Real-time snapping dynamics and nanoscale thickness profiling of salmon keratocyte tunneling nanotubes using quantitative phase microscopy

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Afzal, Marie K. Mikkelborg, Dhivya B. Thiyagarajan, Deanna L. Wolfson, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8172968/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract The highly migratory skin epithelial cells of fish, named keratocytes, display interesting features, including long cellular protrusions resembling tunneling nanotubes (TNTs). It has been shown in other cell types that TNTs may transfer cellular cargo between cells. These TNTs are instrumental not only in cargo transport but also in mediating signaling between cells. Due to the varying height of TNTs, down to 100 nm or less, a highly sensitive quantitative microscopy technique is required for accurate quantification of their morphological and dynamic properties. In this study, a partially spatially coherent quantitative phase microscopy (QPM) system was utilized, providing extremely high spatial sensitivity to track changes in TNT height over time, particularly during the breaking/snapping process. A phase-shifting technique was employed to recover high-resolution phase maps of the TNTs. Observations indicate that TNT height typically ranges from 100 nm to 700 nm. Additionally, the average height-to-width ratio over time suggests that the TNTs adopt a less rounded but flattened shape. Comparisons between intact and broken TNTs further reveal that unbroken TNTs generally exhibit a higher average height, with a maximum observed value of approximately 900 nm. Beyond morphological characterization, this work may open new avenues for understanding TNTs´ potential role in the transfer of intracellular cargo, paving the way for future investigations into cellular communication mechanisms. Biological sciences/Biological techniques Biological sciences/Biophysics Biological sciences/Cell biology Physical sciences/Nanoscience and technology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Atlantic salmon ( Salmo salar ) farming in Norway has progressed to 1.5 million metric tons in 2023 [ 1 ]. Despite this progress, farmed salmon still face nearly 20% mortality on average, largely due to infectious pathogens, physical injuries, and various stressors [ 2 ]. While wound healing on the fish skin occurs following mucosal infection or injury, the cellular communications involved in this process remain poorly understood. Studies on innate immunity in higher vertebrates suggest that cells communicate via ligands and receptors and through more direct cell-to-cell interactions [ 3 ]. Through direct communication, the cells may even exchange organelles such as mitochondria, which is beneficial when it comes to cellular rescue and regeneration [ 4 ]. One such direct path for cell communication is through special extended cytoskeletal protrusions, referred to as tunneling nanotubes (TNTs). These long membrane extensions may be a route of transfer of various small molecules, organelles, and even pathogens, including bacteria and viruses [ 5 ]. TNT-like structures have previously been reported in different human immune cells, including B cells, T cells, and NK cells [ 6 ]. In various human cells, such as kidney cells, monocytes, and Jurkat T cells, TNTs connect cells over distances exceeding 100 µm [ 7 , 8 ]. Furthermore, it has been reported that in cultured rat pheochromocytoma PC12 cells (neuroblastic-like), these TNTs can have diameters ranging from 0.05 µm to 1.5 µm and lengths extending across multiple cell diameters [ 9 ]. TNTs have been documented in zebrafish, where TNT-like structures have been observed facilitating the transfer of Wnt proteins in embryos [ 10 ]. In related research, structures resembling TNTs were identified during a specific developmental stage of zebrafish, notably gastrulation. These structures facilitated the transport of proteins between distant cells [ 11 ]. To our knowledge, there are no reports on TNT-formation by skin primary epithelial cells in any animal species so far. Furthermore, structural and functional knowledge about TNTs in fish is limited. Intriguingly, in Atlantic salmon, we have observed that primary skin epithelial cells (keratocytes) produce TNT-like structures (not published). Live cell imaging is an excellent model for observing cellular changes in real-time and investigating their interactions. Also, the artifacts generated due to the fixation process are eliminated [ 12 ]. Our methodology uses live cell imaging, which can be used to observe dynamic behavior and snapping events of the TNTs during cell migration. Since TNTs are small, slender, and relatively fragile structures, it is essential to use a label-free optical microscopy technique that can provide high-contrast images at high-speed. Otherwise, any other microscopic methods may affect the TNT dynamics. Quantitative phase microscopy (QPM) is a non-invasive, non-contact, and label-free imaging technique highly compatible with live-cell studies. QPM uses the intrinsic refractive index contrast of the specimen with the immersion media to generate high-contrast images. Furthermore, it provides precise, quantitative measurements of various morphological and biophysical parameters, including volume, surface area, dry mass, mean thickness fluctuations, and height variations between cells [ 13 , 14 ]. Over the last two decades, variants of QPM have been developed, which can be classified into two main categories: common-path and non-common-path interferometric configurations [ 15 , 16 ]. A Linnik-based non-common-path interferometric QPM setup is used in this study. QPM encodes biological information, such as optical thickness (geometric thickness × refractive index), in the form of modulated intensity patterns known as interferograms. One of the important parameters of a QPM system is its spatial phase sensitivity, which determines the minimum phase/height detection limit of the system [ 17 – 19 ]. Therefore, the spatial phase sensitivity of the system must be high to detect minuscule variations, e.g., down to 10 nm differences in height, in biological specimens, as in the case of TNTs due to their sub-micrometer height. Here, the choice of the light source plays a crucial role in determining the phase sensitivity of the system [ 18 ]. With a fully coherent laser light source, it is quite easy to form interference fringes in a QPM system, but at the cost of significant speckle noise and parasitic fringe patterns, resulting in reduced spatial phase sensitivity. The highest phase sensitivity in a QPM system can be achieved by implementing low-coherence light sources such as halogen lamps and LEDs [ 17 , 19 ]. However, with this type of light source, obtaining interference fringes is challenging due to their short temporal coherence length (a few microns). The optical path difference between the object and the reference arm of the interferometer must be less than the coherence length of the interferometer to form interference fringes. These issues are addressed by implementing a pseudo-thermal light source (PTLS), which has low spatial coherence and high temporal coherence [ 20 – 23 ]. With PTLS, high spatial phase sensitivity comparable to that of low-coherence light sources can be achieved while also