Optical techniques for detecting and identifying biological-warfare agents

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Citation Greenwood, D.P. et al. “Optical Techniques for Detecting and Identifying Biological-Warfare Agents.” Proceedings of the IEEE 97.6 (2009): 971-989. © 2009 IEEE

As Published http://dx.doi.org/10.1109/JPROC.2009.2013564

Publisher Institute of Electrical and Electronics Engineers

Version Final published version

Citable link http://hdl.handle.net/1721.1/59348

Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. INVITED PAPER Optical Techniques for Detecting and Identifying Biological-Warfare Agents Electro-optical techniques, such as laser-induced fluorescence, can detect and help to identify bio-agents, but laboratory assays are needed before taking medical treatment measures.

By Darryl P. Greenwood, Fellow IEEE, Thomas H. Jeys, Bernadette Johnson, Jonathan M. Richardson, and Michael P. Shatz

ABSTRACT | Rapid and accurate detection and identification of I. INTRODUCTION biological agents is an objective of various national security The defense against the use of biological agents is a programs. Detection in general is difficult owing to natural national security [1] and homeland defense objective [2]. clutter and anticipated low concentrations of subject material. Timely and accurate detection of biological agents is an Typical detection architectures comprise a nonspecific trigger, essential component of the defense because it enables a rapid identifier, and a confirming step, often in a laboratory. protective measures such as masking, evacuation, avoid- High-confidence identification must be made prior to taking ance, and early medical treatment. For technical reasons, action, though this must be traded against regrets stemming detection is exceedingly difficult, since very small quan- from delay. Sensing requirements are best established by tities are infectious and because benign biological matter is positing plausible scenarios, two of which are suggested common in the environment. Understanding the capabil- herein. Modern technologies include the use of elastic scatter ities and limitations of bioagent detection is the subject of and ultraviolet laser-induced fluorescence for triggering and this paper, which surveys the state of the art of optically standoff detection. Optical and nonoptical techniques are used based bioagent detection and gives a prognosis for this routinely in analyzing clinical samples used to confirm infection capability into the future. and illness resulting from a biological attack. Today, environ- Use of biological agents as a means for defeating mental sensing serves at best as an alert to medical authorities enemies has persisted through the centuries [3]. Following for possible action, which would include sample collection and scientific breakthroughs such as the understanding of the detailed analysis. This paper surveys the state of the art of germ theory of disease by Koch in the late nineteenth sensing at all levels. century, bioweapons found increased emphasis, with numerous nation-state programs existing throughout the KEYWORDS | Biological agents; fluorescence; medical diagnos- twentieth century [4], [5], and some into the twenty-first tics; particles; scatter; standoff sensing [6]. Over time, the more that was learned of the potential for bioagents to cause disease and death, the more repug- nant such potential became to most civilized countries and governments. The United States pursued offensive bio- warfare from 1941 until 1969, when President Nixon Manuscript received July 1, 2008; revised October 17, 2008 and December 8, 2008. abruptly terminated the program [7], [8]. Entered into Current version published May 13, 2009. This work was sponsored by the U.S. Air Force under Contract FA8721-05-C-0002, in part by the Defense Threat Reduction Agency force in 1975, the Biological and Toxin Weapons Conven- and the Department of Homeland Security. tion (BTWC), which bans the development, stockpiling, The authors are with MIT Lincoln Laboratory, Lexington, MA 02420 USA (e-mail: [email protected]; [email protected]; [email protected]; production, and use of such weapons, has been ratified by [email protected]; [email protected]). 140 countries. President Gorbachev admitted in 1989 that Digital Object Identifier: 10.1109/JPROC.2009.2013564 the former Soviet program had continued well past the

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date of signing the BTWC [9]. Currently no country openly Prevention1 or to the U.S. Army’s Textbook on Military admits to having a current offensive biowarfare program. Medicine [26]. In October 2001, the United States experienced its most Of importance to this paper are the characteristics of severe bioattack when a perpetrator mailed anthrax spores these agents, since it is the purpose of detection to recog- to the news media and the Congress, resulting in 22 ca- nize microbes for what they are. Physical characteristics sualties, including five deaths [10]. Analysis suggests the include external features such as shape, morphology, and quantity of material used, if dispensed more efficiently, epitopes (particularly antigens and other proteins), sugars, could have caused significantly more casualties. The events and fundamental amino acids. Internal features include of 2001 suggest that in addition to concerns over nation- DNA and RNA. Some techniques look for elemental state bioweaponry, the United States should be concerned makeup at the atomic level. Additional matter may appear over biological agents used by terrorists [11]–[13]. Because in the analyte, including natural biologics and inorganics. of the diversity and potential impact of the threat, the Optics and electro-optical sensors play a role in all of these United States and many other countries maintain defensive approaches. military biological programs, aimed at detecting attacks andprotectingintendedvictimsfromharm[14],[15]. B. Detection and IdentificationVBroadly Speaking A key element of a defensive program is the detection A. The Threat and accurate identification of biowarfare agents at time of A biological agent attack could occur anywhere and use and in postattack. Detection and identification (ID) are through a variety of venues, including food, drinking essential to the defense because they enable individual and water, insect vectors, and air [16]. (This paper does not collective protection, correct and timely medical treat- address attacks by injection or dermal application, nor ment, and identification and apprehension of the perpe- vectors, though one such attack was documented during trator. Regrettably, bioagent detection and ID are difficult World War II [17].) An aerosol attack with biological to achieve for a number of reasons. agents would work optimally as a fine mist of 1–5 m-sized 1) The attack could occur anywhere and through any particles [18] since particles in this size range find number of physical routes. optimum inhalation and retention. According to Pearson 2) Small quantities of material can infect and cause [19], as few as one microbe and as many as 10 000 can great harm. infect an individual, suggesting that aerosol clouds can be 3) Significant benign biological matter exists in the incredibly sparse, thus stressing the limits of detection. environment. Food and water are at risk due to a long and vulnerable 4) Any one of a number of agents could be used. supply chain [20], [21]. Thankfully there is motivation to 5) Natural disease-causing agents could be em- keep the supply safe, and technologies are employed to ployed,thusmaskingwhethertherewasanattack provide security and to sense the presence of contami- or a natural event. nation. An attack on a food supply, as occurred with 6) Bioagents can be engineered to mask detection Salmonella typhimurium in The Dalles, Oregon, in 1984, and delay correct treatment. would be considered today an act of terrorism [22]. On the positive side, disease caused by these agents can Though none of the 751 victims died in that attack, others often be treated if diagnosed or detected in time and if have suggested more potent means of widespread food there are sufficient therapeutic measures available. For all poisoning [23]. Additional research and development are these considerations, it is important to address attack needed to improve the safety of the food and water supply scenarios that are potentially realizable while emphasizing system, including means of improved detection. those incidents that can cause the greatest harm. According to Franz et al. [24] any of a number of bio- The fact that a bioattack may appear as a fine aerosol agents might be employed, ranging from bacteria such as suggests that optical approaches are of great relevance Bacillus anthracis (the causative agent of anthrax), brucella for direct detection. As depicted in Fig. 1, a typical (numerous species), Yersinia pestis (Bthe plague[), and aerosol-detection architecture comprises early warning Franciscella tularensis (tularemia) to viruses such as variola (a Btrigger[), some form of sample collection, an early (the causative agent of smallpox) and Ebola. Also of con- stage identifier, and a late stage confirmer (also an iden- cern are a number of biotoxins, including Staphylococcus tifier, though typically with an alternative technology). Enterotoxin B and botulinum, both of which were re- Early-warning sensing can comprise point detectors that searched in the now-defunct U.S. offensive program. sample the aerosol, or standoff (i.e., remote) sensors that According to Zilinskas [25], Iraq researched and developed scan the air at a distance. In both cases, the sensor assesses anthrax, perfringens, botulinum, aflatoxin, numerous vi- the probability that the suspect air contains threat agent. A ruses, and ricin, though few were carried to a weaponized Bpoint[ trigger sensor is typically a particle counter of state. For a more complete list and understanding of the some sort, with techniques including elastic scatter, physical and medical characteristics of potential bioagents, we refer the reader to the Centers for Disease Control and 1See www.emergency.cdc.gov/agent/agentlist.asp.

