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Abstract Some planktonic patches have markedly higher concentrations of organisms compared to ambient conditions and are <5 m in thickness (i.e. thin layers). Conventional net sampling techniques are unable to resolve this vertical microstructure, while optical imaging systems can measure it for limited durations. Zooglider, an autonomous zooplankton-sensing glider, uses a low-power optical imaging system (Zoocam) to resolve mesozooplankton at a vertical scale of 5 cm while making concurrent physical and acoustic measurements (Zonar). In March 2017, Zooglider was compared with traditional nets (MOCNESS) and ship-based acoustics (Simrad EK80). Zoocam recorded significantly higher vertically integrated abundances of smaller copepods and appendicularians, and larger gelatinous predators and mineralized protists, but similar abundances of chaetognaths, euphausiids, and nauplii. Differences in concentrations and size-frequency distributions are attributable to net extrusion and preservation artifacts, suggesting advantages of in situ imaging of organisms by Zooglider. Zoocam detected much higher local concentrations of copepods and appendicularians (53 000 and 29 000 animals m−3, respectively) than were resolvable by nets. The EK80 and Zonar at 200 kHz agreed in relative magnitude and distribution of acoustic backscatter. The profiling capability of Zooglider allows for deeper high-frequency acoustic sampling than conventional ship-based acoustics.