Tensile Expansion Mass Spectrometry for single cell metabolomics imaging

Key results

1. ShownMultiaxial stretching of hydrogel-adhered OVCAR8-RFP cells enlarged both whole-cell and nuclear areas with cytosolic RFP retained (implying intact membranes), though realized linear expansion fell well below the device's theoretical maximum.

   Mean cell area 349 ± 110 to 1046 ± 366 µm^2 (3.0-fold; ∼1.7x linear; p = 1.02 × 10^-5); nuclear area 148 ± 72 to 255 ± 109 µm^2 (1.7-fold; ∼1.3x linear; p = 1.06 × 10^-11); paired t-test on four independently prepared hydrogels; theoretical device maximum ∼3.8x. · Visualization of OVCAR8-RFP under mechanical…; Figure legends

2. ShownTExMS runs at a 10 µm raster were much shorter than 5 µm microgrid and ∼1 µm t-MALDI acquisitions, while the stretching gave an estimated effective pixel size of about 5.9 µm^2.

   Total acquisition ∼45 minutes (TExMS) vs 1 hour and 9 minutes (fixed cells, 10 µm), 6 hours 13 minutes (MALDI-2 microgrid, 5 µm) and 18 hours and 16 minutes (t-MALDI, ∼1 µm); effective TExMS pixel ∼5.9 µm^2 observed vs ∼2.6 µm^2 expected at ∼3.8x expansion. · Comparison of TExMS with other single cell…; Figure legends

3. SuggestedAfter normalizing to effective pixel area, TExMS gave higher adenine signal per unit area than conventional 5 and 10 µm MALDI-MSI. It also retained L-glutamate, which fixation and washing in conventional preparations appeared to remove.

   Reference ion adenine [M-H]- at m/z 134.0472 tracked in six individual cells per condition across four workflows; TExMS intensities normalized using the ∼1.7x linear expansion; the text gives no numeric intensities or statistical test; L-glutamate comparison is qualitative (Figure 3h). · Comparison of TExMS with other single cell…; Figure legends

4. SuggestedGuided by an in-house LC-MS/MS metabolome library, TExMS mapped more glycolysis, amino acid and TCA-cycle intermediates than t-MALDI. Malic and succinic acid co-localized away from a putative membrane lipid, a pattern the authors interpret as mitochondrial.

   t-MALDI on unexpanded cells annotated ATP and AMP; TExMS annotated glucose-6-phosphate, pyruvate, glycerol 3-phosphate, branched-chain amino acids, and citric, isocitric, α-ketoglutaric, succinic and malic acids; malic m/z 133.0142, succinic m/z 117.0193, lipid m/z 152.8998; 10 µm pixels. · TExMS maps metabolic responses to cellular…; Figure legends

5. SuggestedUnder 24-hour serum starvation, OVCAR8-RFP cells became rounded and non-adherent, and TExMS detected significant shifts in several metabolites, including the glycolytic regulators pyruvic acid and glucose-6-phosphate.

   Serum-starved vs serum-treated (10% FBS) cells; ROI intensities exported, outliers removed by Grubbs' test (α = 0.05), log fold-change computed and Welch's t-test applied; text reports significance but no fold-change magnitudes, p-values or cell counts. · TExMS maps metabolic responses to cellular…; Figure legends