Myeloperoxidase Clearance of Carbon Nanotubes Figure 5. Raman spectroscopic evaluation of ‘‘oxidative’’ defects in SWCNT present in the lungs of w/t and MPO k/o mice at days 1 and 28 post exposure using single point Raman spectroscopy or Raman mapping of different areas within the tissue samples. a. D- band/G- band ratios for single point Raman spectra obtained from samples at days 1 and 28 post exposure. * p,0.05, vs w/t 1 day post exposure, # p,0.05, vs w/t 28 days post exposure. Insert – typical Raman spectra (excitation at 633 nm) of solubilized lungs of w/t mice at days 1 and 28 post exposure. b–d. Raman mapping of SWCNT in different areas of the lung sections. b. Examples of bright-field images with a red box indicating the area where 32632 Raman spectra were acquired. Note that the sizes of the acquired areas were different at day 1 and day 28 (20 mm620 mm and 10 mm610 mm respectively), as significantly smaller SWCNT aggregates were detected at day 28 in w/t mice and the scanned areas were adjusted accordingly. c. Raman maps (excitation at 473 nm excitation) with examples of spectra corresponding to each of the clusters. d. D-band/G-band ratios for Raman spectral maps obtained from the lung of w/t and MPO k/o mice at days 1 and 28 post exposure. doi:10.1371/journal.pone.0030923.g005 concentrated H2SO4 and 30% aqueous H2O2 and sonicated in ultrasonic bath (Branson 1510 SonifierH, output power of 70 W at 40 KHz) for 24 hrs at 0uC. The dispersion was then heated to 70uC for 10 min for ‘‘polishing’’ the nanotubes. This solution was then diluted 10-fold by deionized water and filtered through PTFE membrane (100 mm pore size). The collected sample was thoroughly washed with deionized water and vacuum dried at 110uC for 30 min. Obtained short SWCNT were dispersed in 25 mM HEPES buffer (pH 7.4; containing 150 mM NaCl) by sonication. For purity assessment and characterization of SWCNT we used several standard analytical techniques. TEM was employed to determine the length distribution (Fig. S1a). Raman spectroscopy was implemented to visualize the D and G bands (Fig. S1b). Diffuse reflectance infrared Fourier Transform spectroscopy (DRIFTS) was also performed (methods S1, Fig. S1c). Animals Specific pathogen-free adult (8–10 week) female C57BL/6 mice (w/t) and B6.129X1-MPO mice (MPO k/o) were supplied by Jackson Lab (Bar Harbor, ME) and weighed 20.061.9 g when used. C57BL/6 mice are widely used as background for the PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e30923