Rapid and gentle volumetric imaging of host-pathogen interactions in salmon skin cells using projective oblique plane microscopy

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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 µm3 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 bacteria, 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. Biological sciences/Biological techniques Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Winter ulcers, frequently caused by Moritella viscosa (MV), are a persistent welfare and economic challenge in Atlantic salmon aquaculture, contributing to increased morbidity and mortality during colder seasons[ 1 , 2 ]. Despite advances in vaccination, protection remains incomplete, and skin lesions continue to affect farmed salmonids[ 3 ]. The skin acts as a primary barrier and is populated by migratory skin keratocyte cells (shown in Fig. 1 ) that maintain barrier function, contribute to wound healing, and actively scavenge foreign material. Prior work shows that epidermal keratocytes can ingest bacteria and discriminate between bacterial types, and more recent studies demonstrated uptake of micro- and nanoparticles by salmon skin and corneal epithelial cells[ 4 , 5 ]. However, the timing and dynamics of bacterial uptake, interactions at the cell periphery, and subsequent intracellular processing remain incompletely understood. Quantitative, real-time three-dimensional (3D) imaging of primary cells over extended fields of view (FoV) and time periods is critical to elucidate host–pathogen interactions under physiologically relevant conditions. Imaging methods must balance spatial and temporal resolution against phototoxicity, while accommodating conventional sample mounting and mechanical stability. Point-scanning confocal microscopy provides optical sectioning but is limited by sequential acquisition, photobleaching, and phototoxicity during long imaging sessions[ 6 ]. Spinning disk confocal systems parallelize pinholes and can achieve high frame rates but subject the entire sample to widefield illumination, constraining long-term viability[ 6 , 7 ]. Light-sheet fluorescence microscopy (LSFM) reduces out-of-focus exposure and photodamage via selective plane illumination, but many implementations require nonstandard mounting geometries that complicate live-cell work with marine samples or are sensitive to refractive index mismatches introduced by saline media[ 8 – 19 ]. Oblique plane microscopy (OPM), also known as swept confocally-aligned planar excitation (SCAPE) microscopy, adapts the principles of light-sheet imaging to a single-objective configuration[ 20 – 30 ]. In OPM, illumination and detection use the same high numerical aperture (NA) primary objective, enabling standard mounting while preserving optical sectioning and efficient light collection. A remote focusing system (RFS) re-images the oblique illuminated plane to an intermediate image[ 31 – 33 ], which is then captured by a tilted detection arm to obtain 3D data within a rhomboidal volume without moving the sample. By placing a galvanometric mirror (GM) in a Fourier plane, the effective focal plane (EFP) can be swept rapidly through the sample for high-speed acquisition[ 21 , 26 ]. OPM thus combines the photophysical advantages of light-sheet imaging with a straightforward sample geometry. To further accelerate imaging and minimize dose, we integrated a projective OPM (pOPM) mode producing optically sectioned projections at selectable viewing angles in a single camera exposure as visualized in Fig. 1 c-f[ 34 – 36 ]. pOPM sweeps the EFP through the specimen while imposing a synchronized shear on within the camera plane, equivalent to a shear-warp projection[ 37 ]. In sparse samples, such as bacteria in and around SKCs, two projections acquired at distinct angles localize emitters in 3D by triangulation without collecting a full volumetric stack, reducing acquisition time and light exposure by up to ~ 150× while maintaining comparable detail. Here we present a hybrid OPM + pOPM platform tailored to live imaging of SKC–MV interactions. We characterize system performance across a 20 µm depth window and demonstrate two-colour volumetric imaging of infected primary SKCs, capturing 238 × 158 × 18 µm3 volumes in 5.3 s and acquiring 3 h time-lapse series at 10 min intervals at 4 h, 28 h, and 52 h post-infection. Using an automated pipeline, we quantify bacterial internalization dynamics, revealing an increase in internalized fraction between 4 h and 28 h followed by a plateau at 52 h. These results establish OPM and pOPM as complementary tools for fast, gentle 3D imaging of host–pathogen interactions in marine cell systems. Results System performance characterization We assessed resolution using 120 nm fluorescent beads. At an emission wavelength of 626 nm, the theoretical diffraction limit under the employed conditions is approximately 232 nm laterally. We imaged a 2D bead sample at 5 µm steps in the remote image space; at each position we acquired a 3D stack and fit Gaussian profiles to estimate the point spread function (PSF) across the field. In the nominal focal plane, the measured full width at half maximum (FWHM) was 285 nm (x), 251 nm (y), and 448 nm (z), in close agreement with an OPM simulation (266 nm, 232 nm, and 445 nm, respectively)[ 38 ], with deviations within 7% at the nominal plane. Resolution degraded gradually away from the nominal plane; we constrained the usable depth to maintain resolution within √2 of the nominal centre-resolution across all axes, yielding a practical depth of approximately 20 µm. For our samples, a 20 µm imaging depth provided a suitable balance of volumetric coverage and resolution (Fig. 2 ). Projective OPM enables rapid, optically sectioned projections We compared pOPM projections with computational projections from conventional OPM volumes. Volumetric datasets were acquired at 2 ms per plane (600 planes; volume time 3.6 s with rolling-shutter line delay). pOPM provided optical