scholarly journals HPM Live μ for a full CLEM workflow

2020 ◽  
Author(s):  
Xavier Heiligenstein ◽  
Marit de Beer ◽  
Jérôme Heiligenstein ◽  
Frédérique Eyraud ◽  
Laurent Manet ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Imaging Techniques ◽  
Light And Electron Microscopy ◽  
Environmental Parameters ◽  
Live Imaging ◽  
Chemical Fixation ◽  
Protein Fluorescence ◽  
Spectroscopic Information ◽  
The Many ◽  
Almost All

ABSTRACTWith the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution.Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM.To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live μ instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

scholarly journals Peroxisome development in the regenerating pars recta (P3 segment) of proximal tubules of the rat kidney.

1976 ◽  
Vol 24 (12) ◽  
pp. 1239-1248 ◽  
Author(s):  
J K Reddy ◽  
M S Rao ◽  
D E Moody ◽  
S A Qureshi
Keyword(s):  
Electron Microscopy ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Proximal Tubules ◽  
Cell Multiplication ◽  
Tubular Necrosis ◽  
Mitochondrial Population ◽  
Pars Recta ◽  
Almost All ◽  
Maximum Development

The development of peroxisomes, lysosomes and endocytic vacuoles in regenerating cells of the pars recta (P3 segment) of proximal tubules, in rats given a single interperitoneal injection of d-serine (80 mg/100 g.b.wt), was studied by light and electron microscopy using cytochemical methods. Rapid proliferation of cells occurred between 2 and 5 days after d-serine induced tubular necrosis; by day 6 almost all injured tubules were re-epithelialized with flat or low cuboidal cells. Peroxisomes and lysosomes were not observed during the period of rapid cell multiplication i.e., between 2 and 6 days after d-serine injection. Restitution of mitochondrial population preceded the development of peroxisomes in the newly regenerated cells of P3 tubules. Maximum development of peroxisomes occurred between 9 and 14 days after d-serine injection. The formation of peroxisomes appeared to correlate closely with the differentiation of apical endocytic vacuoles and the brush border. Lysosomes in the regenerated cells of P3 tubules were the last to develop.

scholarly journals Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

2015 ◽  
Vol 259 (2) ◽  
pp. 121-128 ◽  
Author(s):  
K. HÖHN ◽  
J. FUCHS ◽  
A. FRÖBER ◽  
R. KIRMSE ◽  
B. GLASS ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dendritic Cells ◽  
Light And Electron Microscopy ◽  
Protein Fluorescence ◽  
Human Dendritic Cells

scholarly journals A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy

2015 ◽  
Vol 21 (4) ◽  
pp. 902-909 ◽  
Author(s):  
Peta L. Clode
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Plant Tissues ◽  
Chemical Fixation ◽  
High Quality ◽  
Plant Materials ◽  
Low Viscosity ◽  
Transmission Electron ◽  
Plant Sciences ◽  
Very High

AbstractAlthough the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation ofBanksia prionotes(Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

scholarly journals Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Madhumitha Narasimhan ◽  
Alexander Johnson ◽  
Roshan Prizak ◽  
Walter Anton Kaufmann ◽  
Shutang Tan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Constant Curvature ◽  
Imaging Techniques ◽  
Live Imaging ◽  
Unique Role ◽  
Major Route ◽  
Bona Fide ◽  
Endocytic Vesicles

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

scholarly journals Evolutionary unique mechanistic framework of clathrin-mediated endocytosis in plants

2019 ◽  
Author(s):  
Madhumitha Narasimhan ◽  
Alexander Johnson ◽  
Roshan Prizak ◽  
Walter Anton Kaufmann ◽  
Barbara Casillas-Pérez ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Constant Curvature ◽  
Imaging Techniques ◽  
Live Imaging ◽  
Unique Role ◽  
Independent Evolution ◽  
Major Route ◽  
Bona Fide ◽  
Endocytic Vesicles

SUMMARYIn plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME follows the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the surface but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

scholarly journals Methods to study organogenesis in decapod crustacean larvae II: analysing cells and tissues

2021 ◽  
Vol 75 (1) ◽  
Author(s):  
R. R. Melzer ◽  
F. Spitzner ◽  
Z. Šargač ◽  
M. K. Hörnig ◽  
J. Krieger ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Imaging Techniques ◽  
Light And Electron Microscopy ◽  
Decapod Crustacean ◽  
Companion Paper ◽  
Future Directions ◽  
Reflected Light ◽  
Organ Systems ◽  
Adult Organism

AbstractCells and tissues form the bewildering diversity of crustacean larval organ systems which are necessary for these organisms to autonomously survive in the plankton. For the developmental biologist, decapod crustaceans provide the fascinating opportunity to analyse how the adult organism unfolds from organ Anlagen compressed into a miniature larva in the sub-millimetre range. This publication is the second part of our survey of methods to study organogenesis in decapod crustacean larvae. In a companion paper, we have already described the techniques for culturing larvae in the laboratory and dissecting and chemically fixing their tissues for histological analyses. Here, we review various classical and more modern imaging techniques suitable for analyses of eidonomy, anatomy, and morphogenetic changes within decapod larval development, and protocols including many tips and tricks for successful research are provided. The methods cover reflected-light-based methods, autofluorescence-based imaging, scanning electron microscopy, usage of specific fluorescence markers, classical histology (paraffin, semithin and ultrathin sectioning combined with light and electron microscopy), X-ray microscopy (µCT), immunohistochemistry and usage of in vivo markers. For each method, we report our personal experience and give estimations of the method’s research possibilities, the effort needed, costs and provide an outlook for future directions of research.

scholarly journals Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy

2018 ◽  
Vol 272 (2) ◽  
pp. 87-95 ◽  
Author(s):  
M. FUEST ◽  
G.M. NOCERA ◽  
M.M. MODENA ◽  
D. RIEDEL ◽  
Y.X. MEJIA ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Live Imaging

scholarly journals Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM

Microscopy ◽  
2020 ◽  
Author(s):  
Keisuke Ohta ◽  
Shingo Hirashima ◽  
Yoshihiro Miyazono ◽  
Akinobu Togo ◽  
Kei-ichiro Nakamura
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Optical Microscope ◽  
Live Imaging ◽  
Reconstruction Technique ◽  
Electron Microscopic

Abstract 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.

scholarly journals A protocol for Epon-embedding-based correlative super-resolution light and electron microscopy

2019 ◽  
Author(s):  
Pingyong Xu ◽  
Tao Xu ◽  
Mingshu Zhang ◽  
Zhifei Fu ◽  
Dingming Peng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
Ultrathin Section ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Chemical Fixation ◽  
High Quality ◽  
The Core ◽  
Sample Preparation Procedure

Abstract This protocol describes a detailed step-by-step sample preparation procedure for Epon based post-embedding correlative super-resolution light and electron microscopy (SR-CLEM). The newly developed fluorescent protein, mEosEM, is the core in this protocol. The advantage of this method is to simultaneously obtain high-quality LM and EM images from the same ultrathin section of Epon-embedded sample after conventional chemical fixation.

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

Sign in / Sign up

Export Citation Format

Share Document