maintaining the ease of forming interference fringes. In addition, PTLS enables high space-time bandwidth product imaging in the QPM system [ 18 , 24 ]. A high space-time bandwidth is essential for biological applications such as the investigation of TNTs in real-time, where both high-speed and high-spatial sensitivity are required. This study leverages a PTLS-based partial spatial coherence QPM system, utilizing a Linnik interferometer configuration, to analyze and quantify TNTs in keratocytes from Atlantic salmon skin. Central to this study is the monitoring of changes in TNT height over time, with a particular emphasis on observing this change during their breaking process. Additionally, the study examines the morphology of TNTs by analyzing the average height-to-width ratio to better understand their shape. By comparing intact TNTs with those that have ruptured, the investigation explores their resilience and structural integrity, offering insights into the mechanical stability of these structures. Quantifying TNT dimensions also provides an estimate of the size of molecules and organelles capable of traversing these nanotubular conduits. Despite their recognized importance in human cell biology, the role of TNTs in fish physiology remains largely unexplored - an avenue that could serve as a future direction for this work. 2. Materials and Methods 2.1. Sample preparation: Post-smolt Atlantic salmon (500 g to 2 kg, mixed sexes) were obtained from Tromsø Aquaculture Research Station, Kårvika, Norway. The fish were kept in seawater (33–34 ppt salinity, 3–9°C, natural light) and received commercial diets (Skretting Spirit Trout 4.5, Celero 4.5). Ten fish were sampled during different periods of the year, all unvaccinated. After sanitizing equipment with Virkon, the fish were euthanized with a cranial blow and transported to the Norwegian College of Fishery Science, UiT, Tromsø. Scales were collected from one to two fish per experiment. The procedures followed Norwegian regulations for animal experimentation (Forskrift 2015-06-18-761) and EU Directive 2010/63/EU, allowing the use of unregulated post-mortem samples without FOTS applications. For imaging, salmon keratocyte samples were prepared on silicon wafers. A custom-made Polydimethylsiloxane (PDMS) chamber (12 mm × 12 mm, 150 µm thick) was applied to the wafer surface. Fish scales were collected from various skin dorsoventral areas using sterile tweezers and seeded onto the substrate with the interior surface in contact with the substrate. Scales adhered to the surface within approximately six minutes, after which they were exposed to 200 µL of Hank's Balanced Salt Solution (HBSS) (VWR, 21-023-CM) mixed with antibiotics, 100 µg/ml streptomycin, 100IU/ml penicillin (Sigma-P0781), and 1µg/ml of Amphotericin B solution (Sigma-A2942) solution. Samples were incubated at 12°C, cell avalanches were monitored for 2–4 days, and imaging was performed. 2.2. Experimental setup: Imaging data were acquired using a custom-built QPM system to thoroughly investigate TNTs in keratocytes. Figure 1 illustrates the Linnik interferometer-based QPM setup. The details of the setup can be found in Refs. [ 18 , 25 ]. A pseudo-thermal light source (PTLS) was generated by directing a 532 nm laser beam (Cobolt Flamenco laser) onto a rotating diffuser, followed by a multimodal fiber (MMF), as shown in Fig. 1 . The output of MMF was collimated through a lens (L1) and focused by a second lens (L2) at the back focal plane of the sample arm microscope objective lens (60×/1.2NA water immersion, model # UPlanSApo, Olympus). The beam splitter divides the input beam into two, one directed toward the sample arm, known as the sample beam, and the other toward the reference arm, known as the reference beam. The sample beam interacts with the sample, and its information is collected by the same objective lens. The reference beam is passed through the microscope objective lens 10×/0.25NA and is reflected from the reference mirror. Both light beams are recombined at the beam splitter plane to form interference fringes, which are then projected onto a camera using a tube lens. A piezoelectric stage is used in the reference arm to introduce phase stepping between consecutive frames. The acquisition of five phase-shifted frames is performed using a Hamamatsu CMOS camera (C11440-42U) with an effective number of pixels of 2048 (H) × 2048 (V) and a pixel size of 6.5 µm. The acquisition time required to capture five phase-shifted frames is approximately 600 ms at full frame and is performed using Micro-Manager. The sample is mounted on a motorized XYZ translation stage to select a region of interest. Previously, the developed system has demonstrated its effectiveness in studying the association of microplastics with salmon keratocytes [ 26 ]. To capture the dynamic behavior of TNTs, five phase-shifted interferograms of fish keratocytes forming TNTs were recorded and subsequently post-processed to reconstruct corresponding phase maps using the principal component analysis (PCA) algorithm [ 27 ]. The recovered phase is then numerically focused to minimize errors in the calculation of morphological parameters related to TNTs. 2.3. Geometrical height calculation: To quantify TNT height, a line profile with a width of 100 pixels was drawn perpendicular to the TNT length. Phase values were extracted along this line for each frame to monitor changes in the phase map over time. Geometrical height (GH) was then calculated by applying the following equation: $$\:H\left(x,y\right)=\frac{\lambda\:}{4\pi\:\left({n}_{s}-{n}_{m}\right)}\phi\:\left(x,y\right)$$ Where \(\:\phi\:\left(x,y\right)\) represents the measured phase map of the specimen, λ is the wavelength of the illumination light, \(\:{n}_{s}\) is the refractive index of the sample, and \(\:{n}_{m}\) is the refractive index of the surrounding medium. For the calculation of the GH of the TNTs, \(\:{n}_{s}\) =1.38 and \(\:{n}_{m}\) =1.33 were assumed. 3. Results and discussion 3.1. Quantitative phase imaging of salmon keratocyte TNTs Experiments were conducted on salmon keratocytes using a PTLS-QPM system to quantify the GH of TNTs formed between cells. We confirm that collecting fish and removing scales does not require specific ethical approval under the Norwegian Regulations for the Use of Animals in Research ( https://lovdata.no/dokument/SF/forskrift/2015-06-18-761#KAPITTEL_10 ). The procedure is also fully aligned with EU Directive 2010/63/EU ( https://eurlex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010L0063 ). All experimental steps adhered to the applicable guidelines and regulatory frameworks, and the reporting follows the ARRIVE recommendations ( https://arriveguidelines.org ). According to our protocol, this work can be conducted without a separate FOTS (Mattilsynet) approval. The system’s high contrast and nanometric optical path sensitivity enable detailed mapping of TNT morphology and its spatial connectivity between adjacent cells. TNTs appear as thin, elongated connections bridging keratocytes, with measurable phase shifts that reflect variations in height or subcellular mass density along their length. Such label-free visualizations are essential for investigating TNT dynamics and their functional roles in live cell interactions. Videos of up to 1000 timepoints (approximately around 10 minutes total), each with five phase-shifted interferograms, were recorded and post-processed as reported in Materials and Methods (See Supplementary-II Videos). An example of a phase-shifted interferogram is shown in Fig. 2 a. The corresponding reconstructed phase map and gt height map are presented in Figs. 2 b and 2 c, respectively. These images show the presence of TNTs connecting distant keratocytes, with the height of the highlighted TNT around 100 nm. Notably, the height of TNTs varies between cells, potentially reflecting differences in the nature or size of the cargo being transported. Cells may form TNTs with distinct height profiles to facilitate the transfer of specific organelles or molecular complexes, suggesting functional adaptation in their structural morphology [ 28 ]. 