Good practices for reproducibility
Raman spectroscopy requires careful attention to reproducibility in particular with respect to system response and wavelength calibration. It can have profound consequences on longitudinal studies if no proper control measures are implemented. We recommend to perform the following calibration routine prior to any Raman imaging:
Ensuring reproducibility with a specific fixed configuration (laser, spectrometer/CCD, microscope objective, fiber pinhole)
Things to keep an eye on that effect reproducibility
Ensuring reproducibility with a specific fixed configuration (laser, spectrometer/CCD, microscope objective, fiber pinhole)
- Using a power meter, measure and record the laser power at the sample (after the objective).
- Using a silica wafer, optimize the microscope for maximum Raman photon counts at 520 cm-1 and record a spectrum with constant integration time (e.g., 1 sec).
- Using polystyrene or an Argon/Mercury lamp as standard, measure and record a spectrum with constant integration time (e.g., 1 sec).
- Using a NIST fluorescence standard (e.g. SRM2241 or SRM2242) or a calibration lamp measure and record a spectrum with constant integration time.
Things to keep an eye on that effect reproducibility
- In general, keep things constant for a specific experiment (laserpower, laser wavelength, spectrometer/CCD, microscope objective, fiber pinhole, integration time, accumulations)
- Ensure wavelength and intensity calibration are stable. Any deviations can have profound effect on analysis.
- Ensure CCD/camera is properly cooled according to specifications and that there a no new "hot pixels".
- Keep the microscope objective clean and never dip it in chemical solutions.