In this study, we used stimulated Raman spectroscopy (SRS) microscopy to collect Raman signatures from live Saccharomyces cerevisiae cells in the spectral range 2804−3060 cm−1 and 830−2000 cm−1 with a spectral resolution of 8 cm−1. To effect this, we tuned the pump beam to several distinct wavelengths and thus acquired a series of chemical maps in order to reconstruct SRS spectra based on the intensity of the pixels, an approach also referred as hyperspectral SRS (hsSRS). One of the advantages of hsSRS over spontaneous Raman is that it is not overtly plagued by fluorescence and so fluorescent samples like yeast can be analysed. We show however that Raman signatures acquired by this approach may be subject to spectral artefacts that manifest as drops in intensity of Raman signal due to the movement of lipid droplets (LDs) within the yeast cells. To overcome this issue, yeast cells were chemically fixed with 4% formaldehyde and no artefacts were observed in the Raman signatures acquired from ‘stationary’ samples. Our findings indicate that caution must be applied when analysing SRS signatures obtained through hsSRS from mobile LDs and/or any other moving target within a system, whether biological or not.

The development of a label-free, non-destructive and safe analytical method such as Raman spectroscopy for assessing cartilage degradation is highly desirable. Compared to non-optical imaging modalities, Raman mapping offers a more sensitive means of directly assessing the chemical composition of cartilage in three-dimensional space and the potential to monitor cartilage degeneration to inform intervention and treatment strategies. Herein, we report the application of Raman spectroscopic methods ex vivo and at arthroscopy to identify molecular alterations in cartilage specimens containing minor focal lesions characteristic of the early disease phase. Our initial ex vivo analysis, obtained by single-point Raman spectroscopy of cartilage samples, supports previous findings based on S-O stretching vibration bands associated with sulphated glycosaminoglycans (sGAGs). We extended the analyses to the high-wavenumber region where we observed that vibrational bands assigned to C-H and O-H stretching modes discriminated early cartilage alterations from healthy cartilage samples. Furthermore, we performed a proof-of-concept in-clinic study using a custom-built optical probe to acquire Raman spectral measurements for the first time in patients undergoing arthroscopy of knee joints. Spectra were obtained with adequate signal-to-noise ratios that similarly discriminated between lesion and adjacent cartilage sites and identified reductions in sGAGs in apparently healthy cartilage. Building on this, we present initial results from Raman mapping to spatially resolve the molecular constituents of cartilage through its depth and across a lesion. Mapping revealed a non-uniform and reduced sGAG distribution within the lesion and peripheral cartilage that was otherwise visually normal, similar to the in-clinic observations, showing that the degradative influence of the lesion extended beyond its border. This was accompanied by a decreased fluorescence signal intensity, which suggests that fluorescence may provide valuable information as an adjunct to the Raman signal in discriminating normal and degenerating cartilage. This work demonstrates the value of Raman mapping over single-point Raman measurements for the analysis of the anisotropy of articular cartilage and highlights the potential of the technology for in vivo articular joint arthroscopy applications.