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Fig. 1. A typical aerosol-detection architecture, which comprises early warning (a ‘‘trigger’’), some form of sample collection, an early stage identifier, and a late stage confirmer (also an identifier, though typically with an alternative technology). laser-induced-breakdown spectroscopy, fluorescence spec- approaches. As with all detection techniques, immunoas- troscopy, polarization scattering, and Raman spectroscopy. say and PCR are limited by backgrounds, interferents, and Standoff techniques, currently limited to lidars that inter- false readings. rogate clouds of particles, have been developed to measure The objective of the sensing and identification archi- elastic backscatter and fluorescence. While other tech- tecture is to provide sufficiently accurate and timely warn- niques have been considered (including microwave and ing of an exposure so that protective actions can be millimeter wave), the most promising remain optical in employed. Current approaches are limited in sensitivity, nature, since longer wavelength techniques are less sen- timeliness, and accuracy; thus there is room for innovation sitive or less discriminating. Given the presence of back- and advances. In particular, trigger sensors are by design ground aerosols, all such techniques are subject to false generic, trading speed for specificity. A trigger sensor that alarms, and given various limitations in atmospheric pro- could not only detect the presence of potential pathogens pagation (attenuation and scattering), these are invariably but also identify the species and pathogenicity would be range-limited. An additional limitation on point and stand- highly desirable since this would enable rapid and effective off optical sensors is their inability to discriminate live protection and treatment. This paper addresses the state of versus dead microbes, thus opening the potential for false the art and what improvements in the sensors are needed alarms, though this potential objection can also be seen as to provide the needed defense at an affordable level. an advantage in that even a failed attack (e.g., with dead bacteria) would be detected. Optical systems play an important role in identification II. REQUIREMENTS FOR DETECTION of the threat agent, wherein a sample is collected from TECHNOLOGIES AND APPROACHES either the air (or food or water) or infected human subject. To determine requirements for sensing biological agents, Sampling can be triggered by an event such as a positive hit one needs to consider the operational use of such sensing. in an early-warning sensor or presentation of an ill person One must consider not only credible attack scenarios and in a doctor’s office or hospital. In either case, the material specific threat use but also how the information produced is collected and provided to one or more in a class of by the sensors supports a response to obviate, or at least identifiers. The gold standard identifier for many hospital reduce, the effects of the attack and the constraints on the labs and physicians is culture, with optical microscopy the deployment of sensors. There are many possible responses method used to quantify and assess growth of the suspect to a biological attack: avoiding the contaminated area, agent. Culture is, however, slow (days to weeks) and personal and collective physical protection, medical pro- limited in general use since the biomaterial being assayed phylaxis and treatment, and decontamination may all be must be consistent with the culture medium used. More employed either singly or in conjunction. modern techniques include immunoassay and polymerase The different response options impose different re- chain reaction (PCR). Both are much more rapid than quirements on the sensors. For example, measures to avoid culture, with response times in the tens of minutes to exposure require that the sensor detect the attack before it hours. Immunoassay involves the binding of target anti- reaches the people to be protected and respond quickly gens to immobilized antibodies, with binding signaled by enough that protective action can be completed before the an optical or color change (e.g., the physician’s strep test agent reaches the vulnerablepopulation.Ontheother kit). PCR involves a binding of DNA sequences of the hand, a response of treating the population of an urban sample with a known genetic sequence. Many organiza- area does allow time, at least a few hours, for chemical tionsareworkingtoreducePCRtimestoaslittleasa tests (immunoassays or PCR) to detect and identify the minute, with labs-on-a-chip and micropore technologies as agent; however, it has an extremely low tolerance for false

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Fig. 2. Effectiveness of different response options for reducing the impact of an attack in an office building. Plotted are the ratios of the number of lethal exposures expected for a variety of responses relative to those that would occur with no response. Above the zero line are the lethal exposures internal to the facility; below the line are those outside. Note that, in some cases, a response can actually increase the number of fatalities (values > 1). The figure was calculated using a CONTAM model for a low-rise office building. alarm and requires that the agent be identified with response to occur within a few minutes after the attack, enough specificity to determine the correct treatment. and the evacuation to occur within 15 min or so; sensor (The assumed response time of a few hours in this latter responses must be rapid enough to support this. Fig. 2 case is predicated on the assumption there are treatments, shows the ratio of the likely number of lethal exposures available and approved by the FDA. This is generally the that would occur with the indicated response to the case for a bacterial agent, provided potential antibiotic re- number that would occur if the incident were undetected. sistance can be identified. However, viruses and toxins The figure was calculated using a CONTAM2 model for a have few tested approved treatment modalities other than low-rise office building. The tolerance for false alarms in supportive care. For all agents, treatment of a large this scenario is quite low; based on discussions with a populace, with a certain fraction of immunocompromised number of building operators, typical values are about one individuals, is problematic and will heavily stress the per month for HVAC or other changes to slow the spread public health system.) of agent, and something significantly less than once per Wewillfocusontwoscenariosinthissection:ado- year, perhaps once per century, for an evacuation. The mestic facility protection scenario and a military scenario example of fire alarms may suggest a higher tolerance for defending troops on the battlefield. In each case, we re- evacuations, but in the case of fire alarms, there are cognize that the conditions of the release, that is, the techniques for giving a rapid Ball clear[ without causing agent, mass, location, wind, and weather conditions are undue panic. This is unfortunately not the case currently largely under the control of the attacker. Thus, rather than for alarms of biological incidents. The sensitivity require- analyze specific attacks, we generally prefer to analyze ments are more contentious; very small concentrations can classes of attack and attempt to design or develop a system pose a hazard for a population breathing them in over a that protects against as large a fraction of the attacks in the period of an hour or more indoors, and someone wanting class as practical. to preclude such harm would desire sensor sensitivities far For the first type of scenario, we will consider an attack better than can currently be achieved. However, an attack on a domestic facility, which could be a building or using what would seem to be plausible amounts of transportation facility. The responses that we have found material, say, between 1 g and 10 kg of aerosolized agent, to be most effective in a series of analyses have been some released in a brief period, typically results in concentra- facility-dependent response, such as an HVAC shutdown, tions ranging from tens of agents containing particle per to slow the spread of the agent, followed by an evacuation if the attack is confirmed. Typically one would like the first 2www.bfrl.nist.gov/IAQanalysis/.

