Fig. 1: QCL-MIR imaging to guide spatially focused data acquisition in MSI.

a Overview of QCL-based MIR imaging microscope (i), and the QCL-MIR imaging-guided MSI workflow. A_ν: absorbance, ν: wavenumber, CR: coherence reduction. (ii) Selected spectral features (2^nd derivative of absorbance (2^nd)) discriminating cerebellar fiber tracts (FT) and granular layer (GL): 1466 cm⁻¹ (CH₂ bending vibration) and 1742 cm⁻¹ (C = O vibration). (iii) Feature-selective image segmentation and ROI definition. (iv) Co-registration of single wavenumber (1656 cm⁻¹) reference image (v) with hyperspectral dataset (i). (vi) Cerebellar ROI-focused MSI using iprm-PASEF. Scale bar, 2 mm. BioRender. https://BioRender.com/ymashbv. b Multimodal comparison of CCD−1137Sk fibroblast/HT-29 cancer biculture (BCF) versus monoculture fibroblast (MCF) spheroids. (i) Ion images of multiplex-MALDI-IHC⁵² using anti-vimentin (m/z 1230.84; fibroblast) and anti-pan-CK (m/z 1288.71; cancer) antibodies. Mass window ±10 ppm. (ii) MIR data (1742 cm⁻¹; 2^nd) of spheroid section, fibroblast core outline (cyan, dashed) derived by clustering (k = 2) of MALDI-IHC data. (iii) Ion images for m/z 835.54 (PI 34:1[M-H]^-; fibroblasts) and m/z 599.32 (lyso-PI 18:0[M-H]^-; cancer). Scale bar, 200 µm. c QCL-MIR imaging-guided ion image of m/z 722.51 (PE(P-36:4[M-H]^-). Scale bar, 200 µm. d Principal component analysis (PCA) of MSI data distinguishes MCF (dark blue) and BCF (cyan). e (i) MIR image of ARSA−/− mouse kidney (1720 cm⁻¹; 1^st) with putative glomerular ROI (green outline). (ii) QCL-MIR imaging-guided ion image of m/z 1151.71 (GM3 34:1;O2[M-H]^-; mass window ±10 ppm) with highlighted glomerular ROI. Scale bar, 300 µm. f (i) MIR image of EAE mouse spinal cord (1722 cm⁻¹; 1^st) with putative motor neuron ROI (blue outline). (ii) QCL-MIR imaging-guided ion image of m/z 885.549 (PI 38:4[M-H]^-; mass window ±10 ppm) with highlighted neuron ROI. Scale bar, 200 µm. g Sulfatide accumulation in ARSA−/− mice by (i) MSI (sum intensity distribution of 87 sulfatides³²), and (ii) MIR imaging at 988 cm⁻¹ (2^nd, C_β-O vibration). Superimposed kidney inner stripe of outer medulla (ISOM) ROI determined by clustering of MSI data (red and green dashed lines). Scale bar, 2 mm. h Box-plots of Z-score values reveal lipid accumulation in the ISOM region by both MSI and MIR imaging (1466 cm⁻¹; 2^nd) for n = 4 biological replicates. Boxplots indicate median (middle line), 25^th and 75^th percentile (box) and whiskers (1.5 times the interquartile range). i Volcano scatter plot of qTOF-MSI data for ARSA−/− vs. ARSA+ /+ reveals accumulation of sulfatides³² for ARSA−/− (red dots), e.g., I) SM4 34:1;O2[M-H]^-, II) SM4 38:1;O3[M-H]^-, and III) SM3 42:1;O2[M-H]^-. Statistical significance was tested by two-sided standard t-test. P-values are Benjamini–Hochberg-corrected. Source data is provided as Source Data file.