Good practices for calibration
Raman spectroscopy requires careful attention to reproducibility in particular with respect to system response and axis calibration. It can have profound consequences on longitudinal studies if no proper control measures are implemented. We therefore recommend to perform the following calibration routine prior to any Raman imaging:
Protocol for ensuring reproducibility with a specific fixed configuration (laser, spectrometer/CCD, microscope objective, fiber pinhole)
Notes:
Protocol for 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 silicon wafer, optimize the confocal system for maximum photon counts and record a spectrum.
- Using a polystyrene standard measure and record a spectrum.
- Using a NIST fluorescence standard (e.g. SRM785 or SRM532) measure and record a spectrum.
Notes:
- Intensity and wavenumber auto-correction is supported in ORM-IRIS, see the GitHub documentation for more information..
- In order to detect any inconsistency in the calibration, we recommend performing PCA on the three data sets (2,3,4) individually using the available code. This ensures that if on any day there is a deviation in the data, that this will be flagged