In situ semi-quantitative imaging of intracellular metabolic interaction by confocal Raman microscopy.

Non-destructive subcellular metabolite quantification can reveal critical insights into biological interactions (e.g., endosymbiont-host crosstalk). Therefore, we developed a multivariate semi-quantitative imaging method using internal standardization to resolve simultaneous subcellular distributions of multiple metabolites, leveraging confocal Raman microscopy's (CRM's) high spatial resolution. The method was applied to the endosymbiotic mussel Gigantidas platifrons, whose symbiotic interaction mechanism has not been elucidated because symbionts cannot be cultivated. The results showed that the aggregated distribution of distinct phenotypes of symbiont strains was characterized by different glycogen abundances, indicating niche-driven metabolic strategies. Our data may provide direct evidence suggesting that symbionts supply intermediates to the host for cholesterol synthesis, potentially via vesicular trafficking. This work demonstrates CRM's capacity for comparative, spatially resolved metabolite quantification across cellular compartments. While semi-quantitative, CRM emerges as a powerful non-invasive tool for probing metabolic network dynamics and compartmentalization in challenging biological systems where traditional methods are limited.