The following is a description of the key details of the standardized calibration process for Raman microscopy:
Preliminary preparation
1. Equipment status check: Confirm that all components of the instrument are undamaged and the power connection is normal. After turning on the instrument, observe the status of the indicator lights to ensure normal operation.
2. Environmental control: The laboratory temperature is controlled at (15-25) ℃, with a relative humidity of ≤ 65%, to avoid temperature and humidity fluctuations affecting calibration accuracy.
3. Preheating stability: Preheat the laser according to the instructions to ensure stable output of the light source.
Core calibration steps
1. Laser calibration
-Wavelength calibration: Using a low-voltage atomic line lamp as a standard sample, select multiple atomic emission lines of different wavenumbers, measure their full width at half maximum, and calculate the average value to ensure the accuracy of laser wavelength.
-Power adjustment: Select the appropriate laser power based on the characteristics of the sample to avoid excessive burning of the sample or insufficient signal intensity.
2. Spectral resolution calibration
-Using a low-pressure atomic emission line lamp to detect spectral resolution, it is required to have an average of ≤ 10cm ⁻¹ across the entire range. Repeat the measurement six times and take the arithmetic mean to ensure that the instrument's resolution meets the standard.
3. Raman frequency shift calibration
-Indication error and repeatability: Select certified reference materials and measure Raman peak positions in at least five different wavenumber ranges. Repeat the measurement six times and calculate the frequency shift indication error and repeatability. The error should not exceed ± 4cm ⁻¹, and the repeatability should be ≤ 1cm ⁻¹.
-Calibration curve drawing: Plot the measured frequency shift of the standard substance as the vertical axis and the standard value as the horizontal axis for linear fitting, with a correlation coefficient of ≥ 0.995.
4. Intensity calibration
-Use white standard samples to calibrate the instrument response intensity, ensuring comparability of optical signals between different samples.
Operation precautions
1. Optical path adjustment: Adjust the incident angle of the laser beam and the collection optical path to ensure that the optical path is collimated and stable. Focus can be optimized by observing the green bright stripes.
2. Sample preparation: Solid samples should have a smooth and pollution-free surface; Avoid bubbles in liquid samples; Special samples are pre treated as needed.
3. Safety regulations: Wear protective equipment and avoid direct eye contact with laser beams. After calibration, turn off the software and power in order.
Long term maintenance
1. Regular calibration: Conduct periodic calibration according to national standards (such as JJF1544-2015), covering key indicators such as spectral resolution and frequency shift error.
2. Cleaning and maintenance: Regularly clean optical components to prevent dust from affecting the optical path; Keep dry during storage to avoid mold growth on optical components.
By following the above steps, the measurement accuracy and stability of Raman microscopy can be ensured, making it suitable for fields such as scientific research and industrial quality inspection. The specific operation needs to be adjusted according to the instrument manual and actual requirements.