Raman spectroscopy is an optical technique that can be used to evaluate the biomolecular composition of tissue and cell samples in a real-time and non-invasive manner. Subtle differences between datasets of spectra obtained from related cell groups can be identified using multivariate statistical algorithms. Such techniques are highly sensitive to small errors, however, and, therefore, the classification sensitivity of Raman spectroscopy can be significantly impacted by miscalibration of the optical system due to small misalignments of the optical elements and/or variation in ambient temperature. Wavenumber calibration is often achieved by recording the spectrum from a wavenumber reference standard, such as 4-acetamidophenol or benzene, which contains numerous sharp peaks in the fingerprint region. Here, we investigate a commercial polymer slide as a wavenumber reference standard for the calibration of Raman spectra. The Raman spectrum of this slide contains numerous sharp peaks in the fingerprint region. Unlike many other reference standards, the polymer slide is non-hazardous, has an indefinite lifetime, and is designed in the shape of a glass slide used for microscopy. We evaluate this reference in terms of accuracy and repeatability, and we compare with the established 4-Acetamidophenol wavenumber reference.

Wavelength calibration is a necessary first step for a range of applications in spectroscopy. The relationship between wavelength and pixel position on the array detector is approximately governed by a low order polynomial and traditional wavelength calibration involves first-, second-, and third order polynomial fitting to the pixel positions of spectral lines from a well known reference lamp such as Neon. However, these methods lose accuracy for bands outside of the outermost spectral line in the reference spectrum. We propose a fast and robust wavelength calibration routine based on modelling the optical system that is the spectrometer. For spectral bands within the range of spectral lines of the lamp we report similar accuracy to second- and third-order fitting. For bands that lie outside of the range of spectral lines we report an accuracy 12-121 times greater than that of third-order fitting and 2.5-6 times more accurate than second-order fitting. The algorithm is developed for both reflection and transmission spectrometers and tested for both cases. Compared with similar algorithms in the literature that use the physical model of the spectrometer, we search over more physical parameters in shorter time, and obtain superior accuracy. A secondary contribution in this paper is the introduction of new evaluation methods for wavelength accuracy that are superior to traditional evaluation.