The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

• Keywords
• Hordeum vulgare, chromatin, deconvolution microscopy, metaphase chromosome, nanoscopy, photoactivated localization microscopy, stimulated emission depletion microscopy, structured illumination microscopy, topoisomerase II, wide-field microscopy,
• MeSH
• Chromosomes, Plant chemistry   genetics   metabolism   MeSH
• DNA Topoisomerases, Type II metabolism   MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• Indoles chemistry   MeSH
• Hordeum cytology   genetics   MeSH
• Microscopy, Confocal methods   MeSH
• Metaphase genetics   MeSH
• Reproducibility of Results MeSH
• Single Molecule Imaging methods   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• DAPI MeSH Browser
• DNA Topoisomerases, Type II MeSH
• Fluorescent Dyes MeSH
• Indoles MeSH

Light microscopy (LM) approaches are commonly used to attain a description of the cell structure. Even though LM, if compared to electron microscopy (EM), represents a very fast approach, its resolution is, in principle, much lower than in the case of EM. To improve the LM resolution, computational methods based on removal of the image blur are frequently implemented in cell biology studies. One of the standard deblurring approaches is image restoration through deconvolution algorithms. Even though this method of mathematical remodeling of microscopically observed objects represents an efficient tool of current cell biology, it is legitimate to ask what the limits of its use are. We demonstrate that, in the specific case of the fluorescence mapping of active ribosomal genes in HeLa cell nucleoli, restoration generates a biased result. On restoration of model images, we demonstrate the difficulties of one of the most effective deconvolution algorithms during the restoration of ring-shaped fluorescent objects of a diameter comparable to the microscope resolution limit. In the case involving the mapping of nucleolar transcription in HeLa cells, not the restored fluorescence images, but rather the EM images show the true distribution of active ribosomal genes.

• MeSH
• Cell Nucleolus metabolism   MeSH
• Microscopy, Electron methods   MeSH
• Transcription, Genetic MeSH
• HeLa Cells MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Image Processing, Computer-Assisted MeSH
• DNA, Ribosomal * MeSH
• Ribosomes metabolism   MeSH
• Models, Theoretical MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal * MeSH

The future of consumer electronics depends on the capability to reliably fabricate nanostructures with given physical properties. Therefore, techniques to characterize materials and devices with nanoscale resolution are crucial. Among these is magnetic force microscopy (MFM), which transduces the magnetic force between the sample and a magnetic oscillating probe into a phase shift, enabling the locally resolved study of magnetic field patterns down to 10 nm. Here, the progress done toward making quantitative MFM a common tool in nanocharacterization laboratories is shown. The reliability and ease of use of the calibration method based on a magnetic reference sample, with a calculable stray field, and a deconvolution algorithm is demonstrated. This is achieved by comparing two calibration approaches combined with numerical modeling as a quantitative link: measuring the probe's effect on the voltage signal when scanning above a nanosized graphene Hall sensor, and recording the MFM phase shift signal when the probe scans across magnetic fields produced by metallic microcoils. Furthermore, in the case of the deconvolution algorithm, it is shown how it can be applied using the open-source software package Gwyddion. The estimated magnetic dipole approximation for the most common probes currently in the market is also reported.

• Keywords
• Hall nanosensors, calibration, magnetic force microscopy (MFM), magnetic probes, nanoscale,
• Publication type
• Journal Article MeSH

The nanoscale organization of the tropomyosin-related kinase receptor type B (TrkB), a promising therapeutic target for severe neurodegenerative and psychiatric disorders, is examined by stimulated emission depletion (STED) microscopy using the deconvoluted gated STED option. The performed immunofluorescence nanoscopic subdiffraction imaging of the membrane receptor localization reveals that clusters of oligomeric TrkB states and randomly organized nanodomains are formed in the membranes of differentiated human neuroblastoma SH-SY5Y cells, which are studied as an in vitro model of neurodegeneration. Despite that the monomeric (isolated) states of the receptor cannot be distinguished from its dimeric forms in such images, TrkB receptor dimers (or couple of individual monomers) are visualized at super-resolution as single pixels in the magnified Huygens-deconvoluted gated STED images. The clusters of higher-order TrkB oligomers are of dynamic nature rather than of a fixed stoichiometry. The propensity for membrane protein clustering as well as the dissociation of the TrkB receptors nanodomains can be modulated by neurotherapeutic formulations containing ω-3 polyunsaturated docosahexaenoic acid (DHA). Nanomolar concentrations of DHA change the receptor topology and lead to disruption of the cluster phases. This result is of therapeutic importance for TrkB receptor availability upon ligand binding as DHA favours the mobility and the dynamic distribution of the protein populations in the cell membranes.

