. Other nonbacterial materials that aresimilar in color and texture to B. anthracis were alsotested (Table 1).

All bacteriological samples except B. globigii wereprocured from the American Type Culture Collection(ATCC),9 grown, harvested, and washed to producematerial composed of at least 99% vegetative cells.10

Pollen samples were purchased from Greer Labora-tories,11 while the samples of B. globigii were ob-tained directly from the U.S. Army’s EdgewoodChemical and Biological Center (ECBC), as was theArizona road dust. B. globigii, a common simulant forB. anthracis, was measured as received (includingvegetative cells, spores, nutrient, and other uniden-tified contaminants), then washed as described aboveto separate out the vegetative cells. Other materialswere purchased from various sources (Table 1) andwere used as is, and the cigarette ash was collectedfrom a volunteer smoker and sifted to remove largerparticles.

4. Results

A database of the lifetime for each material at eachemission wavelength was developed; these data arepresented here in graphic form. The bacteria, in gen-eral, each exhibited a particular characteristic decaylifetime spectrum. B. globigii, being the prototypicalsimulant for B. anthracis, was investigated the mostthoroughly. As shown in Fig. 2, B. globigii was testedas received (including growth medium) and after sep-aration of the vegetative cells. Certain attributes ofthe graphs can be clearly observed, including fairlyconstant, but somewhat different lifetimes; this par-ticular mixture of B. globigii with nutrient seems tobe distinguishable from the pure samples by the av-erage decay lifetime of 4.5 ns compared to about3.6 ns.

Figure 3 shows the decay lifetimes of the threeother bacteria. B. atrophaeus exhibits a general in-crease in its lifetime from 3 to 5 ns in the 400–500 nm region, then remains constant, while that ofB. subtilis spizizenii decreases from a lifetime of ap-proximately 5.5– 4.3 ns, then maintains that con-stant value. The behavior of B. subtilis is similar tothat of B. atrophaeus in the short wavelength regime,and similarly, exhibits an increase in lifetime forlonger emission wavelengths; however, the increaseis only to about 3.6 ns, rather than the 5 ns of B.atrophaeus. The TI fluorescence spectra of B. subtilisand B. globigii (Fig. 4) and of B. atrophaeus and B.subtilis spizizenii (Fig. 5) are presented.

Fluorescent decay lifetimes were taken for other,nonbacterial materials: Meadow Oat pollen, Mulberrypollen, cigarette ash, and Arizona road dust are re-ported in Fig. 6. Meadow Oat has a fairly constantvalue of about 4 ns, while Mulberry pollen exhibits aslightly increasing lifetime of around 3 ns. The life-times of the cigarette ash are relatively constant at3.5 ns until they decrease very quickly above 570 nmto 2 ns. Arizona road dust similarly decreases, al-though more gradually, above 530 nm to about1.8 ns, but also exhibits short lifetimes at wave-lengths below 400 nm. Figure 7 shows the pollens’ TIspectra, and Fig. 8 shows those of cigarette ash andArizona road dust. Figure 9 shows the lifetime spec-tra for the inorganic materials ammonium sulfate

Fig. 2. B. globigii fluorescence decay lifetimes (�) for samplesadulterated with nutrient and other contaminants and (□) for�99% vegetative cells. The error bar on the left is representative oftypical variations in multiple measurements (see text).

Fig. 3. Fluorescence lifetimes of (�) bacteria B. atrophaeus, (□)B. subtilis, and (Œ) B. subtilis spizizenii.

Table 1. List of Samples

Material SourcePurity

(%)

B. globigii U.S. Army ECBC �99B. subtilis

spizizeniiATCC 6633 �99

B. subtilis ATCC 29056 �99B. atrophaeus ATCC 9372 �99Arizona road dust U.S. Army ECBC �99Mulberry pollen Greer Laboratories # 113 �95Meadow Oat

pollenGreer Laboratories # 19 �95

Ammoniumnitrate

Sigma-Aldrich �99

Ammoniumsulfate

Sigma-Aldrich �99

Carbon black Sigma-Aldrich �99.99Cigarette ash Volunteer 100

6636 APPLIED OPTICS � Vol. 45, No. 25 � 1 September 2006

and ammonium nitrate; however, the time-resolvedtraces recorded were very noisy and the lifetime fits,relatively speaking, poor. Figure 10 shows the TIemission spectra for completeness. Carbon black didnot exhibit fluorescence under 337 nm excitation.

5. Discussion

A number of considerations must be examined to eval-uate the fluorescence lifetime spectrum approach as aviable method for detecting and identifying biologicalmaterials. Because the measured lifetimes are in thenanosecond range, this technique cannot be useful forremote detection of dispersed clouds of these materi-als; the efficacy of the lifetime measurements as ameans of identifying biological materials is clearly lim-ited to point detection. A second concern is that thebacterial samples could be adulterated with someother fluorescent materials. Figure 2 shows the life-time spectra of B. globigii as it was received (withnutrient and other impurities) and after separation ofthe vegetative cells. The error bars were generated asdescribed in Section 2. By virtue of the lack of overlapin the two spectra at mid- and long-emission wave-lengths, this particular mixture of B. globigii with nu-trient seems to be distinguishable from the puresamples. Obtaining the upper curve of Fig. 2 is then afalse-negative result for a test for the presence of B.globigii. Clearly, this implies that adulteration of thesample will mask the true decay lifetimes of any ma-

terial, thus precluding this technique from being usedon unprepared bulk samples. However, if sufficientmaterial is available, separation of the biological com-ponents from the adulterants may be feasible. Alter-natively, Pinnick et al.2 have developed a device thatcan examine individual airborne spores and can, inprinciple, be adapted to make use of the lifetime mea-surement method.

Normally, the fact that the decay parameters ofB. globigii are constant would suggest that only onematerial is fluorescing. Pinnick et al.2 listed a numberof common fluorescent materials in biological samples[tyrosine, tryptophan, reduced nicotinamide adeninedinucleotide (NADH), and riboflavin], and indeed mea-sured their TI emission spectra under 266 nm excita-tion, while Faris et al.12 examined spectra of actualbacteria under a number of conditions. A brief com-parison with the literature is informative. Tyrosinecannot contribute to the spectra measured in thisstudy in that, even at a 266 nm excitation, its emis-

Fig. 4. TI emission spectra of (Œ) B. globigii and (□) B. subtilis.The peaks of the curves have been normalized for easy comparison,but no correction has been made for the spectrometer gratingresponse.

Fig. 5. TI emission spectrum of (□) B. atrophaeus and (Œ)B. subtilis spizizenii (normalized).

Fig. 6. Fluorescence lifetimes of (�) Meadow Oat pollen, (□) Mul-berry pollen, (Œ) Arizona road dust, and (�) cigarette ash.

Fig. 7. TI emission spectra of (□) Meadow Oat pollen, (�)Mulberry pollen, and (�) B. globigii (normalized).

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sion is small above 400 nm. Tryptophan possesses alifetime of 2.6 ns under a 295 nm excitation13 andmay contribute to the emissions below 450 nm. How-ever, neither tryptophan nor tyrosine absorb stronglyat 337 nm. NADH has approximately the same emis-sion spectrum under a 355 nm excitation as for266 nm (Ref. 14); however, with measured lifetimesof less than 1 ns, it is a weak candidate for being asole emitter in these systems. Riboflavin exhibits alifetime of approximately 3 ns (Ref. 15) in the emis-sion range of 500–650 nm, and so is a good candidatefor that region. It should be kept in mind, however,that the dips in the TI emission spectra seen at ap-proximately 450 nm may be almost entirely due tothe monochromator grating response and should notbe interpreted as evidence of two or more emitters.

A comparison of the other three bacteria yieldspromising results. From the decay lifetime spectrain Fig. 3, B. atrophaeus and B. subtilis spizizenii areeasily distinguishable from each other and from B.globigii (Fig. 2). At short-emission wavelengths, thelong decay times ��5 ns� of B. subtilis spizizenii dis-tinguish it from all the other materials in this study.At wavelengths greater than 500 nm, the long life-times of B. atrophaeus ��5 ns� easily distinguish itfrom B. globigii with lifetimes of approximately 3.5ns, a separation sufficiently larger than the esti-mated uncertainty of the measurements. At shortwavelengths, the observed lifetimes of B. subtilis areidentical, within the resolution of this system, tothose of B. atrophaeus. In the region of 500 nm, how-

ever, there is sufficient separation of the curves��1 ns� to discriminate between the two. It is muchmore difficult to differentiate B. subtilis and B. glo-bigii, which possess similar lifetime spectra in theshort-wavelength to midwavelength region, with aslight divergence at long wavelengths that is barelylarger than the resolution of the current apparatus.This potential ambiguity in the identification of thesetwo bacteria can be removed by examining the TIemission spectra (Fig. 4). Here, the two bacteria differmarkedly at short wavelengths. In summation, eachof the four bacteria studied is distinguishable usingthe time-resolved fluorescence technique.

As seen in Fig. 5, the TI emission spectra of B.atrophaeus and B. subtilis spizizenii are similar;B. atrophaeus exhibits slightly more fluorescencearound 400 nm, but is otherwise identical with B.subtilis spizizenii. This demonstrates that the TIemission spectra alone may be insufficient for accu-rate identifications, and that the lifetime spectra areuseful.

