Confocal Raman microscopy was performed using an alpha300 R setup (WITec, Germany). Illumination of the sample was performed using a 532 nm excitation line from a single-mode frequency doubled Nd:YAG laser via a 100 μm single-mode glass fiber. A LD EC Epiplan-Neofluar 50×/0.55 objective (Carl Zeiss AG, Germany) was used and the laser power at the sample behind the objective was 12 mW.
An edge filter was used to separate the Raman signal from the excitation line. Confocality of the Raman signal was achieved via a 50 μm multi-mode fiber glass between the microscope and the Raman spectrometer, where the fiber serves as a pin-hole. The Raman spectrometer was equipped with a holographic grating of 600 lines per mm. For the detector, a Newton 970 EMCCD camera (Andor Technology Ltd, United Kingdom) with 1600 × 200 pixels was used, where this configuration allows a spectral resolution of about 2 cm−1 to be obtained.
An integration time of roughly 0.1 s per spectrum and pixel was used to improve the signal-to-noise ratio (S/N). For Raman depth scans, 100 pixel × 80 pixel scans were used for covering an area of 50 μm × 40 μm. All data sets were analyzed using cluster analysis and non-negative matrix factorization.
An edge filter was used to separate the Raman signal from the excitation line. Confocality of the Raman signal was achieved via a 50 μm multi-mode fiber glass between the microscope and the Raman spectrometer, where the fiber serves as a pin-hole. The Raman spectrometer was equipped with a holographic grating of 600 lines per mm. For the detector, a Newton 970 EMCCD camera (Andor Technology Ltd, United Kingdom) with 1600 × 200 pixels was used, where this configuration allows a spectral resolution of about 2 cm−1 to be obtained.
An integration time of roughly 0.1 s per spectrum and pixel was used to improve the signal-to-noise ratio (S/N). For Raman depth scans, 100 pixel × 80 pixel scans were used for covering an area of 50 μm × 40 μm. All data sets were analyzed using cluster analysis and non-negative matrix factorization.