Abstract
High-speed, low-phototoxicity volumetric imaging is essential to quantify host–pathogen interactions in marine animal cell models, such as internalization of bacteria by Atlantic salmon skin keratocytes (SKCs). We developed an oblique plane microscopy (OPM) platform with an integrated projective OPM (pOPM) mode that preserves standard sample mounting and supports ultra-fast, optically sectioned projections. Leveraging the sparsity of bacterial signal, two pOPM projections at distinct viewing angles allow 3D localization by triangulation, reducing acquisition time and light dose by up to approximately 150-fold relative to full volumetric stacks. The hybrid workflow combines pOPM for rapid, gentle detection with OPM for volumetric context. We characterized system performance using 120 nm fluorescent beads and achieved near-diffraction-limited resolution across a 20 μm depth. In live, two-colour imaging of infected primary SKCs, we acquired 238 × 158 × 18 µm(3) volumes in 5.3 s and performed 3 h time-lapse recordings at 10 min intervals at 4 h, 28 h, and 52 h post-infection. Automated analysis segmented cells, detected bacterial clusters, and classified their spatial relationship to cells, revealing internalization fractions of approximately 18%, 54%, and 52% at 4 h, 28 h, and 52 h post-infection, respectively. These results demonstrate that OPM and pOPM can quantify SKC–bacteria interactions with subcellular resolution over extended fields and timescales, providing a platform to investigate mechanisms of bacterial internalization and clearance relevant to aquaculture health. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-33371-2.