U‑FLIP Multimodal Metabolic Imaging of Human Postnatal Lung in Health and Bronchopulmonary Dysplasia

This dataset contains label‑free multimodal optical images of human postnatal lung tissue from a premature infant with bronchopulmonary dysplasia (BPD) and an age‑matched non‑BPD control, acquired to support the same study. The same donor cohort and tissue blocks as in the lipidomics imaging dataset were used: two donors (one BPD, one control), four left‑upper‑lobe blocks per donor (n = 4 healthy; n = 4 BPD), agarose‑inflated and CMC‑embedded, with 20 µm cryosections mounted on glass slides.

Following MALDI‑FTICR‑MSI and matrix removal, the Ultrafast Focused Light‑based Imaging & Photonics (U‑FLIP) platform was applied to the identical sections. U‑FLIP combines two‑photon fluorescence (TPF), second harmonic generation (SHG), and stimulated Raman scattering (SRS) in a single upright laser‑scanning microscope (25× water objective, 1.05 NA). TPF channels capture autofluorescence from NADH and FAD, enabling computation of a pixel‑wise normalized optical redox ratio, FAD/(NADH + FAD). SRS channels at 2880 and 3011 cm‑¹ report on saturated and unsaturated fatty acids, respectively, allowing calculation of a lipid unsaturation ratio, I3011/(I2880 + I3011). SHG imaging detects fibrillar collagens (types I–III) and supports quantitative analysis of collagen fiber density and thickness. Typical in‑plane sampling is sub‑micrometer for single‑field images, with wide‑field coverage achieved by tile scanning.

To resolve chemical heterogeneity at subcellular scales, hyperspectral SRS (hSRS) stacks (2700–3100 cm‑¹) were acquired and analyzed by variance‑based feature selection, principal component denoising, and k‑means clustering. In addition, PRM‑SRS imaging was used to detect phosphatidylcholine and triglyceride subtypes in alveoli. These measurements revealed BPD‑associated decreases in optical redox ratio across all FTUs, spatially complex alterations in lipid unsaturation (decreased in alveoli, increased in bronchioles), reduced collagen fiber organization around airways, and shifts in alveolar lipid chemistry towards lower unsaturation and increased triglycerides.

H&E staining of the same sections provided pathologist‑guided segmentation of alveoli, bronchi, bronchioles, and vessels. Using the Hierarchical Multimodal Registration network (HiMReg), U‑FLIP images were co‑registered to histology and to the companion “Lipidomics Imaging of Human Postnatal Lung in Health and Bronchopulmonary Dysplasia” MALDI‑MSI dataset. This co‑registration enables pixel‑ and FTU‑level correlation between optical redox state, lipid saturation, collagen microarchitecture, and molecularly specific lipid distributions within the same anatomical context, thereby supporting multiscale interrogation of metabolic remodeling in BPD‑affected human lung.