projections at selected viewing angles with a 50 ms exposure and a frame time of approximately 70.5 ms, yielding an approximately 50-fold reduction in acquisition time per projection relative to a full volume. Intriguingly, pOPM projections preserved structural detail with less background haze than computational projections across multiple angles, including a lab-frame orthogonal view, an oblique view (~ 70°), and a view along the optical axis (Fig. 3 a–i). We validated the hybrid strategy by acquiring a two-colour OPM volume of SKCs with nearby bacteria, followed by two pOPM projections of the bacteria channel at distinct angles. Using a localization script, bacterial positions were triangulated from the projections and registered to the OPM volume, showing good agreement with volumetric ground truth (Fig. 3 j). Because only the in-focus plane contributes signal at any moment during pOPM exposure, projections benefit from optical sectioning and can be acquired at short exposure times with strong signal utilization. Brightest pixels used approximately 95% of the 16-bit well depth, indicating that exposure can be reduced further while maintaining adequate signal. Live two-colour OPM imaging of SKCs infected with M. viscosa For even faster recordings, we imaged primary SKCs exposed to MV using two-colour OPM with a dichroic splitter and two synchronized cameras, enabling simultaneous acquisition of cell (membrane-labelled) and bacteria channels without filter switching. For a 238 × 158 × 18 µm3 volume, the stack was acquired in 5.3 s at 10 ms per plane. We captured multiple cells within a single FoV and resolved subcellular features such as filopodia and lamellipodia with minimal out-of-focus background (Fig. 4 ). The frame rate and gentle illumination enabled long time series of fast-migrating keratocytes Longitudinal quantification of bacterial internalization To quantify internalization dynamics, we acquired tiled time series comprising nine adjacent FoVs per sample at 10 min intervals over 3 h and imaged three independent samples at 4 h, 28 h, and 52 h post-infection. Three matched samples without bacteria served as references. An automated Python pipeline segmented cells, detected bacteria, and classified each bacterium as internalized, membrane-associated, or extracellular by overlap with the cell mask (see Methods and Supplementary Methods S1). At 4 h, the internalized fraction was approximately 18%. By 28 h, both the number of bacteria and the internalized fraction increased, with approximately 54% classified as internalized. At 52 h, the total bacterial count decreased relative to 28 h, and the internalized fraction remained at approximately 52% (Fig. 5 ). Bacteria were frequently observed at cell edges, suggesting active interactions at the periphery prior to internalization. Formal statistical inference across biological replicates will be addressed in future studies. Imaging speed and dose considerations Although pOPM can substantially reduce acquisition time and dose for sparse structures, conventional OPM with dual-camera usage provided sufficient performance for the present live-cell time lapses. Two-colour volumes were acquired at 10 ms per plane, and similar visualization quality was achieved at 6 ms per plane with strong signal and minimal background. Under lower bit depth (11-bit) and with additional optimization, such as leveraging axial aliasing to reduce the number of required planes in the scan direction, the volumetric rate could exceed 2 Hz for comparable volumes. We found that these optimizations were not necessary for SKC imaging in this study but may become relevant in recording internalization events as they occur. Discussion Hybrid OPM + pOPM enables rapid, gentle, and quantitative 3D imaging of host–pathogen interactions in primary salmon skin keratocytes. By combining optically sectioned volumetric OPM with instantaneous, angle‑selectable pOPM projections, the platform supports long‑term, simultaneous two‑colour time‑lapse imaging and fast detection/localization of sparse bacterial signals with markedly reduced acquisition time and light exposure. This is well suited to saline marine media, where refractive‑index mismatches and saltwater compatibility can hinder dual‑objective light‑sheet systems. pOPM produces sectioned projections in a single exposure; for sparse objects, two projections at distinct angles suffice to triangulate 3D positions, reducing time and dose by up to ~ 150× compared to full stacks while preserving detail comparable to computational projections. In practice, pOPM provides a user‑friendly live “survey” mode for navigation and event detection, with immediate switching to OPM for volumetric acquisition when needed. Using this platform, we observe a consistent trajectory in primary SKCs exposed to MV in which peripheral engagement is followed by increased internalization. The internalized fraction rises from approximately 18% at 4 h to about 50% by 28 h, with a sustained plateau thereafter (Fig. 5 ). The concurrent reduction in total bacterial counts by 52 h is compatible with a diminishing extracellular pool, potentially reflecting clearance, reduced adherence, or altered detectability under the labelling conditions used. The frequent presence of bacteria at cell edges is consistent with peripheral capture prior to uptake, although their distribution relative to specific subcellular structures was not quantified in this study. Taken together, these data support a model in which migratory epidermal keratocytes act as active sentinels that sequester bacteria from the skin surface. Before drawing definite conclusions, care must be taken as the used overlap-based internalization classifier can misassign tightly apposed bacteria at ruffled membranes, the 10 min sampling interval may miss short-lived entry or egress events, and imaging at ambient temperature following culture at 4°C may introduce a thermal mismatch that could alter SKC and MV behaviours. Nevertheless, the sustained high