3.2. Height of broken vs unbroken TNTs TNTs exhibited a sensitivity to prolonged light exposure, often resulting in spatial oscillations followed by rupture. However, some TNTs remained stable throughout the imaging process, enabling comparative analysis. A key observation from the experimental data was that TNTs with greater overall height were more likely to remain intact, while thinner TNTs were more prone to breaking. This trend led to a focused comparative analysis of the height distribution between broken and unbroken TNTs. Figure 3 . Comparative Analysis of Average Heights: Unbroken vs. Broken TNTs. This plot illustrates the average heights of six unbroken TNTs against six TNTs prior to breaking, showing a higher range of height for the unbroken TNTs. The maximum value of the unbroken TNTs reaches up to 900 nm, suggesting potential structural resilience in TNTs with greater thickness. This analysis indicates that thicker TNTs may be more resistant to environmental stress. These results suggest a strong correlation between TNT heights and structural resilience. Thicker TNTs may possess increased mechanical stability, potentially due to enhanced cytoskeletal reinforcement or membrane composition, making them more resistant to stress-induced rupture. In human monocyte-derived macrophages, researchers have observed two distinct types of tunneling nanotubules (TNTs): thinner TNTs, approximately 300 nm in diameter, composed solely of F-actin, and thicker TNTs, around 800 nm, which contain both F-actin and microtubules. The thicker TNTs are capable of transporting organelles such as mitochondria, endosomes, and lysosomes [ 28 ]. Thinner TNTs in fish may be involved in the transfer of smaller organelles, calcium ions (Ca²⁺), and the propagation of long-distance electrical signals [ 6 , 7 ]. This trend highlights the significance of geometrical and mechanical parameters in determining TNT integrity and lifespan. The formation and breaking of TNTs are a continuous process as the epithelial cells move around. We observed the height dynamics of thinner TNTs over time, and eventually, they snap. Further research is required to explore the various factors influencing TNT rupture or stability and to fully understand their specific contributions to fish immunology. 3.3. Height measurement of broken TNTs as a function of time The temporal evolution of TNT height was analyzed using a sequence of ten frames from a representative movie: nine frames preceding a TNT rupture event and one frame captured immediately after the break. Figure 4 (a 1 –j 1 ) presents the reconstructed phase maps for each time point, where the yellow box indicates the specific region of interest (ROI) in which the TNT breakage occurs. This ROI was applied across all frames using the ROI Manager in ImageJ, enabling accurate cropping and analysis (see frames in Fig. 4 (a 2 –j 2 ). The evolution of TNT height is visualized through line profiles from frames (Figs. a 3 –j 3 ), revealing a clear trend for this TNT: the height decreases from approximately 0.27 µm in the early frames (Fig. a 3 ) to below 0.1 µm (Fig. i 3 ), just prior to rupture. The dynamic behavior of TNT height varied across different recordings. In some movies, TNT height showed noticeable fluctuations over time (as illustrated in Fig. 4 ), while others exhibited consistent height throughout the sequence (see example in Supplementary I Figure). All TNTs included in this study were analyzed following the same procedure described above. A representative movie is provided in supplementary Video V-IV. 3.4. Statistical analysis of TNT height and height-to-width ratio (H/W) over time The average height variation of TNTs over time is shown in Fig. 5 a, based on measurements from different TNTs across ten frames of six representative movies, all of which included TNT breakage events. Complete video datasets are available in the Supplementary Information. The analysis reveals that TNT thickness in salmon keratocytes can range from approximately 100 nm to 700 nm, reflecting their capacity to transport a variety of subcellular cargo, such as organelles or molecules of different sizes. The results from Fig. 5 a suggest that snapping of TNTs is mostly a sudden phenomenon and not a slow gradual decrease of height. The high sensitivity of the PTLS-QPM system allowed for the detection of the variation in TNT height leading to rupture. After the breaking event, the measured height of the TNTs nearly dropped to the height sensitivity limit, which is around 10 nm. Furthermore, the heights of TNTs are around 300nm, but in some cases, TNTs displayed heights exceeding the typical range, to around 700 nm, which may indicate the presence of larger transported materials or specialized structural adaptations, as it happens in human cell TNTs [ 29 ]. The widths of the six TNTs presented in Fig. 5 b demonstrate minor fluctuations, indicating that the width remains relatively consistent across the samples over time. Further, structural parameters such as the height-to-width (H/W) ratio can provide valuable insight into the functional adaptations and biomechanical integrity of TNTs during dynamic cellular processes. In this analysis, the H/W ratio was quantitatively measured and tracked over 10 s for six individual TNTs. As shown in Fig. 5 c, each TNT exhibits dynamic changes in this ratio. It is obvious that just when it starts to snap, the height reduces drastically, and at the snap, this ratio is undefined. The H/W ratio is less than 1, indicating that the cross-sectional geometry of TNTs deviates from a circular profile and instead is a more flattened or elliptical shape. Notably, a general pattern emerged in which TNTs exhibited a decrease in height following rupture events. The data from "video IV," marked with a light blue line, corresponds to Fig. 4 . 