Dm2700 m confocal microscope

Unpolarized Raman spectra were collected by a Renishaw InVia Micro-Raman spectrometer. This is equipped with a solid-state diode laser source at 532 nm with a nominal output power of nearly 60 mW and a Leica DM2700 M confocal microscope, with a 50X LWD and a 100X objectives. A holographic edge filter determines the high-contrast rejection for the elastically scattered light, and a diffraction grating (1800 grooves/mm) provides a spectral resolution of about 1 cm⁻¹. Scattered photons are detected by a Peltier cooled CCD (1024 × 256 pixel). The laser power at the sample was set by neutral density filters, to prevent photo-damage of tissues. The spot size was set to a few microns. Spectra were collected in the extended scan mode, covering the 100–3800 cm⁻¹ wavenumber range. Wire, LabSpec, MatLab, and Origin software were used to collect, refine, and analyze the raw spectra.

For each sample, we performed several point analyses by Raman spectroscopy, in order to check for possible inhomogeneities. Because of the high sensitivity of Mn and Fe oxides to the laser heating^{44 (link),45 (link)}, Raman measurements were performed with a progressively increasing laser power, in order to avoid degradation of the samples. Raman measurements were performed at the Raman Spectroscopy Laboratory, Department of Science, Roma Tre University, at room temperature using an inVia Renishaw Raman equipped with a diode laser (532 nm, output power 50 mW), an edge filter to select the Raman scattering avoiding the elastic contribution, a 1800 lines/mm diffraction grating and a Peltier cooled 1024 × 256 pixel CCD detector. Samples were mounted on the manual stage of a Leica DM2700 M confocal microscope. Laser beam focusing and collection of Raman signals were realized with a 100x objective (with 2 mW and 5 accumulations of 10 s each, in the range of 200–900 cm⁻¹). The Raman spectrometer was calibrated prior to the measurements using a Si wafer and by performing the automatic offset correction. The spectra acquisition and data analyses were performed using WiRE and Origin softwares. The peak positions are estimated to be accurate to at least ±2 cm⁻¹.

Raman spectra from 4 different spots, covering all the detected colours, were collected with the InVia™ $\mu$ -Raman spectrometer from RENISHAW^®, UK, equipped with a Leica DM2700 M confocal microscope. The spots were analysed with two different solid-state diode laser sources, one at 532 nm and the second at 785 nm, selected according to need. The former source operated at 120 mW power while the latter operated at 250 mW. Neutral filters were used to decrease the power at the sample, taking into account its nature and composition thus avoiding degradation processes. A set of long working distance objectives (namely 20× and 50×) was used, focusing the excitation beam down to few microns.
To achieve enough statistics, 3 to 5 scans, with an integration time of 5 s were acquired. The data were collected with the instrument’s proprietary software and then exported as ASCII files. The output spectra were processed with a graphic analysis software.