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Fig. 3. Latency of potential sensor networks with a probability of detection of 80% as a function of the threshold. Uses same model as Fig. 2 with 1 g releases and shows how the time between the release and point where the concentration at one of the sensors reaches its threshold varies with sensor threshold and density. Sensors are deployed in key portions of the HVAC system, large spaces, and along the corridors of the building.

liter of air (ACPLA)3 to many thousands of ACPLA. An and weather conditions, sensors with limits of detections example is shown in Fig. 3; this figure uses the same model (LODs) ranging up to 500 particles per liter or so have asFig.2with1greleasesandshowshowthetimebetween some utility; sensors with LODs of single particles are the release and point where the concentration at one of the probably unnecessary. To achieve a high Pd in high winds, sensors reaches its threshold varies with sensor threshold the sensors must be close together. The tolerable false- and density. alarm rate depends on the level of perceived threat. If the The second type of scenario is an off-target attack on perceived threat level is very high, false alarms of once troops in the field. Their response in case of an alarm is to every few days or weeks might be tolerable; if the per- employ personal protection (suits and other protective ceived threat level is low, the tolerance for false alarms will gear) and collective protection (closing the hatches on the decline proportionately. vehicles and activating the filters). These responses can Of course, these scenarios do not come close to ex- take about a minute in favorable cases once a sensor sounds hausting the applications of optical sensors. For example, an alarm; this time includes time for a decision to be made, detection of contaminated surfaces, which can be impor- commands to be relayed to the troops, and practiced troops tant both for avoidance and decontamination efforts, to don the gear and engage the collective protection sys- would benefit strongly from optical-based sensing. In tems. The sensors need to be placed reasonably close to the addition, optical sensors are also used to limit the use of protected troops to preclude the attack’s being released consumables in biochemistry-based sensors and to provide downwind of the sensors yet upwind of the troops. The a signal transduction or amplification for biochemical sensors should have rapid responsesVon the order of one binding or recognition. Examples of these applications will minute or less. Alternatively, the sensor could be a standoff be described in subsequent sections. sensor located with the protected troops; in that case, the reaction time requirement will be more stringent if the detection range is short and can be relaxed for long range III. DETECTION AT A POINT detections, since the cloud will take longer to reach the troops. A. Rapid Detection An example of a point sensor deployment is analyzed in The rapid detection of an aerosolized biological agent Fig. 4, which shows contours for 95% network Pd, the dramatically improves the ability to mitigate the effects of probability of detection, for an ensemble of HPAC [27] such an attack. Optical detection is presently the fastest plumes, for a variety of sensor sensitivity and spacings. means for sensing the presence of aerosolized biological These curves represent sensitivity thresholds; anywhere agents and can be divided into individual-particle and above the curves represents aerosolized masses that can be multiple-particle detection. Individual-particle detection is detected at the various thresholds. For a variety of attacks characterized by the detection of signals from one particle at a time, while multiple-particle detection is character- 3An ACPLA is a somewhat imprecise unit, though widely used in this ized by the detection of integrated signals from multiple field. particles. These two categories are exemplified by flow

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Fig. 4. Plots of the minimum amount of aerosolized agent detectable as a function of the wind speed for a linear array of sensors. The curves are for sensors of different limits of detection. The agent release is 2 km upwind of the sensor array. Contours represent different concentration thresholds at a detection probability of 95%. For the 500 m separation at high wind speeds, there is more than a 5% chance that no agent reaches the sensors on either side of the plume, so no level of increased sensitivity would achieive 95% Pd; more sensors are needed. cytometry [28] and lidar (see Section IV). For attacks in ambient particle concentration greatly exceeds the desired which the agent concentration is significantly less than the detection concentration of biological agent. This disparity ambient background concentration, individual-particle requires that the sensor be very good at discriminating detection yields better detection and discrimination of background particles (clay, diesel particulate, pollen, mold, agent particles than multiple-particle detection because etc.) from bioagent particles in order to achieve a low false- the agent particle signals are not mixed with background positive rate. For example, in an urban environment the particle signals. concentration of particles greater than 1 mdiametermay Flow cytometry typically involves the laser illumination range from 1000 to 100 000 per liter depending on many of individual particles traveling in a liquid stream and the parameters (e.g., time of year, traffic conditions). detection of the resulting elastic scattering [29] and The design of a real-time single-particle optically based fluorescence [30] in order to detect and discriminate one biological-agent trigger involves two basic considerations. type of particle from another type of particle. Real-time First, individual aerosol particles must be characterized individual-particle detection of aerosolized biological well enough to discriminate threat-like and non-threat-like agents differs from traditional flow cytometry in two particles. Secondly, the threat-like particle concentration ways. First, the particles are in air, not liquid; and secondly, must be characterized well enough to support a reliable the particles are not modified with fluorescence enhancing trigger threshold that maximizes the probability of chemicals, so only the natural fluorescence from the detection while minimizing false positives. The ability to particle is detected. Because the natural fluorescence from discriminate different types of particles depends upon both unmodified aerosol particles is much less intense and thenativedifferencebetweenparticletypesforthemea- specific than that from fluorescence-enhanced particles, sured properties and the signal-to-noise ratio of these real-time optically based biological agent detectors are measurements. The ability to characterize the threat-like much less discriminating than typical flow cytometers. On particle concentration depends upon the detector air sam- the other hand, these biological agent detectors are more ple rate, the time available to make a concentration mea- rapid and do not require a supply of chemicals that are often surement, and the level of clutter in the environment. toxic. Because elastic scattering and natural fluorescence- The ability to optically detect and discriminate based detection are not sufficiently specific, detectors particles is partially determined by the amount of light based on these phenomena are used not to identify bio- that the particle emits either as elastic scattering or as agents but rather to alert the presence of a threat-like fluorescence. Equation (1) gives the number of detected aerosol. Depending on circumstances, this alert can be photons ðNpÞ that are emitted by a particle, with an optical used, for example, to activate a separate identifier system or cross-section , which is illuminated by a light beam with a to divert air flow in a building ventilation system. The key power of P in a cross-sectional area A for a time .The figures of merit [31] for a biological agent detector are then photon collection and detection efficiencies are c and d sensitivity (minimum detectable agent concentration), pro- respectively, h is Planck’s constant, c is the speed of light, bability of detection, false-alarm rate, and response time. and is the wavelength of the illuminating light

B. Design Considerations An important consideration for bioagent sensors is the P N ¼ : (1) environment in which they will operate. Typically the p c d Ahc

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The number of particles detected (n)isgivenby(2), particles in the measurement threat region dominates the where C is the aerosol particle concentration, T is the number of actual threat particles. time available for counting particles, and is the air sam- Fig. 5 shows a simplified schematic for a real-time ple rate optically based bioagent detector. Light from a source of cross-sectional area d2 is imaged into a sample volume ðd3Þ through which ambient air passes. Particles that pass n ¼ CT: (2) through the sample volume scatter light elastically and emit fluorescence. Some of this radiation is directed to a photodetector with an area d2. Forcontinuoussampling,the air sample rate is given Combining(1)–(3),wecansolveforthedetectable by (3), where A is the cross-sectional area of the sample bioagent concentration as a function of the incident optical volume, L is the length of the sample volume, and is the power, as shown in (4). The light beam cross-sectional area time required for a particle to transit the sample volume. (A or d2), the particle transit time ðÞ through the sample The effective air sample rate (also referred to as volume, and the air sample rate have dropped out of this responsivity) can be calculated by dividing the particle equation count rate by the particle concentration

n Np hc = = : C ¼ : (4) ¼ AL ¼ n ðTCÞ (3) LT cd P

The design of a biological agent detector involves a In addition to elastic scatter, a variety of inelastic compromise between maximizing the signal from each scatter techniques have been pursued, the most prominent particle that enters sample volume and minimizing the of which is laser-induced fluorescence. Biological matter detectable agent concentration. For a given light power, fluoresces due to the presence of aromatic amino acids the maximum particle signal is obtained by focusing that (tryptophan, tyrosine, and phenylalanine), which fluoresce light into the smallest possible cross-sectional area A so as in response to excitation in the near ultraviolet (UV) to increase the particle illumination intensity. However, as (around 260–280 nm), and nicotinamide adenine dinu- thesamplevolumedecreases,theminimumdetectable cleotide (NADH), which can be excited using a relatively concentration increases. broad range of UV wavelengths in the 300–360-nm range. It is rarely possible to perfectly discriminate ambient For a fluorescence-based single-particle detector, Fig. 6 background particles from agent particles. Typically, there shows the detectable concentration as a function of optical is some small fraction of the background aerosol that power for a signal-to-noise ratio of ten and for illumination resembles the agent particles. These particles are referred wavelengths of 280 and 340 nm assuming the values for to as clutter. In a low clutter environment, the number of the other variables as shown in Table 1. The fluorescence threat-like particles in the measurement threat region do- cross-section ðÞ, for a given particle size, is one of the key minates the number of clutter particles in the threat re- parameters that determine the minimum detectable gion. In a high clutter environment, the number of clutter particle concentration. The fluorescence cross-section of