internalized fraction suggests that SKCs contribute to first-line defence by physically removing MV from the epithelial interface. Whether this reduces pathogen burden or, alternatively, provides an intracellular niche depends on the fate of internalized bacteria. Future studies incorporating endosomal/lysosomal markers, physiological temperature control, and event-trigger pOPM high-speed imaging should clarify uptake kinetics and intracellular processing, moving from descriptive sequestration dynamics toward mechanism and, ultimately, toward understanding how SKCs influence MV persistence or clearance in the salmon epidermis. More generally, our imaging approach is broadly applicable to sparse targets in complex cellular contexts (e.g., multi‑strain mixtures, immune cell–pathogen interactions). Simultaneous two‑colour acquisition reduces exposure versus sequential switching, and pOPM’s live projections speed screening and guide targeted volumetric imaging. Future technology-develop work includes higher‑speed volumetric OPM via exploiting axial aliasing, adaptive optics for saline environments, and more flexible temperature control. Methods Cells and bacteria Primary skin keratocyte cells (SKCs) were isolated from Atlantic salmon (Salmo salar) scales as described previously[ 5 ]. Briefly, scales were removed from euthanized post-smolt fish using clean forceps and placed in culture or glass-bottom dishes. After 6–10 min, Hank’s Balanced Salt Solution (HBSS) supplemented with penicillin/streptomycin and amphotericin was added. After 2–3 days at 4°C, cell sheets formed. For bacterial exposure, glycerol stocks of M. viscosa (kind gift from Nofima) were streaked on blood agar plates with 2% NaCl and incubated at 12°C until colony formation (~ 48 h). Single colonies were cultured in FAMP medium to OD ~ 0.6–0.8, pelleted (10,000 rpm, 4°C, 10 min), washed in 0.9% NaCl, and resuspended in HBSS. Bacteria were diluted to 10 − 2 ml − 1 for exposure. Cells were labeled with CellMask Green; live bacteria were labeled with BactoView Red. Full protocols are provided in Supplementary Methods S2. Microscopy and acquisition Imaging was performed on a custom OPM platform with integrated pOPM. A high-NA primary objective (100× silicone immersion, Nikon) provided illumination and detection. A remote focusing system re-imaged the oblique illuminated plane, and a galvanometric mirror (GM1) in a Fourier plane swept the effective focal plane (EFP) through the sample for volumetric OPM. A second galvanometric mirror (GM2) in the detection arm imposed a synchronized shear during pOPM to generate optically sectioned projections at selectable viewing angles. Two-colour OPM used a dichroic splitter to direct channels to two synchronized sCMOS cameras. Representative parameters: For live two-colour OPM, volumes of 238 × 158 × 18 µm3 were acquired in 5.3 s at 10 ms per plane. For pOPM, typical exposures were 50 ms per projection with a frame time of approximately 70.5 ms. For longitudinal studies, nine adjacent FoVs were imaged per sample at 10 min intervals over 3 h at 4 h, 28 h, and 52 h post-infection. Complete system layout, component list, and per-dataset parameters are provided in Supplementary Methods S1. Image processing and analysis OPM volumes were deskewed and rotated to the lab frame using an affine transform based on scan geometry. Two-colour volumes were co-registered via the optical splitter configuration. For bacterial internalization quantification, a Python pipeline performed: (1) segmentation of the cell channel to generate a 3D binary cell mask; (2) detection of bacteria by background thresholding of the bacteria channel; (3) 3D connected-component labeling with size filtering; and (4) overlap-based classification of each bacterium as internalized, membrane-associated, or extracellular based on the fraction of bacterium voxels overlapping the cell mask. Full algorithmic details are provided in Supplementary Methods S1. Ethics statement Atlantic salmon (Salmo salar) post-smolts (approximately 0.5–2 kg) were sourced from the Tromsø Aquaculture Station, Tromsø, Norway, a licensed research aquaculture facility. Fish were clinically healthy and free from pathogens according to routine diagnostics at the facility. Fish were humanely euthanized by a percussive blow to the head prior to sampling; scales were removed post-mortem and transported on ice to UiT The Arctic University of Norway for cell isolation. No chemical anaesthesia was used because aesthetic exposure can alter the scale surface and cell yield; euthanasia preceded any sampling. All procedures were carried out in accordance with the Norwegian Regulations for use of animals in experimentation (FOR-2015-06-18-761[ 45 ]) and the corresponding EU Directive 2010/63/EU[ 46 ]. Under these regulations, the handling described here (sampling of scales from euthanized fish) does not require approval by an institutional animal ethics committee; consequently, formal committee approval was not sought. The study is reported in accordance with the ARRIVE guidelines where applicable. Declarations Competing interests The authors declare no competing interests. Funding This work was supported by the Research Council of Norway (grant numbers 314546, 325159, and 352435) and the Centre for Digital Life Norway. Author Contribution J.R.S.: Instrument design and implementation; data acquisition; image processing; technical validation; manuscript drafting and editing. D.B.T.: Biological sample preparation; SKC isolation and culture; bacterial culture and exposure; methods input. F.S.: Conceptualization; supervision; instrument development; data curation strategy; manuscript drafting and editing; corresponding author. Acknowledgement We thank Nofima for the kind gift of glycerol stocks of M. viscosa. This work was supported by the Research Council of Norway (grant numbers 314546, 325159, and 352435) and the Centre for Digital Life Norway. Data Availability Data supporting the findings of this study are as of now only available from the corresponding author upon reasonable request due to large file sizes, but raw and processed imaging datasets will be made available along with minimal metadata via a public repository in the future. References Ghasemieshkaftaki, M. A. Review of Winter Ulcer Disease and Skin Ulcer Outbreaks in Atlantic Salmon (Salmo salar). Hydrobiology 3 , 224–237 (2024). Singh, G. G., Sajid, Z. & Mather, C. Quantitative analysis of mass mortality events in salmon aquaculture shows increasing scale of fish loss events around the world. Sci. Rep. 14 , 3763 (2024). Norwegian Fish Health Report. 