Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.     

Identification of high explosives using single-particle aerosol mass spectrometry

The application of single-particle aerosol mass spectrometry (SPAMS) to the real-time detection of micrometer-sized single particles of high explosives is described. Dual-polarity time-of-flight mass spectra from 1000 single particles each of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazinane (RDX), and pentaerythritol tetranitrate (PETN), as well as those of complex explosives, Composition B, Semtex 1A, and Semtex 1H, were obtained over a range of desorption/ionization laser fluences between 0.50 and 8.01 nJ/microm2. Mass spectral variability with laser fluence for each explosive is discussed. The ability of the SPAMS system to identify explosive components in a single complex explosive particle ( approximately 1 pg) without the need for consumables is demonstrated.     

Single-particle aerosol mass spectrometry for the detection and identification of chemical warfare agent simulants

Single-particle aerosol mass spectrometry (SPAMS) was used for the real-time detection of liquid nerve agent simulants. A total of 1000 dual-polarity time-of-flight mass spectra were obtained for micrometer-sized single particles each of dimethyl methyl phosphonate, diethyl ethyl phosphonate, diethyl phosphoramidate, and diethyl phthalate using laser fluences between 0.58 and 7.83 nJ/microm2, and mass spectral variation with laser fluence was studied. The mass spectra obtained allowed identification of single particles of the chemical warfare agent (CWA) simulants at each laser fluence used although lower laser fluences allowed more facile identification. SPAMS is presented as a promising real-time detection system for the presence of CWAs.     

Feasibility of detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy

The detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy (LIBS) is investigated using aerosolized Bacillus spores. Spores of Bacillus atrophaeous, Bacillus pumilus, and Bacillus stearothemophilus were introduced into an aerosol flow stream in a prescribed manner such that single-particle LIBS detection was realized. Bacillus spores were successfully detected based on the presence of the 393.4- and 396.9-nm calcium atomic emission lines. Statistical analyses based on the aerosol number density, the LIBS-based spore sampling frequency, and the distribution of the resulting calcium mass loadings support the conclusion of individual spore detection within single-shot laser-induced plasmas. The average mass loadings were in the range of 2-3 fg of calcium/Bacillus spore, which corresponds to a calcium mass percentage of approximately 0.5%. While individual spores were detected based on calcium emission, the resulting Bacillus spectra were free from CN emission bands, which has implications for the detection of elemental carbon, and LIBS-based detection of single spores based on the presence of magnesium or sodium atomic emission was unsuccessful. Based on the current instrumental setup and analyses, real-time LIBS-based detection and identification of single Bacillus spores in ambient (i.e., real life) conditions appears unfeasible.     

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.     

Relative sensitivity factors for alkali metal and ammonium cations in single-particle aerosol time-of-flight mass spectra

A variety of factors have been investigated with regard to the quantitation of chemical species within individual ambient aerosol particles analyzed by laser desorption time-of-flight mass spectrometry. Spectrum to spectrum differences in the interaction of the particle with the ionization laser beam, which affect the absolute peak areas in the mass spectra, can be minimized by using relative peak areas instead of absolute peak areas in each spectrum. Whereas absolute peak areas vary by an average of 59% for a given ion peak in single particle mass spectra of a monodisperse aerosol of particles formed from the same solution, relative peak areas in the same mass spectra vary only by an average of 16%. Relative sensitivity factors (RSF) relating the mass spectral ion intensity of NH4+ and the alkali metal cations Li+, Na+, K+, Rb+, and Cs+ in single particle aerosol time-of-flight mass spectrometry to their bulk concentrations have been determined. The values for Li+/Na+, K+/Na+, Rb+/Na+, Cs+/Na+, and NH4+/Na+ are found to be 0.14, 5.1, 6.0, 7.9, and 0.014, respectively. The higher response for heavier cations of the alkali metals is consistent with the periodic trends of both ionization potential and lattice energies of the species of interest. The response factor for sodium and potassium cations has been used to accurately determine the relative amounts of Na+ and K+ in sea-salt particles, by analyzing a sample of approximately 360 ambient sea-salt particles. The relative amounts of Na+ and K+ are found to be 97 and 3% in particles, respectively, whereas in seawater they are, on average, 98 and 2%.     

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.     

