BACKGROUND: In the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures. RESULTS: Focus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays. CONCLUSIONS: 3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

• Klíčová slova
• Immunofluorescence, Microtubule associated proteins, Microtubules, Structured illumination microscopy,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Chromatin, Histone modification, Image analysis, Nuclear pore complexes, Nucleus, Structured illumination microscopy,
• 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
• Názvy látek
• chromatin MeSH
• heterochromatin MeSH
• histondemethylasy MeSH
• histony MeSH
• KDM1A protein, human MeSH Prohlížeč

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
• Názvy látek
• proteiny huseníčku MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Structured illumination microscopy (SIM) has grown into a family of methods which achieve optical sectioning, resolution beyond the Abbe limit, or a combination of both effects in optical microscopy. SIM techniques rely on illumination of a sample with patterns of light which must be shifted between each acquired image. The patterns are typically created with physical gratings or masks, and the final optically sectioned or high resolution image is obtained computationally after data acquisition. We used a flexible, high speed ferroelectric liquid crystal microdisplay for definition of the illumination pattern coupled with widefield detection. Focusing on optical sectioning, we developed a unique and highly accurate calibration approach which allowed us to determine a mathematical model describing the mapping of the illumination pattern from the microdisplay to the camera sensor. This is important for higher performance image processing methods such as scaled subtraction of the out of focus light, which require knowledge of the illumination pattern position in the acquired data. We evaluated the signal to noise ratio and the sectioning ability of the reconstructed images for several data processing methods and illumination patterns with a wide range of spatial frequencies. We present our results on a thin fluorescent layer sample and also on biological samples, where we achieved thinner optical sections than either confocal laser scanning or spinning disk microscopes.

• MeSH
• kalibrace MeSH
• kapalné krystaly chemie   MeSH
• křemík chemie   MeSH
• mikroskopie přístrojové vybavení   MeSH
• ohebnost (fyzika) MeSH
• osvětlení přístrojové vybavení   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• křemík MeSH

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

• 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

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.

• Klíčová slova
• photoactivation localization microscopy, plant cell biology, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, structured-illumination microscopy, super-resolution microscopy,
• MeSH
• mikroskopie metody   MeSH
• rostlinné buňky chemie   fyziologie   ultrastruktura   MeSH
• zobrazování trojrozměrné MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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.

• Klíčová slova
• Chromatin, Expansion microscopy, Hordeum vulgare, Nucleus, Structured illumination microscopy,
• MeSH
• buněčné jádro ultrastruktura   MeSH
• chromatin ultrastruktura   MeSH
• fluorescenční protilátková technika MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) genetika   MeSH
• mikroskopie metody   normy   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH