A Subpixel Calibration Strategy for Micro-Raman Spectrometer in Subvisible Particles Traceable Detection

Subvisible particles are a major cause of adverse clinical infusion reactions and can lead to serious health problems. Raman spectroscopy is a rapid, nondestructive method for traceable detection of subvisible particles; however, the pixel-level spectral resolution limits the accuracy of the existing micro-Raman spectrometers. Consequently, we propose a subpixel calibration strategy to improve the detection accuracy of micro-Raman spectrometers. First, a combined light source with higher stability and uniformity is selected to improve the standard wavelength coverage of the charge-coupled device (CCD) pixels and the stability of the calibration. Subsequently, a wavelet-based automatic peak localization method is designed to obtain the pixel-integer values of the peak points corresponding to the standard wavelengths. Based on the spectral distribution characteristics, we can obtain a subpixel value corresponding to the peak center of the wavelength by fitting the Gaussian spectral peak curve. Then, a higher resolution subpixel-level calibration is achieved by establishing a mapping function between wavelengths and peak centers. We compare the standard sample detection shift accuracy and stability by using subpixel and pixel calibration. The subpixel-level calibration reduces the accuracy error from 4.3 to 1.3 cm-1 and the stability error from 4.8% to 2.4% compared to the pixel-level calibration, improving the micro-Raman performance indicator from Level III to Level II. Additionally, comparing the results of traceable detection of eight common injectable particles, the average similarity of micro-Raman using subpixel calibration was 96.6%, which is 7.5% higher than the matching accuracy of pixel calibration. These results demonstrate that the improved calibration of micro-Raman enables more accurate and stable detection of particles, meeting the requirements of high-accuracy engineering applications in particle traceable detection for pharmaceutical companies.