Fig. 2: Validating ¹³C-SpaceM by interrogating de novo fatty acid synthesis in spatially heterogeneous normoxia-hypoxia model.

a, Illustration of microscopy and imaging MS data from the model of co-plated primary murine liver cancer cells originally cultured under normoxia (GFP^neg) and hypoxia (GFP^pos). The GFP signal was used for discerning the culturing conditions (white and green cell outlines show normoxic and hypoxic cells, respectively). The normalized intensities of the M+0 isotope of palmitate (representing the fraction of unlabelled palmitate) are shown for MALDI imaging pixels and as assigned to the single cells. b, Mass spectra for individual pixels mapped to cells from a normoxic cell (white outline) and a hypoxic cell (green outline). The peaks corresponding to palmitate isotopologues are marked by grey or green dots. c, Discerning cells cultured under normoxia versus hypoxia using ¹³C-SpaceM for the cells mono-plated for each culturing condition. Scatter-plot and histograms show the values of the GFP reporter (ground truth for telling the condition) and the normalized intensity of the M + 0 isotope of palmitate representing the fraction of unlabelled palmitate for single cells. Different colours are used to show cells with different true state label, known from the growth conditions for the particular well, and different cell state assigned using GFP signal. Prediction accuracy refers to the prediction of the true state based on the isotopologue distributions. d, Same analysis as in c for spatially heterogeneous co-plated cells from both conditions. In this case accuracy refers to prediction of the cell state assigned using GFP signal. e, Confusion matrices for the prediction of culture condition based on ¹³C-SpaceM or GFP in cells grown separately and comparison of the two predictions for co-plated cells. f, Comparison of single-cell versus bulk intensities for the M + 0 isotope of palmitate. Single-cell intensities are from the spatially heterogeneous co-plated model. Bulk intensities are from cells of each mono-cultured condition subjected to total fatty acid analysis by saponification followed by LC–MS. For single-cell data, black lines show average values. For bulk data, data are displayed as mean ± s.d. across three replicates.