• MeSH
• Cell Membrane ultrastructure   MeSH
• Fluorescent Antibody Technique MeSH
• Humans MeSH
• Membrane Glycoproteins chemistry   MeSH
• Microscopy MeSH
• Cell Line, Tumor MeSH
• Neuroblastoma MeSH
• Receptor, trkB chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Glycoproteins MeSH
• Receptor, trkB MeSH
• tropomyosin-related kinase-B, human MeSH Browser

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.

• Keywords
• 4D-STEM, nanoparticle analysis, powder nanobeam electron diffraction,
• Publication type
• Journal Article MeSH

Plants exposed to harmonically modulated irradiance, approximately 1 + cos(omegat), exhibit a complex periodic pattern of chlorophyll fluorescence emission that can be deconvoluted into a steady-state component, a component that is modulated with the frequency of the irradiance (omega), and into at least two upper harmonic components (2omega and 3omega). A model is proposed that accounts for the upper harmonics in fluorescence emission by nonlinear negative feedback regulation of photosynthesis. In contrast to simpler linear models, the model predicts that the steady-state fluorescence component will depend on the frequency of light modulation, and that amplitudes of all fluorescence components will exhibit resonance peak(s) when the irradiance frequency is tuned to an internal frequency of a regulatory component. The experiments confirmed that the upper harmonic components appear and exhibit distinct resonant peaks. The frequency of autonomous oscillations observed earlier upon an abrupt increase in CO(2) concentration corresponds to the sharpest of the resonant peaks of the forced oscillations. We propose that the underlying principles are general for a wide spectrum of negative-feedback regulatory mechanisms. The analysis by forced harmonic oscillations will enable us to examine internal dynamics of regulatory processes that have not been accessible to noninvasive fluorescence monitoring to date.

• MeSH
• Biophysics MeSH
• Biophysical Phenomena MeSH
• Chlorophyll MeSH
• Spectrometry, Fluorescence MeSH
• Fourier Analysis MeSH
• Kinetics MeSH
• Plant Leaves metabolism   MeSH
• Oscillometry * MeSH
• Carbon Dioxide MeSH
• Plants chemistry   metabolism   MeSH
• Spectrophotometry, Infrared MeSH
• Models, Statistical MeSH
• Statistics as Topic MeSH
• Nicotiana metabolism   MeSH
• Microscopy, Video MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Carbon Dioxide MeSH

Blind image deconvolution is required in many applications of microscopy imaging, remote sensing, and astronomical imaging. Unfortunately in a single-channel framework, serious conceptual and numerical problems are often encountered. Very recently, an eigenvector-based method (EVAM) was proposed for a multichannel framework which determines perfectly convolution masks in a noise-free environment if channel disparity, called co-primeness, is satisfied. We propose a novel iterative algorithm based on recent anisotropic denoising techniques of total variation and a Mumford-Shah functional with the EVAM restoration condition included. A linearization scheme of half-quadratic regularization together with a cell-centered finite difference discretization scheme is used in the algorithm and provides a unified approach to the solution of total variation or Mumford-Shah. The algorithm performs well even on very noisy images and does not require an exact estimation of mask orders. We demonstrate capabilities of the algorithm on synthetic data. Finally, the algorithm is applied to defocused images taken with a digital camera and to data from astronomical ground-based observations of the Sun.

• Publication type
• Journal Article MeSH

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

• Keywords
• DNA content, kinetoplast DNA, mitochondrial DNA, protist,
• MeSH
• Euglenozoa genetics   MeSH
• Phylogeny MeSH
• Kinetoplastida genetics   MeSH
• DNA, Mitochondrial genetics   isolation & purification   ultrastructure   MeSH
• Mitochondria genetics   MeSH
• Trans-Splicing genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.

• MeSH
• Cytochalasin D MeSH
• Elastic Modulus MeSH
• Neoplasms * MeSH
• Elasticity MeSH
• Reproducibility of Results MeSH
• Viscosity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochalasin D MeSH