Browsing by Subject "MICROSCOPY"

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  • Kassamakov, Ivan; Lecler, Sylvain; Nolvi, Anton; Leong-Hoi, Audrey; Montgomery, Paul; Haeggström, Edward (2017)
    We present quantitative three dimensional images of grooves on a writable Blu-ray Disc based on a single objective Mirau type interferometric microscope, enhanced with a microsphere which is considered as a photonic nanojet source. Along the optical axis the resolution of this microsphere assisted interferometry system is a few nanometers while the lateral resolution is around 112 nm. To understand the physical phenomena involved in this kind of imaging we have modelled the interaction between the photonic jet and the complex disc surface. Agreement between simulation and experimental results is demonstrated. We underline that although the ability of the microsphere to generate a photonic nanojet does not alone explain the resolution of the interferometer, the nanojet can be used to try to understand the imaging process. To partly explain the lateral super-resolution, the potential role of coherence is illustrated. The presented modality may have a large impact on many fields from bio-medicine to nanotechnology.
  • Rantala, Juha K.; Makela, Rami; Aaltola, Anna-Riina; Laasola, Petra; Mpindi, John-Patrick; Nees, Matthias; Saviranta, Petri; Kallioniemi, Olli (2011)
  • Suleymanova, Ilida; Balassa, Tamas; Tripathi, Sushil; Molnar, Csaba; Saarma, Mart; Sidorova, Yulia; Horvath, Peter (2018)
    Astrocytes are involved in various brain pathologies including trauma, stroke, neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, or chronic pain. Determining cell density in a complex tissue environment in microscopy images and elucidating the temporal characteristics of morphological and biochemical changes is essential to understand the role of astrocytes in physiological and pathological conditions. Nowadays, manual stereological cell counting or semi-automatic segmentation techniques are widely used for the quantitative analysis of microscopy images. Detecting astrocytes automatically is a highly challenging computational task, for which we currently lack efficient image analysis tools. We have developed a fast and fully automated software that assesses the number of astrocytes using Deep Convolutional Neural Networks (DCNN). The method highly outperforms state-of-the-art image analysis and machine learning methods and provides precision comparable to those of human experts. Additionally, the runtime of cell detection is significantly less than that of other three computational methods analysed, and it is faster than human observers by orders of magnitude. We applied our DCNN-based method to examine the number of astrocytes in different brain regions of rats with opioid-induced hyperalgesia/tolerance (OIH/OIT), as morphine tolerance is believed to activate glia. We have demonstrated a strong positive correlation between manual and DCNN-based quantification of astrocytes in rat brain.
  • Linder, Nina; Turkki, Riku; Walliander, Margarita; Martensson, Andreas; Diwan, Vinod; Rahtu, Esa; Pietikainen, Matti; Lundin, Mikael; Lundin, Johan (2014)
  • Diosdi, Akos; Hirling, Dominik; Kovacs, Maria; Toth, Timea; Harmati, Maria; Koos, Krisztian; Buzas, Krisztina; Piccinini, Filippo; Horvath, Peter (2021)
    3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
  • Matlashov, Mikhail E.; Shcherbakova, Daria M.; Alvelid, Jonatan; Baloban, Mikhail; Pennacchietti, Francesca; Shemetov, Anton A.; Testa, Ilaria; Verkhusha, Vladislav V. (2020)
    Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve -40nm resolution in live cells. In living mice we detect -10(5) fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670nm to 720nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.
  • Molnar, Csaba; Jermyn, Ian H.; Kato, Zoltan; Rahkama, Vesa; Ostling, Paivi; Mikkonen, Piia; Pietiainen, Vilja; Horvath, Peter (2016)
    The identification of fluorescently stained cell nuclei is the basis of cell detection, segmentation, and feature extraction in high content microscopy experiments. The nuclear morphology of single cells is also one of the essential indicators of phenotypic variation. However, the cells used in experiments can lose their contact inhibition, and can therefore pile up on top of each other, making the detection of single cells extremely challenging using current segmentation methods. The model we present here can detect cell nuclei and their morphology even in high-confluency cell cultures with many overlapping cell nuclei. We combine the "gas of near circles" active contour model, which favors circular shapes but allows slight variations around them, with a new data model. This captures a common property of many microscopic imaging techniques: the intensities from superposed nuclei are additive, so that two overlapping nuclei, for example, have a total intensity that is approximately double the intensity of a single nucleus. We demonstrate the power of our method on microscopic images of cells, comparing the results with those obtained from a widely used approach, and with manual image segmentations by experts.