Fig. 5. Schematic of generic optically based biological agent particle detector.

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Fig. 6. Calculated detectable particle concentration utilizing light induced fluorescence as a function of optical power for 280- and 340-nm illumination wavelengths. See Table 1 for other variables in calculation.

Table 1 Variables Used in Calculation of Fig. 6

biological particles has been measured by several groups tion, this technique alone has not proven to be very useful [32]–[35] and the values in Table 1 are typical for 1 m for detecting anomalous aerosols that are a minor con- Bacillus subtilis spores. However, it should be noted that stituent of the background aerosol. the cross-section strongly depends on the method of In 1993, Ho et al. [39] showed that the native preparation. fluorescence from individual biological particles could be detected. In this work, they demonstrated the fluores- C. Historical Developments cence detection of Bacillus subtilis spores in a liquid flow The development of real-time optically based bioagent cytometer. They also suggested that this native fluores- sensors goes back to the early 1990s. Yee and Ho [36] cence could be used to directly detect bioagents in the demonstrated that it was possible to detect anomalous atmosphere. In 1996, Ho [40] reported the detection of aerosols by temporally monitoring the concentration and aerosolized individual bioagent simulant particles utiliz- particle-size distribution of the ambient aerosol via an ing an elastic-scattering-based detector [41] that was elastic-scattering-based particle detector (see also modified to include a 325-nm induced fluorescence mea- Stroud et al. [37]). In 1991, Kaye et al. [38] demonstrated surement. This instrument was referred to as the fluo- the detection and discrimination of anomalous aerosol rescence aerodynamic particle sizer (FLAPS). In many particles based on the elastic-sattering measurement of environments, most background aerosols are composed of particle concentration, size, and shape measurements. The inorganic material that fluoresces very weakly as com- particle shape measurement was based on the asymmetry pared to organic particles. Thus, fluorescence greatly in- of elastic scattering. Spherical particles generate a cylin- creases the optical discrimination of biological agent drically symmetric elastic-scattering pattern, while ellip- particles from background aerosols and reduces the false tical particles generate an asymmetric pattern. The degree positive rate of agent detection. The design and perfor- of asymmetry indicates the degree of ellipticity. While mance of the FLAPS was detailed in 1997 [42]. In 1998, elastic-scattering-based detection can discriminate anom- the FLAPS was modified to be smaller, power efficient, alous aerosols when the concentration of these aerosols is a and field portable, and the 325-nm source was replaced significant fraction of the background aerosol concentra- with a 349-nm source [43]. Development of the FLAPS

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Fig. 7. Optical diagram of BAST showing key features of excitation wavelengths and sensing modalities. has continued [44], and it is now available as a Lincoln Laboratory and has proven itself in many sensor commercial product from TSI Inc.4 competitions. It is now part of the U.S. Joint Biological The original FLAPS influenced the development of Point Detection System (JBPDS), which is manufactured many subsequent fluorescence-based bioagent sensors. by General Dynamics Inc.5 TheJBPDSisthefirstfully These include sensors developed at the Naval Research automated military bioagent detection system. In the Laboratory (NRL), the Army Edgewood Chemical and JBPDS, the BAWS performs the function of cueing an air- Biological Center (ECBC), MIT Lincoln Laboratory, and to-liquid particle collector to begin particle collection for the U.K. Defense Science & Technology Laboratory the biological-agent-identifier subsystem. (DSTL). NRL has focused on the development of sensors for measuring the elastic scattering and fluorescence char- D. Looking Forward acteristics of biological aerosols including multiwave- Efforts to improve biological-agent detectors have focused length excitation of these aerosols [45]–[47]. ECBC has on reducing cost and improving the performance of these focused on the development of low-cost sensors, including sensors. These are objectives that can find themselves in providing the initial funding support for the Biological conflict. MIT Lincoln Laboratory has been developing the Agent Warning Sensor (BAWS) and, more recently, Biological Agent Sensor and Trigger (BAST) [59], [60] to developing light-emitting diode (LED)-based sensors meet the needs of low-cost biological agent detection, and the [48]. DSTL has extended the original work of Kaye et al. Rapid Agent Aerosol Detector (RAAD) [61] to meet the needs and developed sensors based on particle shape and of high-performance biological agent detection. fluorescence detection [49]–[51]. In addition to the FLAPS The BAST utilizes low-cost UV LEDs in place of high- produced by TSI Inc., other companies have also developed cost UV lasers. Fig. 7 shows an optical schematic of the fluorescence-based sensors [52]–[55]. BAST. Particles entering the BAST sample volume are The state of the art in a fielded real-time optically based illuminated by both an inexpensive 820-nm diode laser individual-particle biological agent detector is the BAWS and a 365-nm LED. There are four different measurements [56]–[58]. This detector was originally developed at MIT of the particle light emission (820-nm forward elastic

4http://www.tsi.com. 5www.gdatp.com/products/detection_systems/BAWS/BAWS.htm.

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Fig. 8. Optical schematic of the RAAD.

scattering, 365-nm side elastic scattering, 400- to 450-nm IV. STANDOFF BIO-DETECTION fluorescence, and 450- to 600-nm fluorescence). The 820-nm forward scattering measurement detects the pre- A. Early Warning sence of a particle and gives a measure of the particle size. Standoff detection (i.e., detection at a distance) of The 365-nm induced elastic scatter and fluorescence gives biological agents offers many potential capabilities not additional information with which to discriminate threat easily provided by localized (point) detection systems. Al- and nonthreat particles. though point sensors are a more mature technology offer- The RAAD effort is aimed at dramatically improving ing higher localized sensitivity [62], [63], standoff systems the performance and reliability of fluorescence-based can provide sensitivity over a wide area and may be simpler biological agent triggers (see schematic in Fig. 8). The to deploy where this capability is needed. Standoff systems performance is increased by incorporating up to 14 mea- also provide the capability of mapping the course of an surements that are made on each particle that flows aerosol hazard, allowing for advance warning to downwind through the system. Table 2 lists the series of measure- assets. Mapping and tracking of the hazard, particularly ments taken on aerosol particles by RAAD. These addi- when combined with plume modeling, can help to deter- tional measurements dramatically increase the ability to mine what assets have been contaminated, thus guiding discriminate threat and nonthreat particles, therefore re- treatment and decontamination efforts. Lastly, mapping ducing the false positive rate. Reliability is improved by can provide the capability of determining the point(s) of keeping the optics clean with sheath air flow and by in- origin of an attack, which could be vital for identifying creasing laser lifetime by operating lasers only when those responsible. Ultimately, the best solution for protect- particles are present in the sample volume. RAAD is not ing at-risk locations might be an integrated system that specifically intended to be a low-manufacturing-cost instru- combines high-sensitivity point sensors (trigger and con- ment. However, because of a lower false-positive rate and firmatory) with a wide-area coverage standoff detection improved reliability, it should be a low-operating-cost in- system, creating a system-of-systems with the best possible strument. This is important since much of the cost asso- performance [64]. ciated with bioaerosol triggers comes from the cost of The primary technology that has been exploited is reacting to false triggers and from the costs associated with light detection and ranging (lidar), which has the capa- maintaining the sensor. This approach aims to dramatically bility of mapping a threat in space, usually defined in reduce both of these costs. terms of range, azimuth, and angular elevation relative to

