Super‐Multiplexed Label‐Free Raman Imaging Uncovers Novel Testicular Metabolic Couplings for Residual Body and Spermatogonial Differentiation

The biochemical composition underlying cytoplasmic residual body (RB) and arrested spermatogonia remains unresolved in spermatogenesis, in part due to limited approaches for intact-tissue metabolic interrogation. Here we develop Super-multiplexed Label-free Raman Imaging (SLRI) to generate 2D/3D biochemical maps of intact Drosophila testes. SLRI delineates major testicular cell types and provides the first in situ, stage-resolved mapping of cytoplasmic RBs at two discrete phases. We further identify a differentiation-arrest-associated metabolic trajectory in spermatogonial clusters, characterized by coordinated spatiotemporal decay of specific biochemical species. Across 42 spatiotemporally variant metabolites and metabolic pathways, SLRI reveals RB- and arrest-enriched signatures. Notably, RBs and arrested spermatogonia display significant biochemical overlap, alongside unique spectral signatures. Furthermore, we uncovered a ring-like, punctate arrangement of lipids, amino acids, and vitamins encircling RBs, suggesting structured metabolite partitioning during cytoplasmic remodeling. Collectively, SLRI establishes a general framework for high-dimensional metabolic mapping and remodeling analysis in intact tissues.