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Reviews Aquaculture . 12 , 2446–2465 (2020). Karlsen, C., Sørum, H., Willassen, N. P. & Åsbakk, K. Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection. Vet. Microbiol. 154 , 353–362 (2012). Mikkelborg, M. K., Helgestad, A. S., Dalmo, R. A. & Thiyagarajan, D. B. Immune gene expression in salmon keratocytes upon bacterial exposure. BMC Mol. Cell. Biol. 26 , 28 (2025). Krasnov, A. et al. Atlantic salmon scale explants in bacteria-host interaction studies: in vitro challenge model. Fish Shellfish Immunol. 165 , 110466 (2025). Forskrift om bruk av dyr i forsøk - Lovdata. https://lovdata.no/dokument/SF/forskrift/2015-06-18-761#KAPITTEL_10 Directive 63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (Text with EEA relevance, 2010). Additional Declarations No competing interests reported. Supplementary Files Supplementary.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 23 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers agreed at journal 06 Nov, 2025 Reviewers invited by journal 06 Nov, 2025 Editor assigned by journal 06 Nov, 2025 Editor invited by journal 31 Oct, 2025 Submission checks completed at journal 10 Oct, 2025 First submitted to journal 10 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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12:34:19","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92258,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/7a032842b045c5b0528721ab.html"},{"id":96180862,"identity":"a367b42b-d61c-4e66-adb6-b64bcf36bab1","added_by":"auto","created_at":"2025-11-18 12:34:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":401685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eConcept and application overview. a, Two salmon illustrating healthy skin versus winter ulcer lesions. b, Example SKC holotomography image (Nanolive; adapted from prior work). c, Simplified oblique plane microscopy (OPM) schematic with co-moving galvanometric mirrors (GM). d, Volumetric OPM imaging of an infected SKC and representative membrane-labeled data. e, pOPM projection imaging of bacteria with registration to the OPM cell volume. f, Projection formation by sweeping the effective focal plane and imposing a synchronized shear. Scale bar is 10 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/f84b1761efb3cc8a6ba90d8f.png"},{"id":96180861,"identity":"f9e39907-ce0c-41c9-ab23-302e596f9557","added_by":"auto","created_at":"2025-11-18 12:34:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":473738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eResolution characterization with fluorescent beads. a, Lateral and axial resolution across the remote image space from Gaussian fits to 120 nm beads. x denotes light-sheet scan direction; y is the orthogonal transverse axis; z is along the primary objective axis. b, Representative bead images at selected displacements. c, Example XY and XZ zooms at the same axial displacements.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/4e05fbb439f054da6c7fba93.png"},{"id":96180864,"identity":"18fae4a5-5d10-4f84-969e-7bd888dd6b14","added_by":"auto","created_at":"2025-11-18 12:34:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":390809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOptical versus computational projections and triangulation. a–c, pOPM optical projections at orthogonal, oblique (~70°), and axial views. d–f, Computational projections from OPM volumes at matched angles. g–i, Schematic viewing directions. j, Two-colour OPM volume of SKCs with bacteria, with two pOPM projections of the bacteria channel used for triangulation and registration. Sample: fixed SKC stained with Phalloidin–Atto 647N. Scale bars: 30 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/a76dfd4f99deb389933b1af4.png"},{"id":96180865,"identity":"358c2db7-32ed-47bd-991b-3ac862291f8f","added_by":"auto","created_at":"2025-11-18 12:34:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1420112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLive two-colour OPM imaging of infected SKCs. a, Maximum intensity projection along z. b, Single optical section at the glass interface resolving filopodia and lamellipodial features. c, Side view at the position indicated in a. Cyan: SKCs (CellMask Green). Red: bacteria (BactoView Red). Scale bars: 50 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/9c1d6bdb4bf54ab3babc59ff.png"},{"id":96250688,"identity":"ea60e4bc-4eaa-4326-80a7-bb26be2975ea","added_by":"auto","created_at":"2025-11-19 07:38:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":77145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eQuantification of bacterial internalization. a, Counts of internalized bacteria at 4 h, 28 h, and 52 h post-infection aggregated over nine FoVs per sample across 3 h time-lapse. b, Normalized fractions of internalized bacteria; classification performed by automated pipeline.