  • Harjumaki, Riina; Zhang, Xue; Nugroho, Robertus Wahyu N.; Farooq, Muhammad; Lou, Yan-Ru; Yliperttula, Marjo; Valle-Delgado, Juan Jose; Osterberg, Monika (2020)
    Transmembrane protein integrins play a key role in cell adhesion. Cell-biomaterial interactions are affected by integrin expression and conformation, which are actively controlled by cells. Although integrin structure and function have been studied in detail, quantitative analyses of integrin-mediated cell-biomaterial interactions are still scarce. Here, we have used atomic force spectroscopy to study how integrin distribution and activation (via intracellular mechanisms in living cells or by divalent cations) affect the interaction of human pluripotent stem cells (WA07) and human hepatocarcinoma cells (HepG2) with promising biomaterials.human recombinant laminin-521 (LN-521) and cellulose nanofibrils (CNF). Cell adhesion to LN-521-coated probes was remarkably influenced by cell viability, divalent cations, and integrin density in WA07 colonies, indicating that specific bonds between LN-521 and activated integrins play a significant role in the interactions between LN-521 and HepG2 and WA07 cells. In contrast, the interactions between CNF and cells were nonspecific and not influenced by cell viability or the presence of divalent cations. These results shed light on the underlying mechanisms of cell adhesion, with direct impact on cell culture and tissue engineering applications.
  • Aho, Vesa; Myllys, Markko; Ruokolainen, Visa; Hakanen, Satu; Mantyla, Elina; Virtanen, Jori; Hukkanen, Veijo; Kuhn, Thomas; Timonen, Jussi; Mattila, Keijo; Larabell, Carolyn A.; Vihinen-Ranta, Maija (2017)
    Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.
  • Pirhonen, Juho; Arola, Johanna; Sädevirta, Sanja; Luukkonen, Panu; Karppinen, Sanna-Maria; Pihlajaniemi, Taina; Isomäki, Antti; Hukkanen, Mika; Yki-Jarvinen, Hannele; Ikonen, Elina (2016)
    Background and Aims Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. Methods We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. Results We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. Conclusions This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.
  • Ruutila, Minna; Fagerholm, Per; Lagali, Neil; Hjortdal, Jesper; Bram, Thue; Moilanen, Jukka; Kivelä, Tero T. (2021)
    Purpose: To refine the diagnostic criteria for Terrien marginal degeneration (TMD) based on experience in 3 Nordic countries. Methods: This is a retrospective, multicenter, hospital-based cross-sectional study of 49 eyes of 29 white patients in tertiary referral centers in Finland, Sweden, and Denmark from 1998 to January 2018. The median follow-up was 3 years. Symptoms, best corrected visual acuity, astigmatism, corneal thickness, curvature and cavities, stage, and progression were analyzed. Results: TMD was diagnosed equally likely between 15 and 86 years of age (median, 47 years). Twenty patients (69%) had bilateral disease, and 62% were men. Seventeen patients (59%) had symptoms including blurred vision and ocular surface disease symptoms without inflammatory signs. Eight patients (28%) had slightly reduced corneal sensitivity. Median best corrected visual acuity was 20/25 (range, 20/16-20/200) and astigmatism was 2.6 diopters (D) (range, 0-10) with a mean progression of 0.41 D per year (range, 0-5.4). Age and astigmatism were not correlated. All eyes had peripheral vascularization, lipid deposits, and hyperreflectivity throughout thinned peripheral stroma and its anterior edge. The thinning progressed in 15 patients (52%). Of 26 patients, 8 (31%) had single or confluent paralimbal intrastromal cavities, most commonly superiorly. By Suveges classification, the stage was 2 (92%) or 3 (8%). Minimum corneal thickness and corneal curvature were loosely associated, leading to different stages in Wang classification in 34 eyes (69%). Conclusions: TMD is defined by peripheral corneal thinning, superficial neovascularization, lipid deposition at the leading edge, absence of ulceration and inflammation, and frequently cavitation. The most sensitive way to follow its progression is anterior segment optical coherence tomography.