4. Conclusion This study demonstrates the application of partially spatially coherent QPM for high-resolution, label-free analysis of keratocyte TNTs and fine details of their morphological features. By employing a phase-shifting technique, precise measurements of TNT morphology and dynamics were achieved, revealing that TNT thickness typically ranges from 100 nm to 700 nm in these cells, with a maximum observed value of approximately 900 nm in intact TNTs. Temporal analysis indicated an increase in the average height-to-width ratio, suggesting TNTs might have an elliptical shape. Furthermore, comparisons between intact and broken TNTs highlighted a correlation between structural robustness and average height. These findings not only enhance understanding of TNT mechanical stability but also offer a foundation for exploring their functional relevance in fish physiology with a dedicated focus on the transfer of intracellular cargo. As TNTs remain understudied in fish physiology, this work provides a basis for future investigations into intercellular communication mechanisms in fish. Declarations Data availability: All data generated or analysed during this study are included in this article and its supplementary information files. The raw datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Funding: The study was funded by the University of Tromsø, the Arctic University of Norway, and the Research Council of Norway, grant no. 301401 and 325159. A.A. acknowledges FRIPRO Young (project # 345136) funding from the Research Council of Norway. B.S.A. and R.A.D. acknowledge RCN funded ULCER project # 352435 and Converse project # 352764. Contributions: A.A., R.A.D., and B.S.A. conceived the project, conceptualized the study, and secured the funding. B.M.A. performed the experiments and analyzed the data. A.A. developed the reconstruction software. B.M.A. and A.A. primarily wrote the manuscript. B.M.A. and M.K.M. prepared the biological samples and conducted the laboratory work. D.B.T. and D.L.W. supervised the biological laboratory work and provided biological insights. All authors reviewed and edited the manuscript. References Chase, C. Farmed salmon supply will remain flat in 2023, even as demand continues to increase . (2023). Persson, D. et al. Analysing mortality patterns in salmon farming using daily cage registrations. J. Fish Dis. 45 (2), 335–347 (2022). Song, D. et al. Cell–cell communication: old mystery and new opportunity p. 89–93 (Springer, 2019). Qin, Y. et al. 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Supplementary Files Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Dec, 2025 Reviews received at journal 22 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 01 Dec, 2025 Editor invited by journal 01 Dec, 2025 Submission checks completed at journal 28 Nov, 2025 First submitted to journal 28 Nov, 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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09:27:55","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77386,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/17f15422d216aee98b9e35b5.html"},{"id":97451537,"identity":"0d473063-703b-4887-bd66-8848bc826cf5","added_by":"auto","created_at":"2025-12-04 13:44:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the Linnik interferometry-based quantitative phase microscope with a pseudo thermal light source (PTLS-QPM). MMF: multimode fiber; L1 and L2: achromatic doublet lenses; BS: beam splitter; MO: microscope objective; PZT: piezoelectric transducer. \u003c/strong\u003e\u0026nbsp;Panel (A) shows one of the phase-shifted interferograms captured by the camera, and Panel (B) displays the recovered phase map. The color bar indicates the phase values in radians.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/3fa536fed38fae0a15fe835a.png"},{"id":97669225,"identity":"29e339da-83b8-4e9a-9525-e2883087a902","added_by":"auto","created_at":"2025-12-08 09:27:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":384190,"visible":true,"origin":"","legend":"\u003cp\u003eThe high resolution of the PTLS-QPM system allows for the precise visualization of fine connections between cells.\u003cstrong\u003e (a)\u003c/strong\u003e Phase-shifted interferometric image of a salmon TNT connecting two keratocytes, captured using the PTLS-QPM system. \u003cstrong\u003e(b)\u003c/strong\u003eReconstructed phase map from the interferometric image. White arrows point to the TNT connecting cells, and orange arrows show cell bodies. The color bar indicates the phase values in radians. \u003cstrong\u003e(c)\u003c/strong\u003eGeometrical height map displaying the estimated GH of the TNT. The color bar indicates the GH values in mm. The corresponding movie can be found in the supplementary video IV. \u0026nbsp;A 60× / 1.2 NA water immersion objective lens is utilized for imaging TNTs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/1399666fc83b3e84ff182ffe.png"},{"id":97451540,"identity":"ade72506-1447-48cc-a2e4-2fe93221258e","added_by":"auto","created_at":"2025-12-04 13:44:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43381,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Analysis of Average Heights: Unbroken vs. Broken TNTs. This plot illustrates the average heights of six unbroken TNTs against six TNTs prior to breaking, showing a higher range of height for the unbroken TNTs. The maximum value of the unbroken TNTs reaches up to 900 nm, suggesting potential structural resilience in TNTs with greater thickness. This analysis indicates that thicker TNTs may be more resistant to environmental stress.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/ab454ccea6b0851df991741d.png"},{"id":97451538,"identity":"7fd1b7e2-d835-49ef-8641-881a1ee47852","added_by":"auto","created_at":"2025-12-04 13:44:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328553,"visible":true,"origin":"","legend":"\u003cp\u003eChanges over time in the height of 10 frames from a representative movie, including one frame captured immediately after a snapping event. (a\u003csub\u003e1\u003c/sub\u003e – j\u003csub\u003e1\u003c/sub\u003e) Reconstructed phase map images, with a yellow box highlighting the specific ROI measured. (a\u003csub\u003e2\u003c/sub\u003e – j\u003csub\u003e2\u003c/sub\u003e) show the measured area (yellow box) within the specific ROI, with measurements taken across 100 pixels perpendicular to the length to determine the GH. (a\u003csub\u003e3\u003c/sub\u003e – j\u003csub\u003e3\u003c/sub\u003e) Line profiles measured from the ROIs illustrate the variations in thickness over the 10 frames. Notably, the dynamic behavior of TNT thickness varies among different TNTs, and this figure contains only one example.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/fd6717bb0fc76ea535e7eeb1.png"},{"id":97451542,"identity":"fb8ba4e6-7d1c-4bcd-ba51-f99114756baa","added_by":"auto","created_at":"2025-12-04 13:44:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153130,"visible":true,"origin":"","legend":"\u003cp\u003eThe higher phase sensitivity of the PTLS-QPM setup is used to quantify the \u003cdel\u003egt\u003c/del\u003e height variation and shape determination in TNTs. \u003cstrong\u003e(a)\u003c/strong\u003e This graph presents the average height variation across six different TNTs just prior to rupturing, each analyzed for 10 frames. The last frame of each sequence shows the height after snapping, which typically approaches zero. \u003cstrong\u003e(b)\u003c/strong\u003e This graph displays the width of the TNTs across the sequences. (c) displays the ratio of H/W of TNTs. This ratio suggests that the TNTs have a more flattened shape rather than a round.