Label-Free Two-Photon Spectral Microscopy to Track Black Carbon Fate in Live Copepod

Black carbon (BC), a major product of incomplete combustion, is both a potent climate forcer and a widespread marine pollutant. Yet, its biological interactions remain largely unresolved. Here, we report the first application of two-photon (2P) spectral microscopy to track BC uptake in live zooplankton copepods (Acartia spp.). This nonlinear optical technique enables label-free, high-resolution 3D visualization of copepods and uniquely discriminates between their autofluorescence, particulate BC (particulate black carbon (PBC), red-shifted emission ∼650-675 nm), and dissolved/colloidal BC (DBC, broad emission centered near 540 nm) based on intrinsic photophysical fingerprints. By coupling intestinal clearing protocols with spectral mapping, we established, from our knowledge, the first pigment-free intestinal baseline in a live marine zooplankton, eliminating autofluorescence and food-pigment interference. In vivo imaging revealed PBC localized along the digestive tract, accompanied by intestinal swelling and a progressive redistribution of spectral weight from PBC to DBC-like emission toward the posterior gut. These findings indicate that soot aggregates undergo partial reorganization under intestinal conditions, generating colloidal fractions not originally abundant in diesel exhaust. Intestinal swelling was quantified using a dimensionless ratio between the midgut width and the body width at the digestive midgut region (d/D), providing a standardized indicator of gut distension associated with feeding and BC ingestion. By resolving the molecular-level organization within soot aggregates under 2P excitation, this methodological advance introduces a new analytical dimension for pollutant-organism studies, extending beyond the particle-scale changes previously reported. 2P spectral microscopy thus emerges as a powerful, label-free probe of aggregation-dependent photophysics in vivo, providing new insights into the biological transformation of combustion-derived carbon and its implications for marine carbon cycling.