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/65c0ac12182c766940d1e4fe.png"},{"id":99172311,"identity":"d9cae5dd-24b2-4611-8f9e-95c5ed361a38","added_by":"auto","created_at":"2025-12-29 16:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2979573,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/f372f271-f567-4764-91cf-2250c60d035f.pdf"},{"id":96180867,"identity":"14a0363b-9ef8-47d8-9d4e-820a5c00ff0e","added_by":"auto","created_at":"2025-11-18 12:34:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":532830,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7773007/v1/67aee91005324ede970994d8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rapid and gentle volumetric imaging of host-pathogen interactions in salmon skin cells using projective oblique plane microscopy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWinter ulcers, frequently caused by Moritella viscosa (MV), are a persistent welfare and economic challenge in Atlantic salmon aquaculture, contributing to increased morbidity and mortality during colder seasons[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advances in vaccination, protection remains incomplete, and skin lesions continue to affect farmed salmonids[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The skin acts as a primary barrier and is populated by migratory skin keratocyte cells (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) that maintain barrier function, contribute to wound healing, and actively scavenge foreign material. Prior work shows that epidermal keratocytes can ingest bacteria and discriminate between bacterial types, and more recent studies demonstrated uptake of micro- and nanoparticles by salmon skin and corneal epithelial cells[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the timing and dynamics of bacterial uptake, interactions at the cell periphery, and subsequent intracellular processing remain incompletely understood.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuantitative, real-time three-dimensional (3D) imaging of primary cells over extended fields of view (FoV) and time periods is critical to elucidate host\u0026ndash;pathogen interactions under physiologically relevant conditions. Imaging methods must balance spatial and temporal resolution against phototoxicity, while accommodating conventional sample mounting and mechanical stability. Point-scanning confocal microscopy provides optical sectioning but is limited by sequential acquisition, photobleaching, and phototoxicity during long imaging sessions[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Spinning disk confocal systems parallelize pinholes and can achieve high frame rates but subject the entire sample to widefield illumination, constraining long-term viability[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Light-sheet fluorescence microscopy (LSFM) reduces out-of-focus exposure and photodamage via selective plane illumination, but many implementations require nonstandard mounting geometries that complicate live-cell work with marine samples or are sensitive to refractive index mismatches introduced by saline media[\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOblique plane microscopy (OPM), also known as swept confocally-aligned planar excitation (SCAPE) microscopy, adapts the principles of light-sheet imaging to a single-objective configuration[\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In OPM, illumination and detection use the same high numerical aperture (NA) primary objective, enabling standard mounting while preserving optical sectioning and efficient light collection. A remote focusing system (RFS) re-images the oblique illuminated plane to an intermediate image[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which is then captured by a tilted detection arm to obtain 3D data within a rhomboidal volume without moving the sample. By placing a galvanometric mirror (GM) in a Fourier plane, the effective focal plane (EFP) can be swept rapidly through the sample for high-speed acquisition[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. OPM thus combines the photophysical advantages of light-sheet imaging with a straightforward sample geometry.\u003c/p\u003e\u003cp\u003eTo further accelerate imaging and minimize dose, we integrated a projective OPM (pOPM) mode producing optically sectioned projections at selectable viewing angles in a single camera exposure as visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-f[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. pOPM sweeps the EFP through the specimen while imposing a synchronized shear on within the camera plane, equivalent to a shear-warp projection[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In sparse samples, such as bacteria in and around SKCs, two projections acquired at distinct angles localize emitters in 3D by triangulation without collecting a full volumetric stack, reducing acquisition time and light exposure by up to ~\u0026thinsp;150\u0026times; while maintaining comparable detail. Here we present a hybrid OPM\u0026thinsp;+\u0026thinsp;pOPM platform tailored to live imaging of SKC\u0026ndash;MV interactions. We characterize system performance across a 20 \u0026micro;m depth window and demonstrate two-colour volumetric imaging of infected primary SKCs, capturing 238 \u0026times; 158 \u0026times; 18 \u0026micro;m3 volumes in 5.3 s and acquiring 3 h time-lapse series at 10 min intervals at 4 h, 28 h, and 52 h post-infection. Using an automated pipeline, we quantify bacterial internalization dynamics, revealing an increase in internalized fraction between 4 h and 28 h followed by a plateau at 52 h. These results establish OPM and pOPM as complementary tools for fast, gentle 3D imaging of host\u0026ndash;pathogen interactions in marine cell systems.