Influence of saline media on the fluorescence emission of Bacillus spores

Single-particle levitation in conjunction with 264.3-nm laser excitation is used to measure the fluorescence emission of individual particles of Bacillus globigii spores. With precise humidity control, the fluorescence emission of wetted and desiccated Bacillus spore particles is measured from 300 to 450 nm. Comparison of spectra for Bacillus spores suspended in a standard buffer aqueous solution and for a desiccated 10-mum-diameter aggregate Bacillus spore particle shows that the spectra is virtually indistinguishable. However, at 85% relative humidity, corresponding to a 4.5M sodium chloride solution, the spore spectra redshifts by approximately 25 nm. It is postulated that the spectra redshifting is a result of specific interactions between the tyrosine fluorophore of the Bacillus spore and the phosphate moieties in the buffer solution.     

Depth profiling of heterogeneously mixed aerosol particles using single-particle mass spectrometry

Infrared laser evaporation of single aerosol particles in a vacuum followed by vacuum ultraviolet (VUV) laser ionization and time-of-flight mass spectroscopy of the resulting vapor provides a depth profile of the particle's composition. Analyzing glycerol particles coated with 60-150-nm coatings of oleic acid using either a CO2 laser or a tunable optical parametric oscillator as an evaporation laser results in mass spectra that depend on the IR laser power. Low infrared laser powers incompletely vaporize particles and preferentially probe the composition of the surface layers of the particle, but high laser powers evaporate the entire particle and produce spectra representative of the particle's total composition. In the limit of low laser power, the fraction of oleic acid in the mass spectra is as much as 50 times greater than the fraction of oleic acid in the particle, providing a surface-layer-specific characterization. The OPO laser provides even more surface specificity, producing an [oleic acid]/[glycerol] ratio as much as four times larger (for a 60-nm coating) than that obtained using the CO2 laser. The infrared laser power required to sample the core of the particle increases with the thickness of the coating and is sensitive to changes in the coating thickness on the order of 10 nm. In contrast to these intuitively appealing results, high CO2 laser powers (approximately 90 mJ/pulse) produce mass spectra that, at short delays between the CO2 and VUV lasers, show enrichment of the core material rather than the coating. Likewise, tuning the OPO to frequencies that are resonant with the core material but transparent to the coating also results in selective detection of the core. The results suggest that a shattering mechanism dominates the vaporization dynamics in these situations.     

Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of airborne biological particles

We have assembled an aerosol-fluorescence spectrum analyzer (AFS), which can measure the fluorescence spectra and elastic scattering of airborne particles as they flow through a laser beam. The aerosols traverse a scattering cell where they are illuminated with intense (50 kW/cm(2)) light inside the cavity of an argon-ion laser operating at 488 nm. This AFS can obtain fluorescence spectra of individual dye-doped polystyrene microspheres as small as 0.5 μm in diameter. The spectra obtained from microspheres doped with pink and green-yellow dyes are clearly different. We have also detected the fluorescence spectra of airborne particles (although not single particles) made from various biological materials, e.g., Bacillus subtilis spores, B. anthrasis spores, riboflavin, and tree leaves. The AFS may be useful in detecting and characterizing airborne bacteria and other airborne particles of biological origin.     

Quantitative detection of aromatic compounds in single aerosol particle mass spectrometry

Most laser-based aerosol mass spectrometers rely on a single ultraviolet laser to both ablate and ionize the aerosol particle. This technique produces complex and fragmented mass spectra, especially for organic compounds. The approach presented here achieves a more robust and quantitative analysis using a CO2 laser to evaporate the aerosol particle and a vacuum ultraviolet laser to ionize the vapor plume. Vacuum ultraviolet laser ionization produces little fragmentation in the mass spectra, making the identification of an aerosol particle's constituents more straightforward. An analysis of simple, three-component mixtures of aniline, benzyl alcohol, and m-nitrotoluene shows that the technique also provides a quantitative analysis for all the components of the mixture. Furthermore, the detection of predominantly parent ion signal from anthracene particles demonstrates the utility of the technique in the analysis of lower vapor pressure, solid-phase aerosols. Finally, we discuss the potential and limitations of this technique in analyzing organic atmospheric aerosols.     

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.     

Single-particle mass spectrometry of polystyrene microspheres and diamond nanocrystals.

