We have demonstrated that there are many promising and unique techniques which can be applied to the analysis of biological agents to provide biological signatures with high resolution and high sensitivity, including both "bulk" or whole-spore analysis and surface analysis. As an example, we have described their application to a powdered weaponized biological surrogate.
Other high-resolution techniques are currently being applied to this investigation. Environmental scanning electron microscopy (ESEM) is also available for taking high-resolution images under hydrated conditions; this has the advantage of avoiding any artifacts associated with the critical point drying process that is required under normal SEM operations. The ESEM is also equipped with EDX and Backscatter capabilities. Fourier transform mass spectrometry using our 9.4 Tesla magnet is being employed to perform studies of the spore surface and the compounds which have adsorbed to it. Similarly, our nano-SIMS (secondary ion mass spectrometer) will enable studies of elements and their isotopic ratios at ~50 nm resolution.
Because we now have the capability to analyze a single spore or biological entity, it is crucial that our samples be grown in a systematic and homogeneous manner. For techniques which analyze a "bulk population," individual differences between cells or spores in that population are averaged out and the resultant data reflect that average population. However, our ability to analyze a single entity allows resolution of differences between individual cells in a population. Our signature libraries will depend upon highly controlled, systematic growth and processing conditions to produce biological entities to be analyzed by these techniques to identify unique signatures. In the process, large quantities of data will be generated, similar to what is already being "mass produced" by DNA sequencing and gene analysis. Therefore, algorithms need to be developed and implemented for each of these techniques to support the large databases that each technique can and will generate.
As analyses by these techniques continue and signature libraries of correlations between those analyses and growth and processing conditions grow, it will be necessary to develop an information system which combines disparate types of data to determine unique signatures. For example, the system would link weapons manufacturing information and associations with foreign or domestic terrorist groups, weaponization signatures as described in this chapter, DNA sequences, component materials analyses, and metabolic signatures. The information system must provide a transparent and flexible query structure for comparison of new unknown samples with existing data, available for any type of data from SEM images to Raman spectra. Algorithms capable of combining several signatures or pieces of data from individual techniques and generating a distinguishing signature for a growth process will need to be developed.
We have demonstrated that multiple high-resolution, high-sensitivity techniques can and must be applied on the single-spore level. This is the path necessary to link the "how, when, and where" of a bioagent to the perpetrator of a biocrime.
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