Scanning electron microscopy (SEM) provides excellent images of the spore surface. Low beam energies (approximately 1.2 keV) give images of the outer surface detail, while higher beam energies (approximately 15 keV) penetrate the outer exosporium and image the inner coats of the spore. When combined with energy dispersive X-ray microanalysis (EDX), the elemental composition can be determined on the exact same imaged sample, providing a direct connection between the image and its elemental composition. Atomic force microscopy (AFM) allows very-high-resolution imaging of the sample surface, scanning a wide range of fields from 20 nm (0.02 |lm) to 150 |lm. Resolutions on the order of tens of nanometers, with height resolution of 0.1 nm, are routine; by utilizing carbon nanotubes as imaging tips, image resolutions of less than 1 nm can be achieved. The major advantage of AFM is its ability to image directly in a fluid.
Raman and surface-enhanced Raman spectroscopy (SERS) probe molecular bond vibrations and rotations to produce characteristic spectra. Here, electrons in the molecules are excited from a vibrational level to a "virtual level"
lying above the ground state vibrational levels but below the excited electron states and return to a different vibrational level, emitting a characteristic photon indicative of the "Raman shift" as they do so. Raman effects have a low "cross-section" or probability of occurring; however, by placing the molecule near a gold or silver surface, the Raman signal is enhanced by factors as large as 1015! This effect is highly local (as is the electric field) and hence can be taken advantage of to probe surfaces of biological agents such as spores. Protein secondary structure can be determined by its characteristic Raman peaks; by adsorbing gold or silver nanoparticles (~80 nm in diameter) to the surface of a biological agent, only the proteins or other molecules in the local vicinity of the nanoparticle (of order 10 nm) are analyzed.
In general, mass spectrometry measures the masses (i.e., mass-to-charge ratios) of ions and molecules. There are several types of mass spectrometers and a wide variety of sample introduction and ionization methods. Bio-Aerosol Mass Spectrometry (BAMS) is a novel real-time technique for the rapid identification of individual bio-aerosol particles using mass spectrometry. It is the hybrid of a relatively new method of mass spectrometric sample introduction and a novel method of analyzing the resulting data in real-time. BAMS can operate autonomously, consumes only electricity, and is unique in that it allows for the reagentless analysis of a complex environmental sample without any prior sample preparation or discrimination. It is currently capable of detecting and identifying individual cells sampled directly from the air within an aerosol of many background materials in real-time with no reagents. Bacillus spores have been successfully characterized and can be efficiently distinguished from fungal spores, vegetative bacteria, and many other biological and nonbiological background materials in real-time.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) also separates molecular fragments by their size (and, hence, their "time of flight"), but this instrument utilizes an ion beam rather than a laser to generate those fragments. Because it uses an ion beam, the energy can be lowered so as to analyze approximately one atomic layer at a time. The ToF-SIMS captures elemental data (to generate chemical maps) as well as molecular fragments (to generate mass spectra), and does so on a depth-dependent basis (to generate a depth profile).
A third type of mass spectrometry, accelerator mass spectrometry (AMS), is the most sensitive of all techniques included here. AMS combines mass spec-trometry and nuclear detection to measure the concentration of an isotope in a sample. The accelerator is required because the molecules to be broken are only broken at million-electron Volt (MeV) energies, in contrast to the kilo-electron Volt (keV) energies normally used in mass spectrometry. For biological samples, 14C measurements are made by first converting the sample to graphite, followed by ionization and subsequent acceleration to finally become a 35 MeV 14C4+ ion. These ions are resolved by high-energy mass spectrometry and, ultimately, can be quantified in the range of attomoles (i.e., 10-18 moles) of 14C in samples of size less than 1 mg at a precision of better than 1%.
Nuclear microscopy is a combination of particle-induced X-ray emission (PIXE) and scanning transmission ion microscopy (STIM). PIXE uses characteristic X-rays to quantitatively map simultaneous element distributions (for elements greater than sodium) within microscopic regions of a sample, and STIM detects the energy loss of accelerated ions which pass through the sample in the exact same sample region. This loss of energy enables the mass of the sample to be accurately quantified. This technique is relatively nondestructive to the sample, allowing further analysis on the same exact components.
Having described these techniques by the molecular intrinsic properties they measure, we next look at the granularity or spatial resolution each technique can achieve on a sample. Accelerator mass spectrometry reduces the entire sample (less than 1 mg) to carbon before performing the analysis and provides 14C measurements on the bulk sample. Similarly, scanning electron microscopy is useful for characterizing the overall sample through image as well as elemental data. Several of these techniques, however, can analyze a single biological entity such as a (whole) spore. Raman spectroscopy, bioaerosol time-of-flight mass spectrometry, nuclear microscopy, and SEM with energy-dispersive X-ray analysis, while examining just a single spore, derive their data from the signal generated by the whole or entire spore. By comparison, atomic force microscopy, surface-enhanced Raman spectroscopy, and time-of-flight secondary ion mass spectrometry are able to analyze only the surface of a single spore.
By using a combination of these complementary techniques, we can investigate signatures of sample growth, processing, geolocation, and chronometry. The biological signatures will be in the form of images, Raman spectra indicative of molecular composition, and elemental composition due to protein fragment and elemental masses. In addition, this information can be determined on the basis of its spatial distribution in the sample (spore).
In the following paragraphs, we describe each of these analytical techniques in more detail. First, high-resolution imaging by SEM and AFM is addressed, followed by Raman and surface-enhanced Raman spectroscopy. Lastly, we will discuss measurements of mass—bioaerosol time-of-flight mass spectrometry, time-of-flight secondary ion mass spectrometry, particle-induced X-ray emission/scanning transmission ion microscopy, and accelerator mass spectrometry. Results of the application of these techniques to the analysis of Bacillus spores will be presented, concluding with discussion and future directions.
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