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Table 2 Staged Detection Architecture of the RAAD

the position of the sensor. Range information relies on most simply as timing the return optical signal relative to a transmit pulse. It is useful to note that a round-trip of 1 km at the * E A speed of light requires 6.7 s. Using fast digitizers, it is EðR ; ; Þ¼ L 0 OðRÞTðR~; ÞTðR~; Þ 0 1 ~ 2 0 1 easily possible to sample the return signal at a rate equiv- jRj * * alent to 1 m of range, which most likely exceeds the ambðR ;0;1ÞþthrðR ;0;1Þ (5) requirements for biodetection. Angular resolution is determined by the instantaneous field of view (FOV) of the system, which can be as small as 100 rad, giving a where parameters are defined in Table 3. linear resolution of 0.1 m at 1 km, which exceeds the re- Thegoalofthelidarsensorandalgorithmistodetect quirements. (There are still good reasons to limit the FOV, and map regardless of the values of and * thr amb as discussed below.) TðR ;0=1Þ. Where applicable (e.g., for an aerosol compris- A second technology that has been considered is pas- ing similar particles, such as bioparticles), the backscatter sive infrared (IR) spectroscopy (single or multipixel), coefficient can be related to a cross-section ¼ n,where which offers no range information but has been exploited n is the density (in #=m3)and is the backscatter or primarily as a detector for chemical vapor threats.6 To fluorescence cross-section (in m2=Sr). (Note this is a date, passive IR has demonstrated a sensitivity to bioagents differential cross section as compared with the previously that is > 10 times higher (worse) than can be achieved with lidar [65]; however, if it is to be deployed as a Table 3 Parameters in (5) chemical vapor sensor, it would be very useful to determineitsuseasabiosensoraswell.Therehavebeen recent efforts to combine passive IR with lidar to provide a combined standoff detection system for both bio- and chem-agents [66]. Since lidar shows the most promise for standoff (early warning) detection, we limit our technical descriptions in what follows to this technology alone.

B. Design Considerations The signal for a standard backscatter/fluorescence lidar system (in the units of energy/meter) can be expressed

6http://www.jpeocbd.osd.mil/page_manager.asp?pg=7&sub=15.

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defined total cross section in (1).) It should be noted that methods become increasingly difficult as the transmission the concept of a cross-section is less useful for aerosols that becomes low. For a good review, see [67]. have a broad range of sizes such as dusts. It is difficult to Fig. 9 illustrates that the backscatter coefficient of a determine n from lidar data without additional information potentially dangerous aerosol threat could be small when or assumptions concerning the cross-section. compared to that of ambient aerosols. This also demon- Transmission of radiation to and from a target is a key strates that it is important to have a method to discriminate factor in the performance of a system under specific local biological matter from ambient aerosols since simply conditions. The transmission functions can be expressed as looking for an excess of aerosols will not likely offer suf- ficient information in many cases. It is also clearly ad- 0 1 vantageous to be able to detect a threat near its release ZR point, where the density is highest. * @ A TðR ;Þ¼exp ðÞambðz;Þþthrðz;Þ dz (6) As a starting point, one can envision a standoff system 0 that offers aerosol ranging only, leaving discrimination to other (e.g., point) detectors. The choice of wavelength will be determined by several factors: 1) the backscatter cross- where amb=thrðz;Þ are the ambient/threat extinction section of the threat, 2) atmospheric transmission, 3) in- coefficients (with units of m1) at each point. In the case band ambient light, 4) eye-safety limitations, 5) efficiency of elastic scattering, 0 ¼ 1 and the transmission be- of detectors, 6) availability of sources, and 7) unobserved 2 * comes T ðR ;0Þ. When the threat is small and the (e.g., stealthy) operation. For a typical 1–2 m-sized ambient transmission coefficient is sufficiently constant, aerosol-particle threat, the cross-section decreases dra- the total transmission reduces to matically with increased wavelength [68]. At wavelengths much larger than the particle, the rate of decrease 4 approaches the Rayleigh limit (1= ). In the UV-visible 2 * T ðR ;Þexp 2jR~jambðÞ !ðÞ1 2jRjambðÞ (7) near-infrared (NIR) range, there is a tradeoff between atmospheric transmission, which increases at longer wave- length, and cross-section, which is decreasing at longer where the arrow indicates the limiting case for low wavelengths. At longer wavelengths, the solar background extinction. Under such conditions, and with the additional is also decreasing. Finally, there is the concern of eye assumptions about the relationship of and , (5) can be safety. It is not practical to have all personnel wear laser solved for the total and . These so-called lidar inversion safety goggles at all times when the sensor is operating.

Fig. 9. The ratio of the backscatter coefficient of a 1000 particle per liter (kppl) bioaerosol threat to the backscatter from ambient aerosols * * ðthr ðR Þ=ambðR ÞÞ over a range of ambient conditions (clear to hazy). Even under clear conditions, backscatter from this threat is only 2timesthat of the ambient aerosols and molecules. In hazy conditions, the backscatter from this threat drops well below that of ambient aerosols.