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSystem performance characterization\u003c/p\u003e\u003cp\u003eWe assessed resolution using 120 nm fluorescent beads. At an emission wavelength of 626 nm, the theoretical diffraction limit under the employed conditions is approximately 232 nm laterally. We imaged a 2D bead sample at 5 \u0026micro;m steps in the remote image space; at each position we acquired a 3D stack and fit Gaussian profiles to estimate the point spread function (PSF) across the field. In the nominal focal plane, the measured full width at half maximum (FWHM) was 285 nm (x), 251 nm (y), and 448 nm (z), in close agreement with an OPM simulation (266 nm, 232 nm, and 445 nm, respectively)[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], with deviations within 7% at the nominal plane. Resolution degraded gradually away from the nominal plane; we constrained the usable depth to maintain resolution within \u0026radic;2 of the nominal centre-resolution across all axes, yielding a practical depth of approximately 20 \u0026micro;m. For our samples, a 20 \u0026micro;m imaging depth provided a suitable balance of volumetric coverage and resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProjective OPM enables rapid, optically sectioned projections\u003c/p\u003e\u003cp\u003eWe compared pOPM projections with computational projections from conventional OPM volumes. Volumetric datasets were acquired at 2 ms per plane (600 planes; volume time 3.6 s with rolling-shutter line delay). pOPM provided optical projections at selected viewing angles with a 50 ms exposure and a frame time of approximately 70.5 ms, yielding an approximately 50-fold reduction in acquisition time per projection relative to a full volume. Intriguingly, pOPM projections preserved structural detail with less background haze than computational projections across multiple angles, including a lab-frame orthogonal view, an oblique view (~\u0026thinsp;70\u0026deg;), and a view along the optical axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;i).\u003c/p\u003e\u003cp\u003eWe validated the hybrid strategy by acquiring a two-colour OPM volume of SKCs with nearby bacteria, followed by two pOPM projections of the bacteria channel at distinct angles. Using a localization script, bacterial positions were triangulated from the projections and registered to the OPM volume, showing good agreement with volumetric ground truth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Because only the in-focus plane contributes signal at any moment during pOPM exposure, projections benefit from optical sectioning and can be acquired at short exposure times with strong signal utilization. Brightest pixels used approximately 95% of the 16-bit well depth, indicating that exposure can be reduced further while maintaining adequate signal.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLive two-colour OPM imaging of SKCs infected with \u003cem\u003eM. viscosa\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFor even faster recordings, we imaged primary SKCs exposed to MV using two-colour OPM with a dichroic splitter and two synchronized cameras, enabling simultaneous acquisition of cell (membrane-labelled) and bacteria channels without filter switching. For a 238 \u0026times; 158 \u0026times; 18 \u0026micro;m3 volume, the stack was acquired in 5.3 s at 10 ms per plane. We captured multiple cells within a single FoV and resolved subcellular features such as filopodia and lamellipodia with minimal out-of-focus background (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The frame rate and gentle illumination enabled long time series of fast-migrating keratocytes\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLongitudinal quantification of bacterial internalization\u003c/p\u003e\u003cp\u003eTo quantify internalization dynamics, we acquired tiled time series comprising nine adjacent FoVs per sample at 10 min intervals over 3 h and imaged three independent samples at 4 h, 28 h, and 52 h post-infection. Three matched samples without bacteria served as references. An automated Python pipeline segmented cells, detected bacteria, and classified each bacterium as internalized, membrane-associated, or extracellular by overlap with the cell mask (see Methods and Supplementary Methods S1). At 4 h, the internalized fraction was approximately 18%. By 28 h, both the number of bacteria and the internalized fraction increased, with approximately 54% classified as internalized. At 52 h, the total bacterial count decreased relative to 28 h, and the internalized fraction remained at approximately 52% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Bacteria were frequently observed at cell edges, suggesting active interactions at the periphery prior to internalization. Formal statistical inference across biological replicates will be addressed in future studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImaging speed and dose considerations\u003c/p\u003e\u003cp\u003eAlthough pOPM can substantially reduce acquisition time and dose for sparse structures, conventional OPM with dual-camera usage provided sufficient performance for the present live-cell time lapses. Two-colour volumes were acquired at 10 ms per plane, and similar visualization quality was achieved at 6 ms per plane with strong signal and minimal background. Under lower bit depth (11-bit) and with additional optimization, such as leveraging axial aliasing to reduce the number of required planes in the scan direction, the volumetric rate could exceed 2 Hz for comparable volumes. We found that these optimizations were not necessary for SKC imaging in this study but may become relevant in recording internalization events as they occur.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHybrid OPM + pOPM enables rapid, gentle, and quantitative 3D imaging of host–pathogen interactions in primary salmon skin keratocytes. By combining optically sectioned volumetric OPM with instantaneous, angle‑selectable pOPM projections, the platform supports long‑term, simultaneous two‑colour time‑lapse imaging and fast detection/localization of sparse bacterial signals with markedly reduced acquisition time and light exposure. This is well suited to saline marine media, where refractive‑index mismatches and saltwater compatibility can hinder dual‑objective light‑sheet systems. pOPM produces sectioned projections in a single exposure; for sparse objects, two projections at distinct angles suffice to triangulate 3D positions, reducing time and dose by up to ~ 150× compared to full stacks while preserving detail comparable to computational projections. In practice, pOPM provides a user‑friendly live “survey” mode for navigation and event detection, with immediate switching to OPM for volumetric acquisition when needed.