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/a485a2b0977b7e3c18599e2d.png"},{"id":107351028,"identity":"5c4bfac4-3fb6-4f8c-af4a-6f19a8c04c80","added_by":"auto","created_at":"2026-04-20 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1754931,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/217c4107-785b-427c-90b0-2c847f197030.pdf"},{"id":97451544,"identity":"55aebe4a-43b0-4c36-992f-506039bb3bf2","added_by":"auto","created_at":"2025-12-04 13:44:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4436207,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8172968/v1/30983344ecec3f7b438ba6cc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Real-time snapping dynamics and nanoscale thickness profiling of salmon keratocyte tunneling nanotubes using quantitative phase microscopy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAtlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e) farming in Norway has progressed to 1.5\u0026nbsp;million metric tons in 2023 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite this progress, farmed salmon still face nearly 20% mortality on average, largely due to infectious pathogens, physical injuries, and various stressors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While wound healing on the fish skin occurs following mucosal infection or injury, the cellular communications involved in this process remain poorly understood.\u003c/p\u003e\u003cp\u003eStudies on innate immunity in higher vertebrates suggest that cells communicate via ligands and receptors and through more direct cell-to-cell interactions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Through direct communication, the cells may even exchange organelles such as mitochondria, which is beneficial when it comes to cellular rescue and regeneration [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. One such direct path for cell communication is through special extended cytoskeletal protrusions, referred to as tunneling nanotubes (TNTs). These long membrane extensions may be a route of transfer of various small molecules, organelles, and even pathogens, including bacteria and viruses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TNT-like structures have previously been reported in different human immune cells, including B cells, T cells, and NK cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn various human cells, such as kidney cells, monocytes, and Jurkat T cells, TNTs connect cells over distances exceeding 100 \u0026micro;m [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, it has been reported that in cultured rat pheochromocytoma PC12 cells (neuroblastic-like), these TNTs can have diameters ranging from 0.05 \u0026micro;m to 1.5 \u0026micro;m and lengths extending across multiple cell diameters [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. TNTs have been documented in zebrafish, where TNT-like structures have been observed facilitating the transfer of Wnt proteins in embryos [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In related research, structures resembling TNTs were identified during a specific developmental stage of zebrafish, notably gastrulation. These structures facilitated the transport of proteins between distant cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To our knowledge, there are no reports on TNT-formation by skin primary epithelial cells in any animal species so far. Furthermore, structural and functional knowledge about TNTs in fish is limited. Intriguingly, in Atlantic salmon, we have observed that primary skin epithelial cells (keratocytes) produce TNT-like structures (not published).\u003c/p\u003e\u003cp\u003eLive cell imaging is an excellent model for observing cellular changes in real-time and investigating their interactions. Also, the artifacts generated due to the fixation process are eliminated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our methodology uses live cell imaging, which can be used to observe dynamic behavior and snapping events of the TNTs during cell migration. Since TNTs are small, slender, and relatively fragile structures, it is essential to use a label-free optical microscopy technique that can provide high-contrast images at high-speed. Otherwise, any other microscopic methods may affect the TNT dynamics.\u003c/p\u003e\u003cp\u003eQuantitative phase microscopy (QPM) is a non-invasive, non-contact, and label-free imaging technique highly compatible with live-cell studies. QPM uses the intrinsic refractive index contrast of the specimen with the immersion media to generate high-contrast images. Furthermore, it provides precise, quantitative measurements of various morphological and biophysical parameters, including volume, surface area, dry mass, mean thickness fluctuations, and height variations between cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Over the last two decades, variants of QPM have been developed, which can be classified into two main categories: common-path and non-common-path interferometric configurations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A Linnik-based non-common-path interferometric QPM setup is used in this study. QPM encodes biological information, such as optical thickness (geometric thickness \u0026times; refractive index), in the form of modulated intensity patterns known as interferograms.\u003c/p\u003e\u003cp\u003eOne of the important parameters of a QPM system is its spatial phase sensitivity, which determines the minimum phase/height detection limit of the system [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, the spatial phase sensitivity of the system must be high to detect minuscule variations, e.g., down to 10 nm differences in height, in biological specimens, as in the case of TNTs due to their sub-micrometer height. Here, the choice of the light source plays a crucial role in determining the phase sensitivity of the system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. With a fully coherent laser light source, it is quite easy to form interference fringes in a QPM system, but at the cost of significant speckle noise and parasitic fringe patterns, resulting in reduced spatial phase sensitivity. The highest phase sensitivity in a QPM system can be achieved by implementing low-coherence light sources such as halogen lamps and LEDs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, with this type of light source, obtaining interference fringes is challenging due to their short temporal coherence length (a few microns). The optical path difference between the object and the reference arm of the interferometer must be less than the coherence length of the interferometer to form interference fringes. These issues are addressed by implementing a pseudo-thermal light source (PTLS), which has low spatial coherence and high temporal coherence [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. With PTLS, high spatial phase sensitivity comparable to that of low-coherence light sources can be achieved while also maintaining the ease of forming interference fringes. In addition, PTLS enables high space-time bandwidth product imaging in the QPM system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A high space-time bandwidth is essential for biological applications such as the investigation of TNTs in real-time, where both high-speed and high-spatial sensitivity are required.