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This leads to the preferred use of a source of wavelength is capable of near-real-time Bgeneric[ (nonspecific) bio- just above 1.5 m over the more commonly available discrimination over a wide area (up to 3 km from sensor). 1.06-m Nd:YAG, which is transmitted and focused by the An additional capability is detection and tracking of human eye. It is also possible to use an ultraviolet source aerosolsVwithout any specificityVout to 15 km. (G 400 nm) where the eye does not transmit and thus the As a standoff signature, fluorescence has the complica- allowed power level is higher than in the visible. The range tion that it is relatively small (when compared to back- at this wavelength is greatly reduced over the NIR, how- scatter) and lies within the solar band. The return signal can ever. Using an NIR source, aerosol releases in the 1 kppl be either collected into a single detector using a broadband (thousand particle per liter) range have been detected at optical filter or dispersed spectrally into multiple detectors ranges up to 15 km under clear conditions. Regarding the using (for example) a grating [73]. Spectral dispersion offers issue of background light, it is important to reject as much the possibility of additional discrimination between aerosol of the out-of-band signal as possible using an optical filter. species in cases where there is adequate signal to interpret It is also advantageous to narrow the instantaneous FOV as the resulting spectrum. The spectra of many biological much as is practical (matching also to the transmitted species are all quite similar. They are broad and contain no light), thus raising the signal over the background. sharp features and, unfortunately, are also similar to that of Detector efficiency is also a consideration. The goal is to diesel exhaust. The problem of ambient light is particularly detect as many of the received photons as possible with difficult in the case of fluorescence. This is due to the minimal additional noise. In UV-visible wavelengths, one relatively small cross-section and because of the broad can use photomultiplier technology, which offers high fluorescence band. In contrast, elastic backscatter occurs at quantum efficiency (QE) and very low dark current. In the the transmitted wavelength, so that the receiver may include NIR, photomultipliers are no longer responsive, leaving the a narrow-band optical filter that rejects a large fraction of the choice between a photodiode (PD) and an avalanche pho- ambient light. In both cases, it is advantageous to reduce the todiode (APD). These can be silicon through about 1.1 m divergence of the transmitted beam and corresponding FOV but must be InGaAs at longer wavelengths (such as 1.5 m). of the receiver as much as possible. Since the received signal is typically small, the APD is Given the limited range of UV, it is advantageous to typically preferred over the PD, allowing for good QE, but combine IR ranging with UV-LIF, creating a standoff sen- suffering from relatively high dark current. A fairly recent sor with at least three channels: UV scatter, UV-LIF, and development has been the photon-counting (Geiger-mode) IR scatter. This is analogous to point trigger sensor tech- APD, which detects a single photon at a time, providing a nologies described in Section III. Such a sensor can map sensitivity that approaches the ideal Bwhite noise[ limit [69]. aerosols at many kilometers and discriminate at shorter ranges (e.g., 1 km) with greatly improved sensitivity in C. Historical Approaches dark conditions. The backscatter return of the two tran- As in point trigger systems, the most widely studied smitted wavelengths can be compared to get a rough de- standoff biosignature is UV laser-induced fluorescence termination of particle size, and the UV-LIF signal can be (LIF) [70]. The principal standoff fluorescence signature is normalized by either or both of the scatter channels. due to the presence of NADH, which can be excited using UV wavelengths in the 300–360-nm range. The fluores- D. Looking Forward cencelifetimeisontheorderof1–2ns;thusitcanbe In the future, it would be advantageous to develop range-resolved with the backscatter signal [71]. Atmo- methods that do not rely on fluorescence, still emphasizing spheric transmission is a major concern in choosing the methods that suggest improved sensitivity and selectivity. excitation wavelength for a standoff UV-LIF system. A Recent work has demonstrated that polarization-sensitive commonly transmitted wavelength is the third harmonic of infrared lidar may be useful for standoff biodiscrimination Nd:YAG (355 nm), since Nd:YAG is a highly reliable solid- [74]–[77]. Polarization lidar is sensitive to the size, shape, state laser source. Other sources have been suggested as and index of refraction of the aerosols and has been ex- providing a higher signal [72]. Some systems have opted to ploited for differentiating ice crystals from water droplets use shorter wavelengths (such as the fourth harmonic of in clouds [78]. One feature of biospecies is that their Nd:YAG, 266 nm), which excite additional flurophores shape, at least in their natural form, is highly regular. Even (such as tryptophan), but this further limits the range/ inaggregateform,theunderlyingshapeofindividual sensitivity of the system. Of course, one could consider species is retained. The question of whether this signature using multiple excitation wavelengths, but it is not clear is sufficiently selective must be answered through field that the payoff would justify the additional complexity. and laboratory measurements. Another emerging technol- For the past several years, the DoD has been develop- ogy is multiwavelength differential scatter (DISC) in the ing the lidar-based Joint Biological Standoff Detection 9–11-m range [79]. This technology has been demon- System.7 The goal of this effort is to produce a system that strated for detecting chemical vapors and has recently shown promise as a biodetector as well. The method uti- 7 http://www.jpeocbd.osd.mil/page_manager.asp?pg=7&sub=19. lizes a frequency-agile CO2 laser, which transmits a burst

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of 18 different wavelengths in rapid succession, with posited in Section I) stem from those developed for clinical separate range returns from each. The process is fast enough diagnoses. In particular, acquisition of a sample and sub- that the returns from each wavelength can be directly com- sequent culture and examination provide the basis for the pared, giving a backscatter spectrum as a signature. most definitive agent identificationVit is the Bgold Given the wide variety of ambient conditions and the standard.[ Culture is used not only to provide some initial high infectivity or toxicity of some biological threats, it is confirmation of the suspected agent type (by selection of a probably impossible to develop a standoff system that will compatible culture medium) but also to establish the via- have acceptable performance for detecting small, yet still bility of the suspect agent sample and to provide abundant dangerous, releases. Standoff systems are better suited for copy numbers for subsequent assays. While bacterial detecting releases that would inflict massive casualties if agents will propagate in a variety of media, viral agents countermeasures were not taken. Added benefits include can only propagate in a cell culture; thus some a priori rapid deployment, mobile deployment, wide area cover- suspicion of agent type is usually required to select the age, and hazard mapping. appropriate culture medium. Various aspects of the cul- tured colonies, such as their color, shape, surface features, and growth patterns, are all used to aid in identifying the V. ENVIRONMENTAL AND agent and derive primarily from optical microscopic eval- MEDICAL DIAGNOSTICS uation. Despite the wealth of information obtainable from culture, and despite the reality that it is still the prevailing A. Confirmation diagnostic tool in the medical arsenal, it suffers from a It is tempting to speculate that, if we can detect clouds serious drawback. Results can often take days to acquire, of bioaerosol in open air, then we should be able to use that and those days are either spent not treating a sick person or information to determine how to treat an exposed popu- presumptively (and possibly incorrectly) treating a patient lace. While some medically useful information can be without sufficient information to do so. derived from environmental sensing (such as the extent of One way to address this shortcoming has been the exposure and contamination), one drawback to applying development of automated culture machines that can offer the optical techniques that have been developed for aerosol faster turnaround (G 1 day) and culture of hundreds of dif- detection to the area of clinical diagnostics is the relatively ferent bacterial species as well as determine antibiotic nonspecific nature of the optical signatures of agents resistance to tens of common antibiotics [80], [81]. Many themselves. Fluorescence, Raman spectroscopy, and major hospitals have these machines; while they occupy less Fourier transform IR, for example, are all good candidates space than a microbiology laboratory, they are still out of the for biological/nonbiological discrimination of aerosols and price range and availability for most medical facilities. Other even for classification of agents into likely subgroups, such systems have been developed to provide more rapid bacterial asbacteria,viruses,etc.However,theycannotatthistime culture identification on a more limited basis. These rely on provide the specificity required to effect appropriate treat- introduction of a clinical sample to a liquid culture medium ment. Until such time as new signatures and in vivo optical in a small vial, for example [82]. Changes in the medium techniques are developed, treatment-driving diagnostics color are optically sensed as the microorganisms grow, and will rely on acquisition and assay of a sample (the confir- results are often available within several hours. Again, these mation step). That sample may be an aerosol collection into systems are not commonly used, but their existence suggests liquid or onto a filter, a surface wipe from a contaminated that industry recognizes the need. area, a nasal swab from a suspected exposed person, or a clinical sample, such as blood or sputum, from a person C. Immunoassay presenting with symptoms at a hospital or other point of Immunoassay tests rely on immunological recognition care. In general, the assay techniques to perform identifi- of an infectious agent or protein marker, typically through cation fall in the same few categories regardless of the the use of agent-specific antibodies and some form of sample medium, thus, we will focus the subsequent dis- optical signal transduction. The assays are usually rapid cussion on assays in use or development for medical diag- (15 min to an hour) and can vary in sensitivity depending nostics. The techniques described in this section range onthesamplemedium,theagentundersuspicion,andthe from direct optical detection, such as microscopy, to opti- assay format. Common formats include wicking tickets cal signal transduction of chemical or electrochemical (such as is used in a home pregnancy test) and reagent processes. We attempt to summarize the methods in com- color-change (rapid Strep A test). A patient presenting mon use and note where advances are being made or are with flu-like symptoms in a typical U.S. hospital will likely needed to better address the identification problem. be given a rapid influenza test in the Emergency Depart- ment; a sore and reddened throat would prompt a rapid B. Biological Culture Strep test. If a rapid test is negative, a specimen would be Many of the technologies developed for the identifica- sent to a microbiology laboratory (sometimes in the tion and confirmation stages of a biodetection system (as hospital), where the specimen would undergo a screening

