Volumetric electron microscopy can reveal cellular structures in remarkable detail. But when a dataset contains hundreds or thousands of individual 2D images, understanding how those structures fit together in three dimensions can be challenging.
In a recent study published in npj Vaccines, researchers at Radboud University Medical Center (Radboudumc) investigated the early immune response following intranasal administration of an outer membrane vesicle (OMV)-based pneumococcal vaccine candidate in mice. The team combined several imaging and cell-analysis techniques to track where the vaccine travelled and how immune cells responded across the respiratory tract during the first 72 hours after immunization.
A particular focus was understanding how vaccine particles interacted with immune cells in the nasal tissue. To take a closer look, the researchers combined Airyscan fluorescence microscopy, which helped locate vaccine-associated signals and specific immune cells, with focused ion beam-scanning electron microscopy (FIB-SEM), which provided highly detailed views of their cellular structures.
Dragonfly 3D World supported the 3D visualization stage of this workflow. After structures of interest were identified across the FIB-SEM images, the researchers used the software to process the segmented data, reconstruct cellular and subcellular structures, and explore how they were organized in relation to one another throughout the image volume.
Study at a glance
Paper: Tracking spatio-temporal dynamics of early immune responses to an intranasal OMV-based pneumococcal vaccine candidate in mice.
Imaging approach: In vivo imaging, fluorescence microscopy, CLEM, and volumetric FIB-SEM
Analysis approach: Immune-cell analysis, image segmentation, and 3D reconstruction and visualization in 3D World
Tracking the early response to intranasal vaccination
To investigate what happens during the earliest stages following intranasal immunization, the researchers followed both the vaccine candidate and immune response at multiple time points between 1 and 72 hours.
Imaging detected vaccine-associated signals in the nasal cavity within the first hour, where they remained visible for up to 48 hours. The researchers also observed the vaccine moving toward the lower respiratory tract. At the same time, cell analysis showed that immune responses varied between the nasal tissue, lungs, lymph nodes, and spleen.
Neutrophils – immune cells that act as early responders – were particularly prominent in the nasal tissue and emerged as the main cell type interacting with the vaccine particles. In the lungs, several other immune-cell populations were involved, including macrophages and dendritic cells. The researchers also observed changes in T cells, showing that the immune response evolved across different tissues and time points.
Identifying which immune cells were engaging with the vaccine, however, was only one part of the research question. The team also wanted to understand these interactions within their cellular and subcellular context.
Connecting fluorescence with cellular ultrastructure
To investigate these interactions more closely, the researchers used correlative light and electron microscopy (CLEM), an approach that combines information from different types of microscopy to examine the same area at different levels of detail.
Airyscan microscopy was used to locate vaccine-associated signals and identify neutrophils within the nasal tissue. A cell of interest was then selected for volumetric FIB-SEM imaging, allowing the researchers to connect the fluorescence information with a much more detailed view of the cell’s internal structures.
The resulting FIB-SEM dataset contained 1,284 images, each capturing another 5-nanometer section through the sample. While these images provided exceptional detail, understanding how the cell and its internal structures fit together across more than a thousand individual views was much more difficult.
This was where Dragonfly 3D World became an important part of the imaging workflow.
Reconstructing segmented FIB-SEM data in 3D World
After the FIB-SEM images were acquired, key structures (including the cell membrane, nucleus, and internal compartments called vesicles) were identified and separated from the surrounding image data using Arivis software. The segmented data were then imported into 3D World for further processing and 3D visualization.
In 3D World, the researchers could turn the segmented regions into separate 3D objects representing structures such as the cell membrane, nucleus, and vesicles. This made it possible to view each structure individually or bring them together as part of the complete reconstructed cell.
The vaccine-associated fluorescence was also incorporated into the reconstruction, bringing information from the fluorescence and FIB-SEM images together within the same 3D model. This allowed the researchers to see where the vaccine-associated signals appeared in relation to different parts of the cell.
With these elements brought together, the researchers could move beyond inspecting the FIB-SEM dataset one section at a time. By viewing multiple segmented structures simultaneously in 3D World, they could explore how the membrane, nucleus, vesicles, and vaccine-associated signal were positioned relative to one another throughout the complete image volume.
Seeing vaccine–immune cell interactions in 3D
The 3D reconstruction provided a clearer view of the neutrophil and its internal structures than individual FIB-SEM sections alone.
The researchers observed a complex cell shape, including membrane extensions and numerous internal vesicles. When the fluorescence information was added, vaccine-associated signals could be seen near the outer cell membrane, within some of its extensions, close to internal vesicles, and near the nucleus.
Rather than requiring the researchers to piece these spatial relationships together from successive 2D images, 3D World allowed them to explore the complete reconstructed volume. Different structures could be viewed individually or together, making it easier to understand how the different parts of the cell were organized in relation to one another.
This also supported how the complex dataset could be presented. The publication's Figure 7 progresses from the correlated fluorescence and FIB-SEM views to segmented cellular structures and complete 3D renderings, demonstrating how the volumetric data could be transformed into a clearer representation of the cell and its internal structures.
3D reconstruction of the FIB-SEM dataset in Dragonfly 3D World, showing multiple segmented cellular and subcellular structures throughout the reconstructed volume.
Connecting immune responses across scales
Together, the different techniques used in the study allowed the researchers to follow the immune response from the whole organism down to individual cell.
Imaging showed where the vaccine travelled, while cell analysis revealed which immune-cell populations responded over time. Fluorescence microscopy helped identify vaccine-associated signals within specific cells, and FIB-SEM provided a highly detailed view of their internal structures.
3D World completed the visualization stage by bringing the segmented FIB-SEM structures together in 3D. This added the spatial context needed to understand how the detailed features captured in individual images were organized throughout the reconstructed cell.
Overall, the study showed that immune responses varied across different tissues and time points and highlighted the important role of neutrophils in the early response to the vaccine within the nasal tissue.
The same type of workflow could also support research beyond this particular vaccine candidate. Combining detailed volumetric imaging with 3D reconstruction could help researchers investigate how vaccines, pathogens, nanoparticles, and other biological materials interact with specific cells and structures within complex tissues.
Read the paper
Kanwal S, To SV, Uijen R, et al. Tracking spatio-temporal dynamics of early immune responses to an intranasal OMV-based pneumococcal vaccine candidate in mice. npj Vaccines. 2026;11:105. DOI: 10.1038/s41541-026-01430-y.
Researchers
This research was conducted at Radboud University Medical Center (Radboudumc), Nijmegen, under the mentorship of Prof. Marien I. de Jonge, with contributions from the Electron Microscopy Center (EMC) at Radboudumc and Abera Bioscience AB, Uppsala, Sweden.
Authors: Sajida Kanwal, Shaina Vivienne To, Rienke Uijen, Rona Roverts, Bram van Cranenbroek, Fred J. van Opzeeland, Ben Joosten, Bart van den Berg van Saparoea, Christa E. van der Gaast-de Jongh, Esther van Rijssen, Joen Luirink, Dimitri A. Diavatopoulos, Lucille F. van Beek, and Marien I. de Jonge.
Explore your data in 3D with 3D World
Turn complex image data into explorable 3D visualizations with Dragonfly 3D World. Start your free 30-day trial today.