\u003c/p\u003e\u003cp\u003eThis study leverages a PTLS-based partial spatial coherence QPM system, utilizing a Linnik interferometer configuration, to analyze and quantify TNTs in keratocytes from Atlantic salmon skin. Central to this study is the monitoring of changes in TNT height over time, with a particular emphasis on observing this change during their breaking process. Additionally, the study examines the morphology of TNTs by analyzing the average height-to-width ratio to better understand their shape. By comparing intact TNTs with those that have ruptured, the investigation explores their resilience and structural integrity, offering insights into the mechanical stability of these structures. Quantifying TNT dimensions also provides an estimate of the size of molecules and organelles capable of traversing these nanotubular conduits. Despite their recognized importance in human cell biology, the role of TNTs in fish physiology remains largely unexplored - an avenue that could serve as a future direction for this work.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sample preparation:\u003c/h2\u003e\u003cp\u003ePost-smolt Atlantic salmon (500 g to 2 kg, mixed sexes) were obtained from Troms\u0026oslash; Aquaculture Research Station, K\u0026aring;rvika, Norway. The fish were kept in seawater (33\u0026ndash;34 ppt salinity, 3\u0026ndash;9\u0026deg;C, natural light) and received commercial diets (Skretting Spirit Trout 4.5, Celero 4.5). Ten fish were sampled during different periods of the year, all unvaccinated. After sanitizing equipment with Virkon, the fish were euthanized with a cranial blow and transported to the Norwegian College of Fishery Science, UiT, Troms\u0026oslash;. Scales were collected from one to two fish per experiment. The procedures followed Norwegian regulations for animal experimentation (Forskrift 2015-06-18-761) and EU Directive 2010/63/EU, allowing the use of unregulated post-mortem samples without FOTS applications.\u003c/p\u003e\u003cp\u003eFor imaging, salmon keratocyte samples were prepared on silicon wafers. A custom-made Polydimethylsiloxane (PDMS) chamber (12 mm \u0026times; 12 mm, 150 \u0026micro;m thick) was applied to the wafer surface. Fish scales were collected from various skin dorsoventral areas using sterile tweezers and seeded onto the substrate with the interior surface in contact with the substrate. Scales adhered to the surface within approximately six minutes, after which they were exposed to 200 \u0026micro;L of Hank's Balanced Salt Solution (HBSS) (VWR, 21-023-CM) mixed with antibiotics, 100 \u0026micro;g/ml streptomycin, 100IU/ml penicillin (Sigma-P0781), and 1\u0026micro;g/ml of Amphotericin B solution (Sigma-A2942) solution. Samples were incubated at 12\u0026deg;C, cell avalanches were monitored for 2\u0026ndash;4 days, and imaging was performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental setup:\u003c/h2\u003e\u003cp\u003eImaging data were acquired using a custom-built QPM system to thoroughly investigate TNTs in keratocytes. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the Linnik interferometer-based QPM setup. The details of the setup can be found in Refs. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A pseudo-thermal light source (PTLS) was generated by directing a 532 nm laser beam (Cobolt Flamenco laser) onto a rotating diffuser, followed by a multimodal fiber (MMF), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The output of MMF was collimated through a lens (L1) and focused by a second lens (L2) at the back focal plane of the sample arm microscope objective lens (60\u0026times;/1.2NA water immersion, model # UPlanSApo, Olympus). The beam splitter divides the input beam into two, one directed toward the sample arm, known as the sample beam, and the other toward the reference arm, known as the reference beam. The sample beam interacts with the sample, and its information is collected by the same objective lens. The reference beam is passed through the microscope objective lens 10\u0026times;/0.25NA and is reflected from the reference mirror. Both light beams are recombined at the beam splitter plane to form interference fringes, which are then projected onto a camera using a tube lens. A piezoelectric stage is used in the reference arm to introduce phase stepping between consecutive frames. The acquisition of five phase-shifted frames is performed using a Hamamatsu CMOS camera (C11440-42U) with an effective number of pixels of 2048 (H) \u0026times; 2048 (V) and a pixel size of 6.5 \u0026micro;m. The acquisition time required to capture five phase-shifted frames is approximately 600 ms at full frame and is performed using Micro-Manager. The sample is mounted on a motorized XYZ translation stage to select a region of interest. Previously, the developed system has demonstrated its effectiveness in studying the association of microplastics with salmon keratocytes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo capture the dynamic behavior of TNTs, five phase-shifted interferograms of fish keratocytes forming TNTs were recorded and subsequently post-processed to reconstruct corresponding phase maps using the principal component analysis (PCA) algorithm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The recovered phase is then numerically focused to minimize errors in the calculation of morphological parameters related to TNTs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Geometrical height calculation:\u003c/h2\u003e\u003cp\u003eTo quantify TNT height, a line profile with a width of 100 pixels was drawn perpendicular to the TNT length. Phase values were extracted along this line for each frame to monitor changes in the phase map over time. Geometrical height (GH) was then calculated by applying the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:H\\left(x,y\\right)=\\frac{\\lambda\\:}{4\\pi\\:\\left({n}_{s}-{n}_{m}\\right)}\\phi\\:\\left(x,y\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\phi\\:\\left(x,y\\right)\\)\u003c/span\u003e\u003c/span\u003e represents the measured phase map of the specimen, λ is the wavelength of the illumination light, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{s}\\)\u003c/span\u003e\u003c/span\u003e is the refractive index of the sample, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{m}\\)\u003c/span\u003e\u003c/span\u003e is the refractive index of the surrounding medium. For the calculation of the GH of the TNTs, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{s}\\)\u003c/span\u003e\u003c/span\u003e=1.38 and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{m}\\)\u003c/span\u003e\u003c/span\u003e=1.33 were assumed.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Quantitative phase imaging of salmon keratocyte TNTs\u003c/h2\u003e\u003cp\u003eExperiments were conducted on salmon keratocytes using a PTLS-QPM system to quantify the GH of TNTs formed between cells. We confirm that collecting fish and removing scales does not require specific ethical approval under the Norwegian Regulations for the Use of Animals in Research (\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). The procedure is also fully aligned with EU Directive 2010/63/EU (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://eurlex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010L0063\u003c/span\u003e\u003cspan address=\"https://eurlex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32010L0063\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All experimental steps adhered to the applicable guidelines and regulatory frameworks, and the reporting follows the ARRIVE recommendations (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). According to our protocol, this work can be conducted without a separate FOTS (Mattilsynet) approval.