Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 14-15
Author(s):  
Conly L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Light Microscopy ◽  
Living Cell ◽  
Light And Electron Microscopy ◽  
Time Behavior ◽  
Dynamic Interactions ◽  
Positive Identification ◽  
Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

Demonstration of pulmonary vascular perfusion by electron and light microscopy

1993 ◽  
Vol 75 (4) ◽  
pp. 1877-1883 ◽  
Author(s):  
M. F. Konig ◽  
J. M. Lucocq ◽  
E. R. Weibel
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Rabbit Serum ◽  
Capillary Perfusion ◽  
Vascular Perfusion ◽  
Thin Sections ◽  
Gold Particles ◽  
Capillary Recruitment

To estimate the fraction of dense pulmonary capillary network that is perfused under physiological conditions, we developed a new method for the demonstration of in vivo capillary perfusion by light and electron microscopy. Blood plasma was labeled by 8-nm colloidal gold particles coated with rabbit serum albumin. In anesthetized rabbits, 4#x2013;5 ml of this tracer were injected into the right atrium. Two and 15 min later, the circulation was interrupted by a snare around the heart, and the lung was fixed by instillation with glutaraldehyde. Gold particles were found in the plasma space of alveolar capillaries as well as in other organs. A random sample of thin sections studied by electron microscopy revealed that the entire capillary bed of the lung was perfused at least with plasma within 2 min after tracer infusion. Light microscopy of silver-enhanced sections showed areas with different staining intensities but no obviously unperfused capillaries. The concept of capillary recruitment, which would require a significant fraction of capillaries unperfused at rest, may have to be reassessed to consider time factors as well as the two-phase nature of blood; red blood cells and plasma may take different paths.

scholarly journals Dense HRP filling in pre-fixed brain tissue for light and electron microscopy.

1987 ◽  
Vol 35 (10) ◽  
pp. 1127-1136 ◽  
Author(s):  
G H Kageyama ◽  
R L Meyer
Keyword(s):  
Electron Microscopy ◽  
Optic Nerve ◽  
High Resolution ◽  
Light Microscopy ◽  
Glial Cells ◽  
Light And Electron Microscopy ◽  
Polyethylene Tube ◽  
Fixed Tissue ◽  
Cat Brain

The use of neuroanatomical markers in tissues that have been pre-fixed has been virtually ignored, even though this approach could offer certain advantages over in vivo methods, in terms of convenience of application and choice of markers. We have found that HRP can be used on well-fixed brains of cats and goldfish to fill neurons, dendrites, axons, terminals, glial cells, and glial processes for high-resolution light microscopy and electron microscopy. Best results were obtained using brains that were perfusion-fixed with 2.5% depolymerized paraformaldehyde and 1.5% glutaraldehyde. Two methods of HRP application were used: optically guided injections of microliter quantities into various regions of cat brain, and optic nerve fills in goldfish by attaching an HRP-filled polyethylene tube for periods of 1 day to 2 weeks. HRP applied in these ways to pre-fixed tissue was found to fill neurons or glial cells with solid label in the anterograde and retrograde directions.

The Response of Testis Cells to Low Dose X-Irradiation

1981 ◽  
Vol 39 ◽  
pp. 542-543
Author(s):  
D. E. Philpott ◽  
W. Sapp ◽  
C. Williams ◽  
Joann Stevenson ◽  
S. Black
Keyword(s):  
Electron Microscopy ◽  
Stem Cell ◽  
Light Microscopy ◽  
Dose Level ◽  
Cross Sections ◽  
Low Dose ◽  
Light And Electron Microscopy ◽  
Cellular Responses ◽  
Post Irradiation ◽  
X Irradiation

The response of spermatogonial cells to X-irradiation is well documented. It has been shown that there is a radiation resistent stem cell (As) which, after irradiation, replenishes the seminiferous epithelium. Most investigations in this area have dealt with radiation dosages of 100R or more. This study was undertaken to observe cellular responses at doses less than 100R of X-irradiation utilizing a system in which the tissue can be used for light and electron microscopy.Brown B6D2F1 mice aged 16 weeks were exposed to X-irradiation (225KeV; 15mA; filter 0.35 Cu; 50-60 R/min). Four mice were irradiated at each dose level between 1 and 100 rads. Testes were removed 3 days post-irradiation, fixed, and embedded. Sections were cut at 2 microns for light microscopy. After staining, surviving spermatogonia were identified and counted in tubule cross sections. The surviving fraction of spermatogonia compared to control, S/S0, was plotted against dose to give the curve shown in Fig. 1.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

