Spectral artefacts induced by moving targets in live hyperspectral stimulated Raman spectroscopy: The case of lipid droplets in yeast cells

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.