Sample scanning stage

FIGURE 13.8 Schematic illustration of confocal microscope used for the micro-Raman experiments. (a) The 488-nm line of an Ar+ laser is focused onto the sample with a high numerical aperture (NA) objective. The sample is mounted on an xy-piezo stage and raster-scanned over the focused laser beam. Raman scatter is collected with the same high-NA objective and focused onto a confocal aperture. The Rayleigh scatter is removed with a holographic notch filter, and the remaining Raman scatter is focused onto an avalanche photo-diode, the signal which is used to build up the image. Once a feature of interest is located, the laser beam is centered on the object and the Raman scatter is sent into a spectrometer with a back-thinned CCD camera to acquire the Raman spectra. (b) Confocal image of individual bacterial spores dried onto a calcium fluoride substrate. The bright spots correspond to the intrinsic autofluorescence and Raman scatter from the spores. Once the autofluorescence is photobleached, the Raman spectrum from the individual spore is collected. (See color insert.)

Raman Bacteria

FIGURE 13.9 Micro-Raman spectra of individual spores from four different Bacillus species. Each spectra was collected using the 488-nm line from an Ar+ laser. The signal was integrated for 4 minutes for each spectra. The nearly identical spectra can be attributed to the intense signal arising from calcium dipicolinate in the cortex of the spore.

FIGURE 13.9 Micro-Raman spectra of individual spores from four different Bacillus species. Each spectra was collected using the 488-nm line from an Ar+ laser. The signal was integrated for 4 minutes for each spectra. The nearly identical spectra can be attributed to the intense signal arising from calcium dipicolinate in the cortex of the spore.

therefore providing enhanced Raman signals. Enhancements to the Raman signal for species adsorbed onto roughened metal surfaces have been reported to be as high as 106.34 Recently, however, it has been demonstrated that by using metal colloids (50-100 nm in diameter) as the SERS substrate, the enhancement factors can be as large as 1015.34 This enormous enhancement to the Raman signal has made it possible to acquire Raman spectra for single molecules.35 36

Using metal colloids as the SERS substrate also has an additional advantage for biological applications. Because the SERS effect only enhances the signal a few nanometers from the metal surface, the metal nanoparticles act as near-field probes of the structures to which they are attached. This provides a means to probe the local, nanometer-scale, structure, and chemical composition, which would not be possible with conventional Raman spectroscopy. An illustration of using SERS particles as local probes is demonstrated again with Bacillus spores. In this sample the Bacillus spores have been incubated with colloidal silver suspension. The silver colloid is chemisorbed to the outside of o 6

Raman Shift (cm-1)

FIGURE 13.10 Surface-enhanced Raman spectra from colloidal silver particles attached to the outside of the spores. The dashed lines show where calcium-dipicolinate peaks would be if they were present in the spectra. Also highlighted are the regions used to identify the secondary structure of proteins.

Raman Shift (cm-1)

FIGURE 13.10 Surface-enhanced Raman spectra from colloidal silver particles attached to the outside of the spores. The dashed lines show where calcium-dipicolinate peaks would be if they were present in the spectra. Also highlighted are the regions used to identify the secondary structure of proteins.

the spore. Figure 13.10 shows the spectra collected from silver nanoparticles attached to spores of two different Bacillus species. What is evident from the spectra is that they are no longer dominated by the calcium dipicolinate as seen with the conventional Raman spectra in Figure 13.9. This result is consistent with the location of the calcium dipicolinate (DPA) in the cortex of the spore, several hundred nanometers away from the nanoparticle probe, thus providing a means to probe the outside structure of the spore.

In summary, we have shown that micro-Raman spectroscopy and surface-enhanced Raman spectroscopy provide chemical information of biological materials on the sub-micron scale. These are important tools for forensics analyses because of their high spatial resolution, good chemical selectivity, and high sensitivity. We have demonstrated this by presenting spectroscopic results obtained from single, isolated bacterial spores.

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