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panel for standard respiratory viruses. These panels are samples, responds within seconds to a pathogen binding typically some form of enzyme immunoassay, such as and offers sensitivities rivaling that of DNA analysis (see Enzyme-linked Immunosorbent Assay (ELISA), or some Section V-D). The problem of single-agent responsivity can kind of fluorescent-antibody-based stain [83]–[85]. The be addressed by developing multisite microarrays of bound basic mechanisms for signal reporting derive from binding antibodies or other antigenic recognition compounds. of the suspect antigen (which is typically immobilized on a Multisite antibody microarrays looking for protein markers substrate) with enzyme-bound antibodies with fluorescent for parasite infection, for example, have been commer- or colorimetric properties. Trained personnel are required cialized [91]. While the research community has developed to run these assays; and, due to staffing limitations and some multianalyte arrays for bioagent detection [92], the cost, they tend to be batched and conducted only once or a medical diagnostic community has yet to develop broad- few times per day. The specimen is often cultured for spectrum multiagent antibody (or protein) microarrays for bacteria as well, depending on the treating physician’s bioagent screening, and it may be that the only real observations and recommendations. While antibodies, and impetustodosowouldbeiftheyservedamoregeneral corresponding immunoassays, have been developed for respiratory-disease diagnosis function. In other words, most of the biological threat agents, these are not currently there is no real commercial market for bioagent detection part of the screening panels in clinical use. Thus, some assay panels, but a more general panel that included sites other indication that an event had taken place, or the for bioagent markers might inspire investment. progression of illness of the exposed populace, would be required to alert points of care to conduct bioagent testing. D. Nucleic-Acid-Based Assays For environmental sensing, military systems have been The primary mechanism for DNA analysis is via poly- developed that automatically collect an aerosol sample upon merase chain reaction (PCR) [93], which is an amplifica- action of an optical trigger (as described in Section III). The tion scheme for producing abundant copy numbers of collected sample is then injected onto an array of im- agent-specific genetic sequences. When dealing with envi- munoassay tickets, which are then read by an optical reader ronmental or clinical samples, such as blood or sputum, and by visual inspection. These tickets, called handheld some sample preparation is required to separate the DNA assays, are useful for first identification of a suspicious undertestfromtheenzymesandotherchemicalsthat biological release event but are typically neither sensitive nor degrade the DNA and/or inhibit the PCR amplification specific enough for clinical-diagnostic applications. There process.WhenpresentedwithpurifiedDNA,PCRcan are also immunoassay formats where, for example, the provide rapid (on the order of an hour), highly sensitive antibodies are immobilized on fiber-optic probes [86]. These (fewer than ten gene copies), highly specific (subspecies can offer higher sensitivity than the ticket format and have level, in some cases) identification of a biological orga- been developed into field-portable instruments for biological nism. While it is not necessary to know the exact sequence agent detection [87]. of the gene being amplified, it is necessary to know the In general, immunoassays can be limited in sensitivity sequence that flanks the amplified regions (primers). and in the specificity required to provide adequate infor- These sequences have been determined for all the high- mation for appropriate treatment (such as antibiotic re- priority bioagents on the CDC threat list8 and have been sistance), as well as in their utility, given the current developedtoworkwithPCRdevicesrangingfrom assays’ one-agent-per-test designs. There are commercial laboratory-grade to handheld. Originally, PCR devices systems, such as those based on electrochemiluminescence served only to amplify gene copy number; sequence deter- (ECL) and surface plasmon resonance (SPR) [88], [89] mination usually required an additional step, such as gel and developmental systems, such as CANARY [90], that electrophoresis or fluorescent assay. Modern real-time have demonstrated higher sensitivity than typical immu- PCR machines serve both functions, and do so by incor- noassay. ECL systems exploit antibody binding for spe- poration of a fluorescent label (probe) during the ampli- cificity, but the binding reaction is measured using fication process [94]. Thus, as the copy number increases chemiluminescence on an electrode surface, thus the op- (twofold with each PCR thermal cycle), the fluorescent tical signal is generated in the absence of any native back- signal can be tracked in real-time. Specificity derives from ground. SPR refers to the interaction of light (at specific the primer sequence being used (only the correct sequence angles) with delocalized electrons in thin metal films will amplify); sensitivity from the amount of light output (plasmons); analytes bound to the back surface of the film reported in a given number of thermal cycles. Although can alter the local refractive index and thereby change the PCRisaDNAamplification process, RNA can also be deflected angle of the incident light. CANARY (developed analyzed through the process of reverse transcriptase, at MIT Lincoln Laboratory) represents a novel assay that enabling identification of both bacterial and viral agents. consists of genetically modified murine B-cells engineered At this time, PCR is primarily a laboratory analysis, to express antibodies for biological warfare agents and to although the number and use of field-portable devices is emit photons in the event of pathogen binding. The B-cell assay, which is suitable for aerosol as well as clinical 8http://emergency.cdc.gov/agent/agentlist-category.asp.

