Amalgamation of two endocytic probe techniques: fluoresceinated cationized ferritin can show up, sequentially, selected structures, first in living cells and then by electron microscopy

1992 ◽  
Vol 98 (2) ◽  
pp. 141-143
Author(s):  
M. R. Young ◽  
P. D'Arcy Hart
Keyword(s):  
Electron Microscopy ◽  
Living Cells ◽  
Cationized Ferritin

scholarly journals Antibody-induced membrane fusion in Paramecium

1984 ◽  
Vol 65 (1) ◽  
pp. 153-162
Author(s):  
A. Barnett ◽  
E. Steers
Keyword(s):  
Electron Microscopy ◽  
Membrane Fusion ◽  
Phase Contrast ◽  
Immobilized Cells ◽  
Living Cells ◽  
Immune Serum ◽  
Induced Membrane ◽  
Transmission Electron ◽  
Membrane Changes ◽  
Antigen Antibody

Immobilization of cells by specific immune serum involves crosslinking between immunoglobulin G (IgG) and the i-antigen in the cell membrane. Globular material is seen to accumulate at the ciliary tips by phase-contrast and fluorescence microscopy in a manner analogous to ‘capping’ in more typical eukaryotes. When immobilized cells of Paramecium are examined by scanning electron microscopy, the fused ciliary tips are seen to be distended, discoidal membranes. Transmission electron microscopy often reveals several ciliary axonemes enclosed within a single, enlarged membrane that is oriented with the ferritin-labelled second antibody directed against the i-antigen antibody on the outer surface only. Fixed cells or living cells treated with immune Fab do not show membrane changes, but do bind antibody. Membrane fusion occurs only if cells are alive and the i-antigen is directly or indirectly cross-linked by intact immune IgG.

scholarly journals Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

ACS Nano ◽  
2015 ◽  
Vol 10 (1) ◽  
pp. 265-273 ◽  
Author(s):  
Nalan Liv ◽  
Daan S. B. van Oosten Slingeland ◽  
Jean-Pierre Baudoin ◽  
Pieter Kruit ◽  
David W. Piston ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Living Cells

scholarly journals Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
H. M. van der Schaar ◽  
C. E. Melia ◽  
J. A. C. van Bruggen ◽  
J. R. P. M. Strating ◽  
M. E. D. van Geenen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Composition ◽  
Viral Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell ◽  
Genome Replication ◽  
Fluorescent Reporter ◽  
Unique Protein

ABSTRACT Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

scholarly journals Further Observations on the Fine Structure of Chrysochromulina Ericina Parke & Manton

1961 ◽  
Vol 41 (1) ◽  
pp. 145-155 ◽  
Author(s):  
I. Manton ◽  
G. F. Leedale
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Cell Surface ◽  
Light Microscopy ◽  
Flat Plate ◽  
Outer Layer ◽  
Living Cells ◽  
Layered Plates ◽  
Thin Sections ◽  
Micro Anatomy

C. ericina Parke & Manton has been re-investigated to add salient features of micro-anatomy from the electron microscopy of thin sections and also to add photographs of living cells taken with anoptral contrast light microscopy.The most important new observations concern the scales which are shown to be essentially two-layered plates in which the layers in the very large spined scales have become separated except at their edges, with the outer layer greatly hypertrophied to produce a hollow spine with a flared base closed at the bottom by a flat plate. The patterns of external marking on the two layers are very similar in both plate-scales and spines in this species and the orientation of both with respect to the cell surface has been demonstrated by a section of the scales in situ.

scholarly journals A tribute to Shinya Inoue and innovation in light microscopy

2004 ◽  
Vol 165 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Karen R. Dell ◽  
Ronald D. Vale
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Light Microscope ◽  
Light And Electron Microscopy ◽  
Single Molecules ◽  
Living Cells ◽  
Dynamic Processes ◽  
New Techniques

The 2003 International Prize for Biology was awarded to Shinya Inoue for his pioneering work in visualizing dynamic processes within living cells using the light microscope. He and his scientific descendants are now pushing light microscopy even further by developing new techniques such as imaging single molecules, visualizing processes in living animals, and correlating results from light and electron microscopy.

scholarly journals Traffic watch: Live-cell imaging

2003 ◽  
Vol 25 (3) ◽  
pp. 15-17
Author(s):  
David J. Stephens
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Robert Hooke ◽  
The Core ◽  
Biology Research ◽  
Insight Into ◽  
Dynamics Process

Microscopy has been at the core of cell biology research ever since the coining of the term ‘cell’ by Robert Hooke in the 17th Century1. For many years, it has been possible to gain insight into ‘steady-state’ cellular function from the analysis of fixed samples, but it is only relatively recently that imaging of living cells has become a widely used tool to support biochemical and electron microscopy studies. Membrane traffic research, which by its very nature is a highly dynamics process, has benefited hugely from the ability to image specific processes in living cells and tissues.

Correlation of 4Pi- and electron microscopy to study transport through single Golgi stacks in living cells with super resolution

Traffic ◽  
2009 ◽  
Author(s):  
Giuseppe Perinetti ◽  
Tobias Müller ◽  
Alexander Spaar ◽  
Roman Polishchuk ◽  
Alberto Luini ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Super Resolution ◽  
Living Cells

The Study of Stalk Rot and Causal Organisms via Scanning Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 240-241
Author(s):  
Judith A. Murphy
Keyword(s):  
Electron Microscopy ◽  
Living Cells ◽  
Economic Importance ◽  
Positive Correlation ◽  
Stalk Rot ◽  
Host Parasite Interactions ◽  
Parenchyma Cells ◽  
Host Parasite ◽  
Natural Cell ◽  
Scanning Electron

Cooperative SIU Research Includes: A. J. Pappelis, W. E. Schmid, O. Myers, Jr. (Botany); J. N. Bemiller, C. Hinckley (Chemistry); and J. Murphy (Center for Electron Microscopy).Stalk rot of corn is a disease costing millions of dollars annually. Because of the economic importance of this disease, many studies have been undertaken on the nature of resistance to stalk rot, host-parasite interactions, as well as studies of the various pathogens causing stalk rot.In studying the number of mechanisms for stalk rot resistance, A. J. Pappelis discovered a positive correlation between stalk rot susceptibility to Diplodia maydis and the pattern of natural cell death of parenchyma cells in the stalk and a positive correlation between disease resistance and the presence of living cells in that tissue.

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