Fluorescent decay lifetimes for Mulberry andMeadow Oat pollens are shown in Fig. 6. Althoughthe two pollens exhibit similar flat lifetime spectra,their values are separated by more than the uncer-tainty in the system. However, B. globigii has a sim-ilar lifetime spectrum (Fig. 1) that lies immediatelybetween those of the two pollens and may be confusedwith either. The additional information provided bythe TI emission spectra (Fig. 7) resolves any possibleambiguity. In a similar manner, the lifetime spec-trum of Mulberry pollen, although slightly fasterthan that of B. subtilis, does not differ by an amountmuch greater than the uncertainty of the measure-ments. Comparison of their TI emission spectra (re-plotted in Fig. 11) again removes any ambiguity.

Cigarette ash and Arizona road dust (Fig. 6) bothexhibit rapidly decreasing decay constants at longerwavelengths. While cigarette ash is very similar to B.subtilis in terms of its decay spectrum for wavelengthsbetween 380 and 570 nm, their behaviors above 570nm are very different, and the materials are easilydistinguished. Additionally, Fig. 8 indicates that the TIemission spectrum for cigarette ash is very differentfrom that of B. subtilis (Fig. 4). The very short decayconstants of Arizona road dust seen at short-emission

Fig. 8. TI emission spectra of (Œ) Arizona road dust and (□)cigarette ash (normalized).

Fig. 9. Fluorescence lifetimes of (□) ammonium nitrate and (�)ammonium sulfate.

Fig. 10. TI emission spectra of (□) ammonium nitrate and (�)ammonium sulfate (normalized).

6638 APPLIED OPTICS � Vol. 45, No. 25 � 1 September 2006

wavelengths uniquely distinguish it from all other ma-terials in this study; its TI emission behavior is in-cluded in Fig. 8 for completeness. Figure 9 shows thelifetime spectra for the inorganic materials ammoniumsulfate and ammonium nitrate. As might be expected,the curves are similar to each other, but are differentfrom the other materials; however, the time-resolvedtraces recorded were very noisy, resulting in poor life-time fits and a corresponding increase in the estimateduncertainty to about �0.6 ns.

6. Conclusion

Emission wavelength-dependent, time-resolved fluo-rescence lifetime measurements appear to have prom-ise as a method for identifying and distinguishingsome varieties of bacteriological materials from eachother and from similarly appearing materials, such aspollens. A database for the fluorescence lifetime decayspectra of four species of bacteria that possess at leastpassing similarities to B. anthracis, as well as for othernonbacteriological materials, has been constructed.Sufficient differences are evident among these fourbacteria such that identification can be made with con-fidence. TI emission spectra add an additional compo-nent of information that may be used to remove anyambiguities and, with an excitation source at 337 nm,are a useful complement to the work already per-formed by Pinnick et al.2 In principle, the two typesof measurement could be performed simultaneouslyif a streak camera were to be used as the detector;indeed, all emission wavelengths couldin this manner be recorded at once, obviating theneed to scan a monochromator, and thus greatlyreduce the time necessary to conduct these mea-surements. Future work should include similarmeasurements with different excitation wave-lengths, such as 266 nm, which would provide moreinformation than the current study in that (i) moreemission wavelengths (e.g., 285–640 nm) could be ex-amined, and (ii) higher energy excitation should re-sult in different contributions of emissions from thevarious lumophores within the cells that will be

reflected in changing decay time constants. Shorterexcitation pulses would make possible the deconvo-lution of the several lifetime contributions to the de-cay curves, possibly allowing identification of theindividual lumophores.

This research was funded in part by the U.S.Army’s Edgewood Chemical and Biological Center,Aberdeen Proving Ground, Maryland 21010, and bythe Dean’s Office of Goucher College. The authorsthank Azusa Tananka for her assistance with thesample preparation.

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Laboratory Response Network,” Centers for Disease ControlandPrevention,revised28January2001,http://www.bt.cdc.gov/agent/anthrax/lab-testing/approvedlrntests.asp.

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6. PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin,Germany. Available in the U.S. from Toptica Photonics, Incor-porated, 94 North Elm Street, Suite 101, Westfield, Mass.01085.

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14. J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, and M. L.Johnon, “Fluorescence lifetime imaging of free and protein-bound NADH,” Proc. Natl. Acad. Sci. USA 89, 1271–1275(1992).

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Fig. 11. TI emission spectra of (□) B. subtilis and (�) Mulberrypollen (normalized) replotted.

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