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growing, especially by the military in environmental and treatment information so that appropriate therapeutics can clinical sample analyses.9 be administered. A rapid diagnosis of a bacterial infection is As with immunoassay, a limitation to PCR is that one useless without a concurrent understanding of what anti- must know which agent one is looking for (aprioriknowl- biotics that bacteria is responsive to. In addition, the limits edge of the primer sequences) and that each reaction is of detection of the assay must correspond to clinically re- usually specific to a single agent (although there are multi- levant levels, ideally even those found in exposed but pre- plex PCR processes that query for several possible agents at symptomatic individuals. An ideal medical system would once). There are DNA microarrays comprising thousands comprise a diagnostic device cheap enough to be used at of probes labelled with complementary DNA (cDNA) that every point of care (POC), that screened for many common have been developed for (originally) gene expression and uncommon (including bioagent-based) infectious di- studies and (eventually) also for host expression studies seases, that offered direct patient benefit while a presenting [95]–[97]. The microarray format could permit hundreds patient was still at the POC (say, 15 min or less), and that to thousands of concurrent pathogen screening assays to be relayed relevant information to local and regional networks conducted, provided that one has enough organism-specific in real time, so that infectious disease patterns would be knowledge with which to select the cDNA probes. A small detected as they occurred. An ideal environmental moni- pilot study, funded by the U.S. Air Force and the Defense toring system would perform an identification assay in real- Threat Reduction Agency, called Epidemiological Outbreak time, following a trigger event, or frequently enough to Surveillance, developed a pathogen DNA microarray that influence a treatment distribution decision in a timely man- was used to test for respiratory diseases among recruits in ner. Although there are many novel approaches under way to training at a U.S. Air Force base [98]. Results were encour- address these requirements (including single-molecule aging, in that early diagnoses of highly contagious diseases chemical detection, nanoparticle detection techniques, such as adenovirus were enabled, thereby providing infor- chip-based mass spectrometry, etc.), optical technologies mation to recommend isolation of infected individuals and that are suitable for use under a variety of environmental limit the subsequent spread of the disease. To our knowl- conditions, that are minimally invasive to the patient, that edge, however, the Holy Grail of the bioagent or even use simple chemistries and optical sources (such as LEDs), pathogen genome chip has yet to be realized. The chips are and that offer robust high-throughput operation will be at the expensive, the optical readers are expensive, and the assay vanguard of the next generation of rapid diagnostics. times for the large-site-number chips are often several hours. Thus, while DNA microarrays are promising means of conducting large numbers of concurrent tests from a VI. SUMMARY single clinical sample, they have not yet matured to the Biological agents have features that an adversary may find practical level of common use. attractive as an agent of war or terrorism. To protect potential victims from infection and harm requires rapid E. Looking Forward and accurate detection and identification of bioagents. The aforementioned schemes for identifying biological Since such agents could be used at any time or place, it is agents from environmental and clinical samples have many well to consider plausible scenarios that help to guide variations,designedtomakethemmoresensitive,faster, sensor system design. Two such scenarios were outlined cheaper, etc. In general, there is a tradeoff between the here. For detection and identification of biological-warfare speed of the assay and the specificity/sensitivity. The faster agents in the environment and in the laboratory, a correct diagnosis is made, the sooner the patient can electrooptical techniques have found widespread applica- receive appropriate treatment and the less likely that a tion. The principal approach to early warning sensing patient will die. Furthermore, the first diagnosis of a bio- today is laser-induced fluorescence, taking advantage of logical warfare agent will set in motion a public health ubiquitous biological constituents that emit light in response to determine if the patient was the victim of an response to excitation in the UV (particularly tryptophan attack or of a natural exposure. Thus, timely diagnosis is andNADH).However,sinceUV-LIVsensingisnot critical not only for the presenting patients but also to specific, additional steps of identification must take place. minimize fatalities in the (possibly much larger) exposed Even the most robust present-day field sensors require a populace. Techniques to increase assay speed need to be final confirmation step prior to taking any medical developed, but it is essential that the assays also provide measures. Today, that final confirmation is accomplished 9For example, the R.A.P.I.D and RAZOR systems from Idaho through laboratory assays that may involve culture, PCR, Technologies Inc. immunoassay, or a combination of these. h

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988 Proceedings of the IEEE |Vol.97,No.6,June2009 Greenwood et al.: Optical Techniques for Detecting and Identifying Biological-Warfare Agents

ABOUT THE AUTHORS Darryl P. Greenwood (Fellow, IEEE) was born in Bernadette Johnson received the B.S. degree in Dallas, TX, in 1945. He received the B.S. (summa physics from Dickinson College, Carlisle, PA, in cum laude), M.S., and Ph.D. degrees from the 1979, the M.S. degree in condensed matter theory University of Texas at Austin in 1968, 1969, and from Georgetown University, Washington, DC, in 1971, all in electrical engineering. 1981, and the Ph.D. degree in plasma physics from He served in the U.S. Air Force from 1971 to Dartmouth College, Hanover, NH, in 1986. 1975attheRomeAirDevelopmentCenter,Griffiss She joined MIT Lincoln Laboratory, Lexington, AFB, NY, honorably discharged as a Captain in MA, in 1985, where she is now Assistant Division 1975. Since 1975, he has been with MIT Lincoln Head in the Homeland Protection and Tactical Laboratory, Lexington, MA. He has held various Systems Division. She has been involved in a technical and leadership positions at the laboratory, including Head of number of programs related to laser-based propagation and sensing and the Optics Division. From 1994 to 1996, he served on an Intergovern- biodefense. Examples of past work include experiments in adaptive mental Personnel Act assignment as Chief Scientist, Air Force Rome optics to facilitate high-energy-laser propagation through the atmo- Laboratory. His research interests for many years were in adaptive optics sphere, and the adaptation and installation of an adaptive optics system and laser propagation. More recently, he developed and led the lab’s on the 60-in telescope at Mt. Wilson Observatory. From 1993 to 1996, she biological and chemical defense mission. He is currently a Principal directed the Environmental Monitoring project. She subsequently Laboratory Researcher responsible for initiatives in the energy area. He became involved in experiments to investigate microlaser-induced was a Reviewer and past Associate Editor of Optics Letters.He breakdown spectroscopy for in situ elemental analysis. She then was member of the U.S. Air Force Scientific Advisory Board from 1998 developed and managed a program to investigate the feasibility and to 2002. utility of active hyperspectral imaging for a variety of military and civilian Dr. Greenwood is a Fellow of the Optical Society of America. In 2002, applications, including unexploded ordnance and landmine sensing. In he was received the Exceptional Civilian Service decoration from the 1999, she became involved with Lincoln Laboratory’s growing biodetec- Secretary of the Air Force. tion program area. In 2001, she became the founding Leader in the Biodefense Systems Group to address critical defense needs against biological and chemical threat agents. Her responsibilities include direction of multiple programs in sensor development, laboratory and field measurements, biodetection forensics, and systems analyses. Dr. Johnson received the 2007 Lincoln Laboratory’s Technical Excellence Award.

Jonathan M. Richardson received the B.A. degree from the University of Pennsylvania, Philadelphia, in 1985 and the Ph.D. degree in physics from Harvard University, Cambridge, MA, in 1994. He joined MIT Lincoln Laboratory, Lexington, Thomas H. Jeys was born in Long Beach, CA, in MA, in 2003, where he is a Member of the Sensor 1950. He received the B.S. and M.S. degrees in Technology and System Applications Group. While physics from the University of Houston, Houston, at Lincoln, he has contributed to a variety of TX,in1972and1975,respectively,andthePh.D. chemical and biological defense (CBD) counter- degree in atomic physics from William Marsh Rice measure efforts, including lidar signature identification, bistatic CBD University, Houston, in 1982. detection, and in-flow CBD remediation. He performed his doctoral His Ph.D. dissertation concerned the electric research at the National Institute of Standards and Technology, where he field ionization of sodium Rydberg atoms. From subsequently held a National Research Council Postdoctoral Fellowship. 1982 to 1985, he was an Assistant Professor of Prior to joining Lincoln Laboratory, he worked for several years in private physics at Rice University. In 1986, he joined MIT industry developing medical imaging and other technologies. Lincoln Laboratory, Lexington, MA, as a Member of Technical Staff. From 1986 to 1995, he worked on developing Nd:YAG sum-frequency sources Michael P. Shatz received the S.B. degree from of sodium resonance radiation for both the Department of Defense and the Massachusetts Institute of Technology, astronomy adaptive optics applications. Since 1995, he has worked on Cambridge, in 1979 and the Ph.D. degree from the detection of aerosolized biological agents. He led the early California University of Technology, Pasadena, in development of the Biological Agent Warning Sensor, which in 2000 1984, both in physics. was transitioned to the Joint Biological Point Detection System. In 2004, He Joined MIT Lincoln Laboratory, Lexington, he cochaired the Chemical and Biological Sensor Standards Study for the in 1984 as a Member of Technical Staff in the Defense Advanced Research Projects Agency. Since 2002, he has been Systems and Analysis Group, where he worked on investigating new biological agent detection techniques that will enable analysis of air defense systems and counter- the development of low-cost biological agent sensors through use of low- measures to precision guided munitions. He is cost components such as UV LED sources and charge-coupled device currently Leader of the Advanced System Concepts Group, which detectors. He is now a Senior Member of Technical Staff in the Laser analyzes a wide range of military and homeland protection systems, Technology and Applications Group, MIT Lincoln Laboratory. including biodefense systems.

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