  • Toth, Timea; Balassa, Tamas; Bara, Norbert; Kovacs, Ferenc; Kriston, Andras; Molnar, Csaba; Haracska, Lajos; Sukosd, Farkas; Horvath, Peter (2018)
    To answer major questions of cell biology, it is often essential to understand the complex phenotypic composition of cellular systems precisely. Modern automated microscopes produce vast amounts of images routinely, making manual analysis nearly impossible. Due to their efficiency, machine learningbased analysis software have become essential tools to perform single-cell-level phenotypic analysis of large imaging datasets. However, an important limitation of such methods is that they do not use the information gained from the cellular micro-and macroenvironment: the algorithmic decision is based solely on the local properties of the cell of interest. Here, we present how various features from the surrounding environment contribute to identifying a cell and how such additional information can improve single-cell-level phenotypic image analysis. The proposed methodology was tested for different sizes of Euclidean and nearest neighbour-based cellular environments both on tissue sections and cell cultures. Our experimental data verify that the surrounding area of a cell largely determines its entity. This effect was found to be especially strong for established tissues, while it was somewhat weaker in the case of cell cultures. Our analysis shows that combining local cellular features with the properties of the cell's neighbourhood significantly improves the accuracy of machine learning-based phenotyping.
  • Cenev, Zoran; Zhang, Hongbo; Sariola, Veikko; Rahikkala, Antti Tuomas Antero; Almeida Santos, Helder; Liu, Dongfei; Zhou, Quan (2018)
    Selective, precise, and high-throughput manipulation of individual superparamagnetic microparticles has profound applications in performing location-tailored in vitro biomedical studies. The current techniques for manipulation of microparticles allow only a single particle in the manipulation workspace, or simultaneous transportation of multiple microparticles in batches. In this work, a method based on a robotized electromagnetic needle for manipulation of individual superparamagnetic microparticles within a microparticle population is introduced. By automatically controlling the highly localized magnetic field of the needle, a single microparticle is selectively picked when its neighboring particle is few micrometers away. Supported by the nanometer resolution of the robotic positioner, particles are placed at sub-micrometer precision. This manipulation technique allows the creating of arbitrary patterns, sorting of microparticles based on size and morphology, and transporting of individual microparticles in 3D space. Therefore, this approach has the potential to enable more deterministic and quantitative microanalysis and microsynthesis using superparamagnetic microparticles.
  • Li, Lei; Hsu, Hsun-Chia; Verkhusha, Vladislav V.; Wang, Lihong V.; Shcherbakova, Daria M. (2021)
    Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of approximate to 3 mu m in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of approximate to 150 mu m with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.
  • Shcherbakova, Daria M.; Stepanenko, Olesya V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V. (2018)
    Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescentproteins(FPs) engineeredfrombacterialphytochromeshave become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.
  • Rasse, Tobias M.; Hollandi, Reka; Horvath, Peter (2020)
    Various pre-trained deep learning models for the segmentation of bioimages have been made available as developer-to-end-user solutions. They are optimized for ease of use and usually require neither knowledge of machine learning nor coding skills. However, individually testing these tools is tedious and success is uncertain. Here, we present the Open Segmentation Framework (OpSeF), a Python framework for deep learning-based instance segmentation. OpSeF aims at facilitating the collaboration of biomedical users with experienced image analysts. It builds on the analysts' knowledge in Python, machine learning, and workflow design to solve complex analysis tasks at any scale in a reproducible, well-documented way. OpSeF defines standard inputs and outputs, thereby facilitating modular workflow design and interoperability with other software. Users play an important role in problem definition, quality control, and manual refinement of results. OpSeF semi-automates preprocessing, convolutional neural network (CNN)-based segmentation in 2D or 3D, and postprocessing. It facilitates benchmarking of multiple models in parallel. OpSeF streamlines the optimization of parameters for pre- and postprocessing such, that an available model may frequently be used without retraining. Even if sufficiently good results are not achievable with this approach, intermediate results can inform the analysts in the selection of the most promising CNN-architecture in which the biomedical user might invest the effort of manually labeling training data. We provide Jupyter notebooks that document sample workflows based on various image collections. Analysts may find these notebooks useful to illustrate common segmentation challenges, as they prepare the advanced user for gradually taking over some of their tasks and completing their projects independently. The notebooks may also be used to explore the analysis options available within OpSeF in an interactive way and to document and share final workflows. Currently, three mechanistically distinct CNN-based segmentation methods, the U-Net implementation used in Cellprofiler 3.0, StarDist, and Cellpose have been integrated within OpSeF. The addition of new networks requires little; the addition of new models requires no coding skills. Thus, OpSeF might soon become both an interactive model repository, in which pre-trained models might be shared, evaluated, and reused with ease.
