Structured-illumination microscopy
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UNLABELLED: SIMToolbox is an open-source, modular set of functions for MATLAB equipped with a user-friendly graphical interface and designed for processing two-dimensional and three-dimensional data acquired by structured illumination microscopy (SIM). Both optical sectioning and super-resolution applications are supported. The software is also capable of maximum a posteriori probability image estimation (MAP-SIM), an alternative method for reconstruction of structured illumination images. MAP-SIM can potentially reduce reconstruction artifacts, which commonly occur due to refractive index mismatch within the sample and to imperfections in the illumination. AVAILABILITY AND IMPLEMENTATION: SIMToolbox, example data and the online documentation are freely accessible at http://mmtg.fel.cvut.cz/SIMToolbox. CONTACT: ghagen@uccs.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
- MeSH
- buňky Hep G2 MeSH
- fluorescence * MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- mikrofilamenta ultrastruktura MeSH
- osvětlení metody MeSH
- počítačové zpracování obrazu MeSH
- software * MeSH
- zobrazování trojrozměrné metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.
- MeSH
- Arabidopsis MeSH
- intravitální mikroskopie metody MeSH
- rostlinné buňky * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.
- MeSH
- Arabidopsis metabolismus MeSH
- epidermis rostlin cytologie metabolismus MeSH
- hypokotyl cytologie metabolismus MeSH
- konfokální mikroskopie MeSH
- mikroskopie metody MeSH
- mikrotubuly metabolismus MeSH
- mutace genetika MeSH
- osvětlení * MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.
- MeSH
- buněčné jádro metabolismus MeSH
- chromatin chemie metabolismus MeSH
- epigeneze genetická MeSH
- fluorescenční mikroskopie MeSH
- HeLa buňky MeSH
- heterochromatin chemie MeSH
- histondemethylasy metabolismus MeSH
- histony chemie MeSH
- jaderný obal metabolismus MeSH
- jaderný pór metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Background: Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings: Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions: Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.
- MeSH
- algoritmy MeSH
- Bayesova věta MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- fluorescenční mikroskopie MeSH
- králíci MeSH
- lidé MeSH
- počítačové zpracování obrazu metody MeSH
- software MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.
In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders' colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders' colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.
Mitotic cell division in plants is a dynamic process playing a key role in plant morphogenesis, growth, and development. Since progress of mitosis is highly sensitive to external stresses, documentation of mitotic cell division in living plants requires fast and gentle live-cell imaging microscopy methods and suitable sample preparation procedures. This chapter describes, both theoretically and practically, currently used advanced microscopy methods for the live-cell visualization of the entire process of plant mitosis. These methods include microscopy modalities based on spinning disk, Airyscan confocal laser scanning, structured illumination, and light-sheet bioimaging of tissues or whole plant organs with diverse spatiotemporal resolution. Examples are provided from studies of mitotic cell division using microtubule molecular markers in the model plant Arabidopsis thaliana, and from deep imaging of mitotic microtubules in robust plant samples, such as legume crop species Medicago sativa.
Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.
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.