High-resolution mass spectra of single submicrometer-sized particles are obtained using an electrospray ionization source in combination with an audio frequency quadrupole ion-trap mass spectrometer. Distinct from conventional methods, light scattering from a continuous Ar-ion laser is detected for particles ejected out of the ion trap. Typically, 10 particles are being trapped and interrogated in each measurement. With the audio frequency ion trap operated in a mass-selective instability mode, analysis of the particles reveals that they all differ in mass-to-charge ratio (m/z), and the individual peak in the observed mass spectrum is essentially derived from one single particle. A histogram of the spectra acquired in 10(2) repetitions of the experiment is equivalent to the single spectrum that would be observed when an ion ensemble of 10(3) particles is analyzed simultaneously using the single-particle mass spectrometer (SPMS). To calibrate such single-particle mass spectra, secular frequencies of the oscillatory motions of the individual particle within the trap are measured, and the trap parameter qz at the point of ejection is determined. A mass resolution exceeding 10(4) can readily be achieved in the absence of ion ensemble effect. We demonstrate in this work that the SPMS not only allows investigations of monodisperse polystyrene microspheres, but also is capable of detecting diamond nanoparticles with a nominal diameter of 100 nm, as well.     

Capabilities of femtosecond laser ablation inductively coupled plasma mass spectrometry for depth profiling of thin metal coatings

The capabilities of ultraviolet femtosecond laser ablation inductively coupled plasma mass spectrometry (UV-fs-LA-ICPMS) for depth profile analysis of thin metal coatings were evaluated. A standard sample consisting of a single Cr thin layer of 500 nm +/- 5% on a Ni substrate was used. A fast washout was obtained by a high-efficiency aerosol dispersion ablation cell (V approximately 1 cm3), which allowed single-shot analysis with increased depth resolution. Laser ablation was performed in helium at atmospheric pressure conditions. A laser repetition rate of 1 Hz and low laser fluence (<0.5 J/cm2) were used. Very low ablation rates (<10 nm/pulse) were determined by atomic force microscopy (AFM). Information about the crater geometry and morphology was investigated using scanning electron microscopy and AFM. The depth resolution, calculated via the maximum slope of the tangent in the layer interface region, was smaller than 300 nm. Our data indicate that UV-fs-LA-ICPMS represents a powerful combination of high lateral and depth resolution for the analysis of thin metal coatings. Moreover, an overall ion yield, defined as the ratio of detected ions and ablated atoms, of approximately 5 x 10-5 was estimated for the chromium layer under the operating conditions chosen. The absolute amount of ablated material per laser pulse was approximately 1 pg, which corresponds to a detection limit of 180 microg/g.     

Online laser desorption-multiphoton postionization mass spectrometry of individual aerosol particles: molecular source indicators for particles emitted from different traffic-related and wood combustion sources

Direct inlet aerosol mass spectrometry plays an increasingly important role in applied and fundamental aerosol and nanoparticle research. Laser desorption/ionization (LDI) based techniques for single particle time-of-flight mass spectrometry (LDI-SP-TOFMS) are a promising approach in the chemical analysis of single aerosol particles, especially for the detection of inorganic species and distinction of particle classes. However, until now the detection of molecular organic compounds on a single particle basis has been difficult due to the high laser power densities which are required for the LDI process as well as due to the inherent matrix effects associated with this ionization technique. By the application of a two-step approach, where an IR desorption laser pulse is applied to perform a gentle desorption of organic material from the single particle surface and a second UV-laser performs the soft ionization of the desorbed species, this drawback of laser based single particles mass spectrometry can be overcome. The postionization of the desorbed molecules has been accomplished in this work by resonance enhanced multiphoton ionization (REMPI) using a KrF excimer laser (248 nm). REMPI allows an almost fragmentation free trace analysis of polycyclic aromatic hydrocarbons (PAHs) and their derivatives from individual single particles (laser desorption-REMPI postionization-single particle-time-of-flight mass spectrometry or LD-REMPI-SP-TOFMS). Crucial system parameters of the home-built aerosol mass spectrometer such as the power densities and the relative timing of both lasers were optimized with respect to the detectability of particle source specific organic signatures using well characterized standard particles. In a second step, the LD-REMPI-SP-TOFMS system was applied to analyze different real world aerosols (spruce wood combustion, gasoline car exhaust, beech wood combustion, and diesel car exhaust). It was possible to distinguish the particles from different sources by their molecular signature. Finally, exemplary ambient aerosol measurements have been carried out, which demonstrate the potential of the method for investigating urban aerosol and making contributions to source attribution studies.     

