In this chapter, conventional techniques are described for the preparation of plant samples at room temperature before examination in the high resolution scanning electron microscopy. Protocols are given on how to collect, to fix, to dehydrate, and to dry plant samples. Subsequently, it is described how to stick them to stubs and cover with a thin conductive layer. These methods are suitable for a wide variety of plant specimens, ranging from microalgae to higher plants.

• Klíčová slova
• Chemical fixation, Critical point drying, Dehydration, High-resolution scanning electron microscope, Metal coating, Plant sample, Sample storage, t-Butanol freeze drying,
• MeSH
• fixace tkání metody   MeSH
• lyofilizace metody   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• rostliny ultrastruktura   MeSH
• teplota MeSH
• vysoušení metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We present a new method of multiple immunolabeling that is suitable for a broad spectrum of biomedical applications. The general concept is to label both sides of the ultrathin section with the thickness of 70-80 nm with different antibodies conjugated to gold nanoparticles and to distinguish the labeled side by advanced imaging methods with high resolution scanning electron microscopy, such as by correlating images acquired at different energies of primary electrons using different signals. From the Clinical Editor: The use of transmission electron microscopy has become an indispensible tool in the detection of cellular proteins. In this short but interesting article, the authors described their new method of labeling and the identification of four different proteins simultaneously, which represents another advance in imaging technique.

• Klíčová slova
• BSE imaging, Gold nanoparticles, High resolution SEM, Multiple immunolabeling, STEM imaging,
• MeSH
• akrylové pryskyřice chemie   MeSH
• barvení a značení metody   MeSH
• imunohistochemie MeSH
• kovové nanočástice chemie   ultrastruktura   MeSH
• mikrotomie metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• skenovací elektrochemická mikroskopie metody   MeSH
• vylepšení obrazu metody   MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylové pryskyřice MeSH
• zlato MeSH

Submolecular imaging by atomic force microscopy (AFM) has recently been established as a stunning technique to reveal the chemical structure of unknown molecules, to characterize intramolecular charge distributions and bond ordering, as well as to study chemical transformations and intermolecular interactions. So far, most of these feats were achieved on planar molecular systems because high-resolution imaging of three-dimensional (3D) surface structures with AFM remains challenging. Here we present a method for high-resolution imaging of nonplanar molecules and 3D surface systems using AFM with silicon cantilevers as force sensors. We demonstrate this method by resolving the step-edges of the (101) anatase surface at the atomic scale by simultaneously visualizing the structure of a pentacene molecule together with the atomic positions of the substrate and by resolving the contour and probe-surface force field on a C60 molecule with intramolecular resolution. The method reported here holds substantial promise for the study of 3D surface systems such as nanotubes, clusters, nanoparticles, polymers, and biomolecules using AFM with high resolution.

• Klíčová slova
• Noncontact atomic force microscopy (NC-AFM), high-resolution imaging, submolecular resolution, three-dimensional dynamic force spectroscopy,
• MeSH
• analýza selhání vybavení MeSH
• design vybavení MeSH
• fullereny chemie   MeSH
• krystalografie metody   MeSH
• mikroskopie atomárních sil přístrojové vybavení   MeSH
• molekulární konformace MeSH
• molekulární sondy - techniky přístrojové vybavení   MeSH
• molekulární zobrazování přístrojové vybavení   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• vylepšení obrazu přístrojové vybavení   MeSH
• zobrazování trojrozměrné přístrojové vybavení   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fullerene C60 MeSH Prohlížeč
• fullereny MeSH

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.

• MeSH
• DNA ultrastruktura   MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• mikrosféry MeSH
• mikroskopie * přístrojové vybavení   metody   MeSH
• počítačové zpracování obrazu MeSH
• senzitivita a specificita MeSH
• teoretické modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva MeSH

Electron microscopy in situ hybridization (EM-ISH) represents a powerful method that enables the localization of specific sequences of nucleic acids at high resolution. We provide here an overview of three different nonisotopic EM-ISH approaches that allow the visualization of nucleic acid sequences in cells. A comparison of various methods with respect to their sensitivity and the structural preservation of the sample is presented, with the aim of helping the reader to choose a convenient hybridization procedure. The post-embedding EM-ISH protocol that currently represents the most widely used technique is described in detail, with a special emphasis on the organization of the cell nucleus.

• MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• denaturace nukleových kyselin MeSH
• deoxyribonukleasa I MeSH
• DNA genetika   ultrastruktura   MeSH
• elektronová kryomikroskopie metody   MeSH
• elektronová mikroskopie metody   MeSH
• endopeptidasa K MeSH
• fixativa MeSH
• geny rRNA MeSH
• HeLa buňky MeSH
• HIV-1 genetika   MeSH
• hybridizace in situ metody   MeSH
• lidé MeSH
• mikrotomie metody   MeSH
• molekulární sondy MeSH
• pankreatická ribonukleasa MeSH
• RNA virová genetika   ultrastruktura   MeSH
• RNA genetika   ultrastruktura   MeSH
• zalévání tkání plastickou hmotou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• deoxyribonukleasa I MeSH
• DNA MeSH
• endopeptidasa K MeSH
• fixativa MeSH
• molekulární sondy MeSH
• pankreatická ribonukleasa MeSH
• RNA virová MeSH
• RNA MeSH

A modern scanning electron microscope equipped with a pixelated detector of transmitted electrons can record a four-dimensional (4D) dataset containing a two-dimensional (2D) array of 2D nanobeam electron diffraction patterns; this is known as a four-dimensional scanning transmission electron microscopy (4D-STEM). In this work, we introduce a new version of our method called 4D-STEM/PNBD (powder nanobeam diffraction), which yields high-resolution powder diffractograms, whose quality is fully comparable to standard TEM/SAED (selected-area electron diffraction) patterns. Our method converts a complex 4D-STEM dataset measured on a nanocrystalline material to a single 2D powder electron diffractogram, which is easy to process with standard software. The original version of 4D-STEM/PNBD method, which suffered from low resolution, was improved in three important areas: (i) an optimized data collection protocol enables the experimental determination of the point spread function (PSF) of the primary electron beam, (ii) an improved data processing combines an entropy-based filtering of the whole dataset with a PSF-deconvolution of the individual 2D diffractograms and (iii) completely re-written software automates all calculations and requires just a minimal user input. The new method was applied to Au, TbF3 and TiO2 nanocrystals and the resolution of the 4D-STEM/PNBD diffractograms was even slightly better than that of TEM/SAED.

• Klíčová slova
• 4D-STEM, nanoparticle analysis, powder nanobeam electron diffraction,
• Publikační typ
• časopisecké články MeSH

Fluorescence microscopy has become one of the most rapidly developing observation techniques in the field of molecular biology, since its high sensitivity, contrast and labeling specificity together with being non-invasive fulfill the most important requirements of live cell imaging. The biggest limitation of the technique seems to be the spatial resolution which is, based on Abbe's diffraction law, restricted to some hundreds of nanometres. Recently, various approaches have been developed that overcome the limit imposed by the diffraction barrier and these methods currently lead the development in the field of fluorescence microscopy. In this contribution, we present dynamic saturation optical microscopy (DSOM)--a new technique that monitors the temporal decay of the excited singlet state due to a dark state formation. By mapping the intensity dependent decay kinetics, enhanced resolution images can be obtained. Generally, any dark state of fluorescent molecules can be employed in DSOM. Here, we focus our attention on triplet state formation.

• MeSH
• fluorescenční mikroskopie metody   MeSH
• kinetika MeSH
• konfokální mikroskopie metody   MeSH
• lipidové dvojvrstvy analýza   MeSH
• mikrosféry MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidové dvojvrstvy MeSH

The egg plays a pivotal role in the reproduction of our species. Nevertheless, its fundamental biology remains elusive. Transmission electron microscopy is traditionally used to inspect the ultrastructure of female gametes. However, two-dimensional micrographs contain only fragmentary information about the spatial organization of the complex oocyte cytoplasm. Here, we employed the Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to explore human oocyte intracellular morphology in three dimensions (3D). Volume reconstruction of generated image stacks provided an unprecedented view of ooplasmic architecture. Organelle distribution patterns observed in nine donor oocytes, representing three maturational stages, documented structural changes underlying the process by which the egg acquires developmental competence. 3D image segmentation was performed to extract information about distinct organelle populations, and the following quantitative analysis revealed that the mitochondrion occupies ∼ 4.26% of the maturing oocyte cytoplasm. In summary, this proof-of-concept study demonstrates the potential of large volume electron microscopy to study rare samples of delicate female gametes and paves the way for applying the FIB-SEM technique in human oocyte research.

• Klíčová slova
• 3D ultrastructure, FIB-SEM, electron microscopy, human oocyte, oocyte maturation, volume microscopy,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• 3D STED, alcohol dehydration, high refractive index medium, large biological objects, oocyte, sample preparation, super‐resolution,
• MeSH
• ethanol chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• myši MeSH
• odběr biologického vzorku * metody   MeSH
• oocyty * cytologie   ultrastruktura   MeSH
• refraktometrie metody   MeSH
• zobrazování trojrozměrné * metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ethanol MeSH

Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm-1) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.

• MeSH
• 53BP1 chemie   MeSH
• apoptóza MeSH
• dvouřetězcové zlomy DNA * MeSH
• fibroblasty účinky záření   MeSH
• fluorescenční protilátková technika MeSH
• fosforylace MeSH
• histony chemie   MeSH
• ionizující záření MeSH
• konfokální mikroskopie * MeSH
• kultivované buňky MeSH
• lidé MeSH
• lineární přenos energie * MeSH
• oprava DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 53BP1 MeSH
• H2AX protein, human MeSH Prohlížeč
• histony MeSH
• TP53BP1 protein, human MeSH Prohlížeč