\u003c/p\u003e\u003cp\u003eUsing this platform, we observe a consistent trajectory in primary SKCs exposed to MV in which peripheral engagement is followed by increased internalization. The internalized fraction rises from approximately 18% at 4 h to about 50% by 28 h, with a sustained plateau thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The concurrent reduction in total bacterial counts by 52 h is compatible with a diminishing extracellular pool, potentially reflecting clearance, reduced adherence, or altered detectability under the labelling conditions used. The frequent presence of bacteria at cell edges is consistent with peripheral capture prior to uptake, although their distribution relative to specific subcellular structures was not quantified in this study. Taken together, these data support a model in which migratory epidermal keratocytes act as active sentinels that sequester bacteria from the skin surface. Before drawing definite conclusions, care must be taken as the used overlap-based internalization classifier can misassign tightly apposed bacteria at ruffled membranes, the 10 min sampling interval may miss short-lived entry or egress events, and imaging at ambient temperature following culture at 4°C may introduce a thermal mismatch that could alter SKC and MV behaviours.\u003c/p\u003e\u003cp\u003eNevertheless, the sustained high internalized fraction suggests that SKCs contribute to first-line defence by physically removing MV from the epithelial interface. Whether this reduces pathogen burden or, alternatively, provides an intracellular niche depends on the fate of internalized bacteria. Future studies incorporating endosomal/lysosomal markers, physiological temperature control, and event-trigger pOPM high-speed imaging should clarify uptake kinetics and intracellular processing, moving from descriptive sequestration dynamics toward mechanism and, ultimately, toward understanding how SKCs influence MV persistence or clearance in the salmon epidermis. More generally, our imaging approach is broadly applicable to sparse targets in complex cellular contexts (e.g., multi‑strain mixtures, immune cell–pathogen interactions). Simultaneous two‑colour acquisition reduces exposure versus sequential switching, and pOPM’s live projections speed screening and guide targeted volumetric imaging. Future technology-develop work includes higher‑speed volumetric OPM via exploiting axial aliasing, adaptive optics for saline environments, and more flexible temperature control.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCells and bacteria\u003c/p\u003e\u003cp\u003ePrimary skin keratocyte cells (SKCs) were isolated from Atlantic salmon (Salmo salar) scales as described previously[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, scales were removed from euthanized post-smolt fish using clean forceps and placed in culture or glass-bottom dishes. After 6–10 min, Hank’s Balanced Salt Solution (HBSS) supplemented with penicillin/streptomycin and amphotericin was added. After 2–3 days at 4°C, cell sheets formed. For bacterial exposure, glycerol stocks of M. viscosa (kind gift from Nofima) were streaked on blood agar plates with 2% NaCl and incubated at 12°C until colony formation (~ 48 h). Single colonies were cultured in FAMP medium to OD ~ 0.6–0.8, pelleted (10,000 rpm, 4°C, 10 min), washed in 0.9% NaCl, and resuspended in HBSS. Bacteria were diluted to 10 − 2 ml − 1 for exposure. Cells were labeled with CellMask Green; live bacteria were labeled with BactoView Red. Full protocols are provided in Supplementary Methods S2.\u003c/p\u003e\u003cp\u003eMicroscopy and acquisition\u003c/p\u003e\u003cp\u003eImaging was performed on a custom OPM platform with integrated pOPM. A high-NA primary objective (100× silicone immersion, Nikon) provided illumination and detection. A remote focusing system re-imaged the oblique illuminated plane, and a galvanometric mirror (GM1) in a Fourier plane swept the effective focal plane (EFP) through the sample for volumetric OPM. A second galvanometric mirror (GM2) in the detection arm imposed a synchronized shear during pOPM to generate optically sectioned projections at selectable viewing angles. Two-colour OPM used a dichroic splitter to direct channels to two synchronized sCMOS cameras.\u003c/p\u003e\u003cp\u003eRepresentative parameters: For live two-colour OPM, volumes of 238 × 158 × 18 µm3 were acquired in 5.3 s at 10 ms per plane. For pOPM, typical exposures were 50 ms per projection with a frame time of approximately 70.5 ms. For longitudinal studies, nine adjacent FoVs were imaged per sample at 10 min intervals over 3 h at 4 h, 28 h, and 52 h post-infection. Complete system layout, component list, and per-dataset parameters are provided in Supplementary Methods S1.\u003c/p\u003e\u003cp\u003eImage processing and analysis\u003c/p\u003e\u003cp\u003eOPM volumes were deskewed and rotated to the lab frame using an affine transform based on scan geometry. Two-colour volumes were co-registered via the optical splitter configuration. For bacterial internalization quantification, a Python pipeline performed: (1) segmentation of the cell channel to generate a 3D binary cell mask; (2) detection of bacteria by background thresholding of the bacteria channel; (3) 3D connected-component labeling with size filtering; and (4) overlap-based classification of each bacterium as internalized, membrane-associated, or extracellular based on the fraction of bacterium voxels overlapping the cell mask. Full algorithmic details are provided in Supplementary Methods S1.