Ramanscopeiii

Trypomastigotes (2 x 10⁶ cells per mL) were stimulated for 1h with 7.5 μM AA as above and centrifuged. Cells were then resuspended in 3.7% formaldehyde overnight, pelleted and analyzed by Raman spectroscopy without any labeling. Raman spectra of cells were recorded using a FT-Raman spectrometer model RFS 100S coupled to RamanScopeIII (Bruker Optik GmbH, Ettlingen, Germany), equipped with a ND:YAG laser with an excitation line at 1064 nm. For acquisition of the spectra, the laser power was adjusted to 500 mW (at source) and a good signal/noise ratio was obtained by performing 2048 scans in the region of 3500–50 cm^-1 with a spectral resolution of 4 cm^-1. The acquisition of Raman spectra was performed by OPUS 6.0 software (Bruker).
To investigate the presence of AA directly in LBs, purified LBs from untreated and 7.5 μM AA-treated groups were placed over 20 mm CaF₂ windows (cat. number 63207; Edmund Optics, Barrington, NJ, USA) and the data collected with a laser power of 20 mW, 50 s integration time and 5 co-additions, without any labeling. The Raman spectra were obtained in a Senterra Raman spectrometer (Bruker) based in a 180° backscattering configuration and using a 50x objective and the 632.8 nm wavelength of He-Ne laser output as excitation. A spectral resolution of 3–5 cm^-1 and slit width of 50x1000 μm were chosen.

The organic and inorganic contents of residual dentin after the treatments were measured (n = 6/group = 84). The specimens for the FT-Raman analysis were randomly assigned from the ones used for the CSH measurements. An FT-Raman microscope (Raman Scope III, Bruker Optics Inc., Karlsruhe, Germany; serial number: DE84716930) with a germanium detector and laser source was used. The dentin specimens were irradiated using an Nd: YAG laser (λ = 1064 nm) at 200 mW with a spectral resolution of 4 cm⁻¹. To each specimen, one spectrum with a 40 μm thickness and another with a 200 μm thickness were recorded via 150 scans. Changes on the organic and inorganic contents of the dentin were analyzed by comparing the integrated areas of the Raman shifts, centered in 350–542 cm⁻¹ (phosphate), 870–1125 cm⁻¹ (carbonate) and 2750–3100 cm⁻¹ (C-H bonds) [47 (link)]. The integrated area of the shifts was calculated using the LabSpec 6 Spectroscopy Suite software (Horiba Instruments Brasil Ltda., Jundiai, SP, Brazil).