  • Stape, Thiago Henrique Scarabello; Tjäderhane, Leo; Abuna, Gabriel; Sinhoreti, Mário Alexandre Coelho; Martins, Luís Roberto Marcondes; Tezvergil-Mutluay, Arzu (2018)
    Objective. To determine whether bonding effectiveness and hybrid layer integrity on acid-etched dehydrated dentin would be comparable to the conventional wet-bonding technique through new dentin biomodification approaches using dimethyl sulfoxide (DMSO). Methods. Etched dentin surfaces from extracted sound molars were randomly bonded in wet or dry conditions (30 s air drying) with DMSO/ethanol or DMSO/H2O as pretreatments using a simplified (Scotchbond Universal Adhesive, 3M ESPE: SU) and a multi-step (Adper Scotchbond Multi-Purpose, 3M ESPE: SBMP) etch-and-rinse adhesives. Untreated dentin surfaces served as control. Bonded teeth (n=8) were stored in distilled water for 24 h and sectioned into resin-dentin beams (0.8 mm(2)) for microtensile bond strength test and quantitative interfacial nanoleakage analysis (n = 8) under SEM. Additional teeth (n = 2) were prepared for micropermeability assessment by CFLSM under simulated pulp ar pressure (20 cm H2O) using 5 mM fluorescein as a tracer. Microtensile data was analyzed by 3-way ANOVA followed by Tukey Test and nanoleakage by Kruskal-Wallis and Dunn-Bonferroni multiple comparison test (alpha = 0.05). Results. While dry-bonding of SBMP produced significantly lower bond strengths than wet-bonding (p Conclusion. DMSO pretreatments may be used as a new suitable strategy to improve bonding of water-based adhesives to demineralized air-dried dentin beyond conventional wetbonding. Less porous resin-dentin interfaces with higher bond strengths on air-dried etched dentin were achieved; nonetheless, overall efficiency varied according to DMSO's co-solvent and adhesive type. Clinical significance. DMSO pretreatments permit etched dentin to be air-dried before hybridization facilitating residual water removal and thus improving bonding effectiveness. This challenges the current paradigm of wet-bonding requirement for the etch-and-rinse approach creating new possibilities to enhance the clinical longevity of resin-dentin interfaces. (C) 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.
  • Vurpillot, Francois; Parviainen, Stefan; Djurabekova, Flyura; Zanuttini, David; Gervais, Benoit (2018)
    The ideal picture of a near-perfect 3D microscope often presented regarding Atom Probe Tomography faces several issues. These issues degrade the metrological performance of the instrument and find their roots in the phenomena acting at the atomic to the mesoscopic level in the vicinity of the surface of a field emitter. From the field evaporation process at the atomic scale, to the macroscopic scale of the instrument, the path to model the imaging process and to develop more accurate and reliable reconstruction algorithms is not a single lane road. This paper focused on the numerical methods used to understand, treat, and potentially heal imaging issues commonly affecting the data in atom probe experiments. A lot of room for improvement exists in solving accuracy problems observed in complex materials by means of purely electrostatic models describing the image formation in a classical approach. Looking at the sample at the atomic scale, the phenomena perturbing the imaging process are subtle. An examination of atomic scale modifications of the sample surface in the presence of a high surface electric field is therefore mandatory. Atomic scale molecular dynamic models integrating the influence of the high surface electric are being developed with this aim. It is also demonstrated that the complex behavior of atoms and molecules in high fields, and consequences on collected data, can be understood through the use of accurate ab-initio models modified to include the impact of the extreme surface electric field.
  • Li, Lei; Shemetov, Anton A.; Baloban, Mikhail; Hu, Peng; Zhu, Liren; Shcherbakova, Daria M.; Zhang, Ruiying; Shi, Junhui; Yao, Junjie; Wang, Lihong V.; Verkhusha, Vladislav V. (2018)
    Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphPPCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-mu m spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.
  • Hankaniemi, Minna M.; Baikoghli, Mo A.; Stone, Virginia M.; Xing, Li; Väätäinen, Outi; Soppela, Saana; Sioofy-Khojine, Amirbabak; Saarinen, Niila V. V.; Ou, Tingwei; Anson, Brandon; Hyöty, Heikki; Marjomäki, Varpu; Flodström-Tullberg, Malin; Cheng, R. Holland; Hytönen, Vesa P.; Laitinen, Olli H. (2020)
    Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is similar to 2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.