\u003c/p\u003e\u003cp\u003eEthics statement\u003c/p\u003e\u003cp\u003eAtlantic salmon (Salmo salar) post-smolts (approximately 0.5–2 kg) were sourced from the Tromsø Aquaculture Station, Tromsø, Norway, a licensed research aquaculture facility. Fish were clinically healthy and free from pathogens according to routine diagnostics at the facility. Fish were humanely euthanized by a percussive blow to the head prior to sampling; scales were removed post-mortem and transported on ice to UiT The Arctic University of Norway for cell isolation. No chemical anaesthesia was used because aesthetic exposure can alter the scale surface and cell yield; euthanasia preceded any sampling.\u003c/p\u003e\u003cp\u003eAll procedures were carried out in accordance with the Norwegian Regulations for use of animals in experimentation (FOR-2015-06-18-761[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]) and the corresponding EU Directive 2010/63/EU[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Under these regulations, the handling described here (sampling of scales from euthanized fish) does not require approval by an institutional animal ethics committee; consequently, formal committee approval was not sought. The study is reported in accordance with the ARRIVE guidelines where applicable.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Research Council of Norway (grant numbers 314546, 325159, and 352435) and the Centre for Digital Life Norway.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.R.S.: Instrument design and implementation; data acquisition; image processing; technical validation; manuscript drafting and editing. D.B.T.: Biological sample preparation; SKC isolation and culture; bacterial culture and exposure; methods input. F.S.: Conceptualization; supervision; instrument development; data curation strategy; manuscript drafting and editing; corresponding author.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Nofima for the kind gift of glycerol stocks of M. viscosa. This work was supported by the Research Council of Norway (grant numbers 314546, 325159, and 352435) and the Centre for Digital Life Norway.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the findings of this study are as of now only available from the corresponding author upon reasonable request due to large file sizes, but raw and processed imaging datasets will be made available along with minimal metadata via a public repository in the future.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGhasemieshkaftaki, M. A. 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Atlantic salmon scale explants in bacteria-host interaction studies: in vitro challenge model. \u003cem\u003eFish Shellfish Immunol.\u003c/em\u003e \u003cb\u003e165\u003c/b\u003e, 110466 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eForskrift om bruk av dyr i fors\u0026oslash;k - Lovdata. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lovdata.no/dokument/SF/forskrift/2015-06-18-761#KAPITTEL_10\u003c/span\u003e\u003cspan address=\"https://lovdata.no/dokument/SF/forskrift/2015-06-18-761#KAPITTEL_10\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDirective \u003cem\u003e63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes\u003c/em\u003e (Text with EEA relevance, 2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7773007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7773007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh-speed, low-phototoxicity volumetric imaging is essential to quantify host\u0026ndash;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 \u0026micro;m depth. In live, two-colour imaging of infected primary SKCs, we acquired 238\u0026times;158\u0026times;18 \u0026micro;m3 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 bacteria, 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\u0026ndash;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.\u003c/p\u003e","manuscriptTitle":"Rapid and gentle volumetric imaging of host-pathogen interactions in salmon skin cells using projective oblique plane microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 12:34:14","doi":"10.21203/rs.3.rs-7773007/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T10:26:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-23T05:53:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T02:34:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20484739515572153505736933521471783978","date":"2025-11-13T14:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25562592414344047322930152292331278778","date":"2025-11-13T14:24:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173678684619228328914868246444733094830","date":"2025-11-12T10:22:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92855796405444711970921923802358731884","date":"2025-11-11T21:24:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60527449250245977377416824279159837392","date":"2025-11-11T17:36:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14374122142518632564725833125557633524","date":"2025-11-11T16:33:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130898950346998408737646865949425835392","date":"2025-11-07T01:56:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-07T00:25:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T23:01:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-31T17:50:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T09:14:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-10T09:11:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8edce22-cc3f-4712-8891-4542eec47d06","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58147656,"name":"Biological sciences/Biological techniques"},{"id":58147657,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-29T16:01:42+00:00","versionOfRecord":{"articleIdentity":"rs-7773007","link":"https://doi.org/10.1038/s41598-025-33371-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-22 15:57:57","publishedOnDateReadable":"December 22nd, 2025"},"versionCreatedAt":"2025-11-18 12:34:14","video":"","vorDoi":"10.1038/s41598-025-33371-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-33371-2","workflowStages":[]},"version":"v1","identity":"rs-7773007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7773007","identity":"rs-7773007","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}