\u003c/p\u003e\u003cp\u003eThe system\u0026rsquo;s high contrast and nanometric optical path sensitivity enable detailed mapping of TNT morphology and its spatial connectivity between adjacent cells. TNTs appear as thin, elongated connections bridging keratocytes, with measurable phase shifts that reflect variations in height or subcellular mass density along their length. Such label-free visualizations are essential for investigating TNT dynamics and their functional roles in live cell interactions. Videos of up to 1000 timepoints (approximately around 10 minutes total), each with five phase-shifted interferograms, were recorded and post-processed as reported in Materials and Methods (See Supplementary-II Videos).\u003c/p\u003e\u003cp\u003eAn example of a phase-shifted interferogram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The corresponding reconstructed phase map and gt height map are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, respectively. These images show the presence of TNTs connecting distant keratocytes, with the height of the highlighted TNT around 100 nm. Notably, the height of TNTs varies between cells, potentially reflecting differences in the nature or size of the cargo being transported. Cells may form TNTs with distinct height profiles to facilitate the transfer of specific organelles or molecular complexes, suggesting functional adaptation in their structural morphology [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Height of broken vs unbroken TNTs\u003c/h2\u003e\u003cp\u003eTNTs exhibited a sensitivity to prolonged light exposure, often resulting in spatial oscillations followed by rupture. However, some TNTs remained stable throughout the imaging process, enabling comparative analysis. A key observation from the experimental data was that TNTs with greater overall height were more likely to remain intact, while thinner TNTs were more prone to breaking. This trend led to a focused comparative analysis of the height distribution between broken and unbroken TNTs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Comparative Analysis of Average Heights: Unbroken vs. Broken TNTs. This plot illustrates the average heights of six unbroken TNTs against six TNTs prior to breaking, showing a higher range of height for the unbroken TNTs. The maximum value of the unbroken TNTs reaches up to 900 nm, suggesting potential structural resilience in TNTs with greater thickness. This analysis indicates that thicker TNTs may be more resistant to environmental stress.\u003c/p\u003e\u003cp\u003eThese results suggest a strong correlation between TNT heights and structural resilience. Thicker TNTs may possess increased mechanical stability, potentially due to enhanced cytoskeletal reinforcement or membrane composition, making them more resistant to stress-induced rupture. In human monocyte-derived macrophages, researchers have observed two distinct types of tunneling nanotubules (TNTs): thinner TNTs, approximately 300 nm in diameter, composed solely of F-actin, and thicker TNTs, around 800 nm, which contain both F-actin and microtubules. The thicker TNTs are capable of transporting organelles such as mitochondria, endosomes, and lysosomes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thinner TNTs in fish may be involved in the transfer of smaller organelles, calcium ions (Ca\u0026sup2;⁺), and the propagation of long-distance electrical signals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This trend highlights the significance of geometrical and mechanical parameters in determining TNT integrity and lifespan. The formation and breaking of TNTs are a continuous process as the epithelial cells move around. We observed the height dynamics of thinner TNTs over time, and eventually, they snap. Further research is required to explore the various factors influencing TNT rupture or stability and to fully understand their specific contributions to fish immunology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Height measurement of broken TNTs as a function of time\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe temporal evolution of TNT height was analyzed using a sequence of ten frames from a representative movie: nine frames preceding a TNT rupture event and one frame captured immediately after the break. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a\u003csub\u003e1\u003c/sub\u003e\u0026ndash;j\u003csub\u003e1\u003c/sub\u003e) presents the reconstructed phase maps for each time point, where the yellow box indicates the specific region of interest (ROI) in which the TNT breakage occurs. This ROI was applied across all frames using the ROI Manager in ImageJ, enabling accurate cropping and analysis (see frames in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a\u003csub\u003e2\u003c/sub\u003e\u0026ndash;j\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\u003cp\u003eThe evolution of TNT height is visualized through line profiles from frames (Figs. a\u003csub\u003e3\u003c/sub\u003e\u0026ndash;j\u003csub\u003e3\u003c/sub\u003e), revealing a clear trend for this TNT: the height decreases from approximately 0.27 \u0026micro;m in the early frames (Fig. a\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) to below 0.1 \u0026micro;m (Fig. i\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), just prior to rupture.\u003c/p\u003e\u003cp\u003eThe dynamic behavior of TNT height varied across different recordings. In some movies, TNT height showed noticeable fluctuations over time (as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while others exhibited consistent height throughout the sequence (see example in Supplementary I Figure). All TNTs included in this study were analyzed following the same procedure described above. A representative movie is provided in supplementary Video V-IV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Statistical analysis of TNT height and height-to-width ratio (H/W) over time\u003c/h2\u003e\u003cp\u003eThe average height variation of TNTs over time is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, based on measurements from different TNTs across ten frames of six representative movies, all of which included TNT breakage events. Complete video datasets are available in the Supplementary Information. The analysis reveals that TNT thickness in salmon keratocytes can range from approximately 100 nm to 700 nm, reflecting their capacity to transport a variety of subcellular cargo, such as organelles or molecules of different sizes. The results from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea suggest that snapping of TNTs is mostly a sudden phenomenon and not a slow gradual decrease of height. The high sensitivity of the PTLS-QPM system allowed for the detection of the variation in TNT height leading to rupture. After the breaking event, the measured height of the TNTs nearly dropped to the height sensitivity limit, which is around 10 nm. Furthermore, the heights of TNTs are around 300nm, but in some cases, TNTs displayed heights exceeding the typical range, to around 700 nm, which may indicate the presence of larger transported materials or specialized structural adaptations, as it happens in human cell TNTs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The widths of the six TNTs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb demonstrate minor fluctuations, indicating that the width remains relatively consistent across the samples over time.