Abstract 530: Hemostasis Revisited: a Combined Light and Electron Microscopy Analysis of Hemostatic Plug Formation in vivo

2018 ◽  
Vol 38 (Suppl_1) ◽  
Author(s):  
Maurizio Tomaiuolo ◽  
Chelsea N Matzko ◽  
Izmarie Poventud-Fuentes ◽  
John W Weisel ◽  
Lawrence F Brass ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Plug Formation

scholarly journals An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorothea Pinotsi ◽  
Simona Rodighiero ◽  
Silvia Campioni ◽  
Gabor Csucs
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Wide Field ◽  
Set Up ◽  
Bio Compatibility ◽  
Scanning Electron

Abstract A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

scholarly journals Electron Microscopy in Rat Brain Slices Reveals Rapid Accumulation of Cisplatin on Ribosomes and Other Cellular Components Only in Glia

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Lidia Zueva ◽  
Yomarie Rivera ◽  
Lilia Kucheryavykh ◽  
Serguei N. Skatchkov ◽  
Misty J. Eaton ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Organic Cation ◽  
Nuclear Dna ◽  
Staining Method ◽  
Brain Slices ◽  
High Electron ◽  
Rapid Accumulation ◽  
Cellular Components ◽  
Direct Attack

Cisplatin is a widely used, effective anticancer drug. Its use, however, is associated with several side effects including nephrotoxicity and neurotoxicity. It is known that cisplatin is accumulated in cells by the organic cation transport system and reacts with nucleotides, damaging them, but the precise target of cisplatin-induced neurotoxicity remains obscure. Here we report direct visualization of cisplatin inside brain cells using in vivo “cisplatin staining,” a technique that takes advantage of the high electron density of cisplatin, which contains platinum (atomic mass=195). After applying 0.1% cisplatin to living brain slices for 30 min, we fixed the tissue and observed the accumulated cisplatin using electron microscopy. We found that cisplatin was localized mainly to ribosomes associated with endoplasmic reticulum (EPR) in glial cells and to the myelin sheath formed by oligodendrocytes around neuronal axons. Staining of nuclear DNA was moderate. Our in vivo “cisplatin staining” method validated that the main target of cisplatin is a direct attack on myelin and the RNA contained in ribosomes.

scholarly journals THE GLOMERULUS IN EXPERIMENTAL RENAL DISEASE IN RATS AS OBSERVED BY LIGHT AND ELECTRON MICROSCOPY

1955 ◽  
Vol 102 (5) ◽  
pp. 573-580 ◽  
Author(s):  
Carolyn F. Piel ◽  
Luther Dong ◽  
F.W.S. Modern ◽  
Joseph R. Goodman ◽  
Roger Moore
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Basement Membrane ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
Crescent Formation ◽  
Colloidal Iron ◽  
Narrow Channels ◽  
Glomerular Capillary

Nephrotoxic serum disease in rats has been studied by light and electron microscopy from 1 hour to 10 weeks after production of the disease. By light microscopy leucocytic infiltration of the glomerular capillary was observed between the 3rd and 6th hour. At 6 hours an increase in colloidal iron-positive material was observed coating the extraluminal surface of the capillaries. Also at this time swelling of the endothelial cells becomes prominent. By 72 hours, thickening of the basement membrane was observed. Glomerular capillary thrombi were observed in approximately half the tissue examined in the first 2 weeks of disease. 50 per cent of the animals showed severe chronic lesions, exudation into the capsular space, crescent formation, and obliteration of glomeruli. At 1 hour electron microscopic pictures showed that osmophilic material may line the foot processes of the epithelial cells and obliterate all but narrow channels of the space between the feet. By 6 hours thickening of the basement membrane was prominent. This change persisted throughout 10 weeks of observation. The tissue from animals which had severe chronic alterations by light microscopy revealed changes which could not be interpreted at this time.

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.

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