Conditional-sampling spectrograph detection system for fluorescence measurements of individual airborne biological particles

We report the design and operation of a prototype conditional-sampling spectrograph detection system that can record the fluorescence spectra of individual, micrometer-sized aerosols as they traverse an intense 488-nm intracavity laser beam. The instrument's image-intensified CCD detector is gated by elastic scattering or by undispersed fluorescence from particles that enter the spectrograph's field of view. It records spectra only from particles with preselected scattering-fluorescence levels (a fiber-optic-photomultiplier subsystem provides the gating signal). This conditional-sampling procedure reduces data-handling rates and increases the signal-to-noise ratio by restricting the system's exposures to brief periods when aerosols traverse the beam. We demonstrate these advantages by reliably capturing spectra from individual fluorescent microspheres dispersed in an airstream. The conditional-sampling procedure also permits some discrimination among different types of particles, so that spectra may be recorded from the few interesting particles present in a cloud of background aerosol. We demonstrate such discrimination by measuring spectra from selected fluorescent microspheres in a mixture of two types of microspheres, and from bacterial spores in a mixture of spores and nonfluorescent kaolin particles.     

Investigation of the utility of laser-secondary neutral mass spectrometry for the detection of polyaromatic hydrocarbons in individual atmospheric aerosol particles

The distribution of polyaromatic hydrocarbons (PAHs) in ambient aerosol particles is of importance to both human health and climate forcing. Although time-of-flight secondary ion mass spectrometry (ToF-SIMS) has proven useful for studying the distribution of organic compounds in individual aerosol particles, it is difficult to detect PAHs at relevant concentrations in individual aerosol particles because of their low ion yield. In this study, we explore the potential of using laser secondary neutral mass spectrometry (Laser-SNMS) to study three PAHs: pyrene, anthracene, and naphthalene. Because of the high volatility of PAHs, a cryostage was required for the analysis to prevent sublimation of the molecules into the vacuum chamber. We studied two laser systems, a 157 nm excimer laser, which is capable of single-photon ionization of the PAHs, and a 193 nm laser, which requires multiphoton ionization. Under optimized conditions for laser power density and primary ion pulse length, 193 nm postionization resulted in a 2-50-fold increase in ion yield over ToF-SIMS. Using the 157 nm laser, the yield was increased by more than 3 orders of magnitude for all 3 PAHs studied. The single-photon postionization process proved superior in terms of both yield enhancement and reduced fragmentation. By using the optimized 157 nm laser system and a cryostage, we were able to detect PAHs on the surface of 2 μm diameter ambient aerosol particles.     

Thermal vaporization-vacuum ultraviolet laser ionization time-of-flight mass spectrometry of single aerosol particles

Single aerosol particles of ethylene glycol and oleic acid are vaporized on a heater at temperatures between 500 and 700 K, and the resulting vapor plume is ionized by a 10.5-eV vacuum ultraviolet (VUV) laser. The mass spectra are compared to those obtained by CO2 laser vaporization followed by VUV laser ionization. The relative intensities of the parent and fragment ion peaks are remarkably similar for the two modes of vaporization. A Maxwell-Boltzmann distribution of speeds accurately describes the dependence of the signal as a function of the VUV laser pulse timing. The signal levels obtained with this design are sufficient to obtain good-quality mass spectra.     

Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced. This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer. At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (approximately 5 mg) cocrystallized with alpha-cyano-4-hydroxycinnamic acid. Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides. The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape. Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin. Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides. Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (approximately 5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin.     

Near-infrared surface-enhanced-Raman-scattering-mediated detection of single optically trapped bacterial spores

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) surface-enhanced Raman scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on approximately 60-nm-diameter gold colloids bound to 3-aminopropyltriethoxysilane derivatized glass. The light from a 787-nm laser diode was used to trap and manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores. Comparison of normal Raman and SERS spectra reveals not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.