Techniques for Selective Labeling of Molecules and Subcellular Structures for Cryo-Electron Tomography.

With the continuous advent of new types of electron microscopes and the continuous improvement and development of electron microscope technique , biological electron microscope tech-nique has become one of the important experimental means in the fields of biology , molecular biology, basic medicine and clinical medicine , Department of Pathology of College of Basic Medical Sciences in Jilin University has reviewed the teaching feature and teaching status of biological electron microscope technique based on the training objectives of postgraduate education. By finding the problems and analyz-ing them, they have added the teaching link of review discussion in teaching process and elaborated on its implementation process and teaching results , aiming to provide practical basis for application of review discussion for postgraduate teaching. Key words: Biological electron microscope technique; Review discussion; Postgraduates

Since the earliest description of Alzheimer’s disease (AD) at the beginning of the last century, the intra-and extracellular deposition of proteins and the alterations in neuronal and glial morphology have been consistently detected in the brains of demented patients. During the following decades with the enormous developments in molecular biology and electron microscopy, the most obvious histopathological elements of AD have been described in more details on the molecular level and the topographical distribution. While intracellular neurofibrillary tangles and extracellular amyloid deposits are found in varying amounts in neuropathological samples of AD, numeric loss of synapses and neuronal cell death are most closely correlated to cognitive decline. However, despite the extensive characterisation of intracellular neurofibrillary tangles and extracellular amyloid deposits on the molecular level, the pathophysiological relationship between these four type of lesions — neuronal tangles, amyloid plaques, synaptic loss, and neuronal death — are not well understood (Hull et al. 2000).

The paper analyses the different systems of corpus labeling, such as TODI, TOBI, and IVIE, which are three kinds of speech labeling system, and the paper puts forward a suitable labeling system of multi-modal corpus for teaching Chinese as a foreign language. The corpus includes eight levels of character description and labeling of speech tagging system. The system of labeling is applied to the improvement of the labeling method of TIMIT, which has the layer of phonetic transcription, the layer of phoneme layer, the layer of intermittent index, the lay of stress, the layer of target, the layer of phonology, and the layer of the behavior (includes the layer of posture and the layer of movement, and the layer of miscellanea. Finally, we label on the experimental corpus, and it can obtain better results in the process of teaching Chinese phonetic than before.KeywordsCorpusLabelingTeachingChinese as a foreign language

The coming of age of biological electron microscopy

We present a description of our work in recent years on imaging in confocal microscopy in the context of biological applications. The first system presented considers a Nipkow disc type arrangement where a detailed investigation of optimal aperture arrangements and spacings is performed. The effect of varying these parameters on the optical sectioning characteristics and on the light throughput is evaluated. Novel routes to achieving alternative multi-aperture configurations are presented. A programmable array microscope demonstrator is described using a ferroelectric liquid crystal SLM. A novel system is also proposed which uses variable focus microlenses in a confocal imaging system. We also discuss current trends in confocal microscopy in biology.

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Here we report carboxy-eosin as a fluorescent marker for in vivo visualization of DNA in the correlative (fluorescence and electron) biological microscope. For that purpose a fluorescent thymidine analog 2’,4’,5’,7’-tetrabromofluorescein-aminoallyl-dUTP (eosin-aminoallyl-dUTP) was synthesized and incorporated into the genome of Escherichia coli. The fluorescent dye eosin contains four bromine atoms, which enable electron spectroscopic imaging in the transmission electron microscope. Labeled cells were prepared and directly observed by fluorescence and electron microscope without any preliminary fixation step. We demonstrated that eosin-aminoallyl-dUTP is a suitable marker for imaging of cell’s ultrastructures by correlative biological microscopy.

The rapid pace of innovation in biological microscopy has produced increasingly large images, putting pressure on data storage and impeding efficient data sharing, management and visualization. This trend necessitates new, efficient compression solutions, as traditional coder–decoder methods often struggle with the diversity of bioimages, leading to suboptimal results. Here we show an adaptive compression workflow based on implicit neural representation that addresses these challenges. Our approach enables application-specific compression, supports images of varying dimensionality and allows arbitrary pixel-wise decompression. On a wide range of real-world microscopy images, we demonstrate that our workflow achieves high, controllable compression ratios while preserving the critical details necessary for downstream scientific analysis.

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

Protein adsorption at oil-water interfaces interfacial structure and function of proteins in emulsions

With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein-ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein-ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of the Plasmodium falciparum 20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human and P. falciparum 20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of the icr3d software used for three-dimensional reconstruction.

Biological electron microscopy (EM) of specimens prepared using chemical fixation, heavy metal staining, and resin embedding has advanced the biosciences for over half a century. These chemical preparation methods are applied every day to research and clinical transmission EM, the rapidly growing discipline of 3D volume EM, for correlative EM with light and X-ray microscopy, and for freeze-substitution cryogenic preparation EM. While EM imaging and image analysis are becoming increasingly automated, most research labs still perform their specimen preparation manually using commodity labware not designed for EM of biospecimens. This report shows how the purpose-built labware for microscopy, the mPrep™ System and the ASP™ family of automated specimen processors, improves preparation reliability, cuts reagent consumption and user exposure, and reduces specimen preparation time and effort by up to 10-fold. This report examines how the Multiscale Microscopy Core at Oregon Health & Science University, a core lab specializing in cancer and biomedical research, uses the mPrep™ ASP™-2000 automated specimen processor to streamline and speed specimen preparation workflows for volume EM (including serial block face, focused ion beam, and array tomography SEM) and for transmission EM.

The reason the oxide replica gives a more resolved image in electron microscopy than Formvar or collodium replica is because the small oxide particles (50-100 Å.) composing replica film are oriented according to the orientation of the metal crystals of substrates. The electrodeposited replica and sulphide film formed on metals give the resolved images in microscopy. This is explained in the same way as for oxide replica.

Validation of non-uniform illumination correction techniques in microscopic digital TB images using image sharpness measures