Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.
- Keywords
- 3D STED, alcohol dehydration, high refractive index medium, large biological objects, oocyte, sample preparation, super‐resolution,
- MeSH
- Ethanol chemistry MeSH
- Microscopy, Fluorescence methods MeSH
- Mice MeSH
- Specimen Handling * methods MeSH
- Oocytes * cytology ultrastructure MeSH
- Refractometry methods MeSH
- Imaging, Three-Dimensional * methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Ethanol MeSH
Chromatin organization and its interactions are essential for biological processes, such as DNA repair, transcription, and DNA replication. Detailed cytogenetics data on chromatin conformation, and the arrangement and mutual positioning of chromosome territories in interphase nuclei are still widely missing in plants. In this study, level of chromatin condensation in interphase nuclei of rice (Oryza sativa) and the distribution of chromosome territories (CTs) were analyzed. Super-resolution, stimulated emission depletion (STED) microscopy showed different levels of chromatin condensation in leaf and root interphase nuclei. 3D immuno-FISH experiments with painting probes specific to chromosomes 9 and 2 were conducted to investigate their spatial distribution in root and leaf nuclei. Six different configurations of chromosome territories, including their complete association, weak association, and complete separation, were observed in root meristematic nuclei, and four configurations were observed in leaf nuclei. The volume of CTs and frequency of their association varied between the tissue types. The frequency of association of CTs specific to chromosome 9, containing NOR region, is also affected by the activity of the 45S rDNA locus. Our data suggested that the arrangement of chromosomes in the nucleus is connected with the position and the size of the nucleolus.
- Keywords
- 3D immuno-FISH, chromosome painting, chromosome territory, microscopy, rice, spatial organization,
- Publication type
- Journal Article MeSH
Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson's correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.
- Keywords
- CD9, Juno, MD simulation, STED, docking, oocyte, oolemma compartments, protein interaction,
- Publication type
- Journal Article MeSH
Previously, a number of ~ 1.4 of mitochondrial DNA (mtDNA) molecules in a single nucleoid was reported, which would reflect a minimum nucleoid division. We applied 3D-double-color direct stochastic optical reconstruction microscopy (dSTORM), i.e. nanoscopy with ~ 25-40 nm x,y-resolution, together with our novel method of Delaunay segmentation of 3D data to identify unbiased 3D-overlaps. Noncoding D-loops were recognized in HeLa cells by mtDNA fluorescence in situ hybridization (mtFISH) 7S-DNA 250-bp probe, containing biotin, visualized by anti-biotin/Cy3B-conjugated antibodies. Other mtFISH probes with biotin or Alexa Fluor 647 (A647) against ATP6-COX3 gene overlaps (1,100 bp) were also used. Nucleoids were imaged by anti-DNA/(A647-)-Cy3B-conjugated antibodies. Resulting histograms counting mtFISH-loci/nucleoid overlaps demonstrated that 45% to 70% of visualized nucleoids contained two or more D-loops or ATP6-COX3-loci, indicating two or more mtDNA molecules per nucleoid. With increasing number of mtDNA per nucleoid, diameters were larger and their distribution histograms peaked at ~ 300 nm. A wide nucleoid diameter distribution was obtained also using 2D-STED for their imaging by anti-DNA/A647. At unchanged mtDNA copy number in osteosarcoma 143B cells, TFAM expression increased nucleoid spatial density 1.67-fold, indicating expansion of existing mtDNA and its redistribution into more nucleoids upon the higher TFAM/mtDNA stoichiometry. Validation of nucleoid imaging was also done with two TFAM mutants unable to bend or dimerize, respectively, which reduced both copy number and nucleoid spatial density by 80%. We conclude that frequently more than one mtDNA molecule exists within a single nucleoid in HeLa cells and that mitochondrial nucleoids do exist in a non-uniform size range.
- MeSH
- DNA-Binding Proteins * genetics metabolism MeSH
- HeLa Cells MeSH
- In Situ Hybridization, Fluorescence MeSH
- Humans MeSH
- DNA, Mitochondrial * genetics metabolism MeSH
- Mitochondrial Proteins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA-Binding Proteins * MeSH
- DNA, Mitochondrial * MeSH
- Mitochondrial Proteins MeSH
Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.
- Keywords
- 3D organization, Nucleus, RNA Pol II, SIM, STED imaging, chromatin, chromosome, crossovers, image analysis, meiosis, metaphase, mitosis, nuclear bodies, nuclear speckles, oligo FISH, pachytene, quantification, segmentation, spatial distribution, transcription factories,
- MeSH
- Cell Nucleus * MeSH
- Chromatin * MeSH
- Microscopy, Fluorescence MeSH
- In Situ Hybridization, Fluorescence MeSH
- Humans MeSH
- Workflow MeSH
- Green Fluorescent Proteins MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromatin * MeSH
- Green Fluorescent Proteins MeSH
Alzheimer's disease (AD) involves the propagation of filaments of tau protein throughout the cerebral cortex. Imaging tau filaments and oligomers in human brain at high resolution would help contribute insight into the mechanism and progression of tauopathic diseases. STED microscopy is a nano-scale imaging technique and we aimed to test the abilities of this method for resolving tau structures within human brain. Using autopsied 50μm AD brain sections, we demonstrate that STED microscopy can resolve immunolabelled tau filaments at 77nm resolution. Ribbon-like tau filaments imaged by STED appeared smooth along their axis with limited axial undulations. STED also resolved 70-80nm wide tau puncta. Of the fluorophores tested, STAR635p was optimal for STED imaging in this tissue. This was in part due to brain tissue autofluorescence within the lower wavelength ranges (488-590nm). Further, the stability and minimal photobleaching of STAR635p allowed STED z-stacks of neurons packed with tau filaments (neurofibrillary tangles) to be collated. There was no loss of x-y image resolution of individual tau filaments through the 20μm z-stack. This demonstrates that STED can contribute to nano-scale analysis and characterisation of pathologies within banked human autopsied brain tissue. Resolving tau structures at this level of resolution provides promising avenues for understanding mechanisms of pathology propagation in the different tauopathies as well as illuminating what contributes to disease heterogeneity.
- Keywords
- AD, Alzheimer’s disease, Human tissue, PHF, STED, Super resolution, Tau filament, Tau protein,
- MeSH
- Alzheimer Disease diagnostic imaging pathology MeSH
- Staining and Labeling MeSH
- Humans MeSH
- Neurofibrillary Tangles pathology ultrastructure MeSH
- Optical Imaging MeSH
- tau Proteins chemistry ultrastructure MeSH
- Gray Matter diagnostic imaging pathology MeSH
- Imaging, Three-Dimensional MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- MAPT protein, human MeSH Browser
- tau Proteins MeSH
Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted.
- Keywords
- photoactivation localization microscopy, plant cell biology, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, structured-illumination microscopy, super-resolution microscopy,
- MeSH
- Microscopy methods MeSH
- Plant Cells chemistry physiology ultrastructure MeSH
- Imaging, Three-Dimensional MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