\u003c/p\u003e\u003cp\u003eFurther, structural parameters such as the height-to-width (H/W) ratio can provide valuable insight into the functional adaptations and biomechanical integrity of TNTs during dynamic cellular processes. In this analysis, the H/W ratio was quantitatively measured and tracked over 10 s for six individual TNTs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, each TNT exhibits dynamic changes in this ratio. It is obvious that just when it starts to snap, the height reduces drastically, and at the snap, this ratio is undefined. The H/W ratio is less than 1, indicating that the cross-sectional geometry of TNTs deviates from a circular profile and instead is a more flattened or elliptical shape. Notably, a general pattern emerged in which TNTs exhibited a decrease in height following rupture events. The data from \"video IV,\" marked with a light blue line, corresponds to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates the application of partially spatially coherent QPM for high-resolution, label-free analysis of keratocyte TNTs and fine details of their morphological features. By employing a phase-shifting technique, precise measurements of TNT morphology and dynamics were achieved, revealing that TNT thickness typically ranges from 100 nm to 700 nm in these cells, with a maximum observed value of approximately 900 nm in intact TNTs. Temporal analysis indicated an increase in the average height-to-width ratio, suggesting TNTs might have an elliptical shape. Furthermore, comparisons between intact and broken TNTs highlighted a correlation between structural robustness and average height. These findings not only enhance understanding of TNT mechanical stability but also offer a foundation for exploring their functional relevance in fish physiology with a dedicated focus on the transfer of intracellular cargo. As TNTs remain understudied in fish physiology, this work provides a basis for future investigations into intercellular communication mechanisms in fish.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article and its supplementary information files. The raw datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by the University of Troms\u0026oslash;, the Arctic University of Norway, and the Research Council of Norway, grant no. 301401 and 325159. A.A. acknowledges FRIPRO Young (project # 345136) funding from the Research Council of Norway. B.S.A. and R.A.D. acknowledge RCN funded ULCER project # 352435 and Converse project # 352764.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.A., R.A.D., and B.S.A. conceived the project, conceptualized the study, and secured the funding. B.M.A. performed the experiments and analyzed the data. A.A. developed the reconstruction software. B.M.A. and A.A. primarily wrote the manuscript. B.M.A. and M.K.M. prepared the biological samples and conducted the laboratory work. D.B.T. and D.L.W. supervised the biological laboratory work and provided biological insights. All authors reviewed and edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChase, C. \u003cem\u003eFarmed salmon supply will remain flat in 2023, even as demand continues to increase\u003c/em\u003e. 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Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (20), 12545 (2022).\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-8172968/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8172968/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe highly migratory skin epithelial cells of fish, named keratocytes, display interesting features, including long cellular protrusions resembling tunneling nanotubes (TNTs). It has been shown in other cell types that TNTs may transfer cellular cargo between cells. These TNTs are instrumental not only in cargo transport but also in mediating signaling between cells. Due to the varying height of TNTs, down to 100 nm or less, a highly sensitive quantitative microscopy technique is required for accurate quantification of their morphological and dynamic properties. In this study, a partially spatially coherent quantitative phase microscopy (QPM) system was utilized, providing extremely high spatial sensitivity to track changes in TNT height over time, particularly during the breaking/snapping process. A phase-shifting technique was employed to recover high-resolution phase maps of the TNTs. Observations indicate that TNT height typically ranges from 100 nm to 700 nm. Additionally, the average height-to-width ratio over time suggests that the TNTs adopt a less rounded but flattened shape. Comparisons between intact and broken TNTs further reveal that unbroken TNTs generally exhibit a higher average height, with a maximum observed value of approximately 900 nm. Beyond morphological characterization, this work may open new avenues for understanding TNTs\u0026acute; potential role in the transfer of intracellular cargo, paving the way for future investigations into cellular communication mechanisms.\u003c/p\u003e","manuscriptTitle":"Real-time snapping dynamics and nanoscale thickness profiling of salmon keratocyte tunneling nanotubes using quantitative phase microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 13:44:11","doi":"10.21203/rs.3.rs-8172968/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-29T04:13:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T15:58:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T01:14:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T20:04:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98198530825961672332310505070983204824","date":"2025-12-02T22:47:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307705390868436537847519656296893011933","date":"2025-12-02T18:44:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170617794604861843026766520882255830006","date":"2025-12-02T17:10:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79211349112059007427204405301667727569","date":"2025-12-02T15:56:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T15:31:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T16:21:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-01T10:40:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-28T13:08:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-28T13:01:15+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":"163675ee-4f70-444c-b5d9-092ce433bbeb","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58989801,"name":"Biological sciences/Biological techniques"},{"id":58989802,"name":"Biological sciences/Biophysics"},{"id":58989803,"name":"Biological sciences/Cell biology"},{"id":58989804,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2026-04-20T16:05:50+00:00","versionOfRecord":{"articleIdentity":"rs-8172968","link":"https://doi.org/10.1038/s41598-026-46064-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-17 15:58:57","publishedOnDateReadable":"April 17th, 2026"},"versionCreatedAt":"2025-12-04 13:44:11","video":"","vorDoi":"10.1038/s41598-026-46064-1","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46064-1","workflowStages":[]},"version":"v1","identity":"rs-8172968","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8172968","identity":"rs-8172968","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}