Correlative Light and Electron Microscopy of Rare Cell Populations in Zebrafish Embryos Using Laser Marks

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-SEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-SEM provides a large quantifiable overview of 200 nm tissue sections without the presence of grid bars, and thicker sections enhance the immunofluorescent labelling.

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Mouse Hepatoblastomas: A Histologic, Ultrastructural, and Immunohistochemical Study

Veterinary Pathology ◽

10.1177/030098588802500407 ◽

1988 ◽

Vol 25 (4) ◽

pp. 286-296 ◽

Cited By ~ 26

Author(s):

T. Nonoyama ◽

F. Fullerton ◽

G. Reznik ◽

T. J. Bucci ◽

J. M. Ward

Keyword(s):

Electron Microscopy ◽

Tumor Cells ◽

Immunohistochemical Study ◽

Light And Electron Microscopy ◽

Alpha Fetoprotein ◽

Cell Populations ◽

Bile Canaliculi ◽

Cytoplasmic Organelles ◽

Dense Bodies ◽

Hepatocellular Adenomas

Hepatoblastomas from B6C3F1 and BALB/c mice were examined by light and electron microscopy and by immunohistochemical reactions for alpha-fetoprotein, keratin, and vimentin. Tumors occurred in one group of a chronic bioassay for the interaction of diet, genetic strain, and the carcinogen, 2-acetylaminofluorene. Tumors had several populations (including epithelial and mesenchymal cells) in various stages of differentiation. Neoplastic epithelial cells had features of embryonal hepatocytes, such as sparse cytoplasmic organelles, absence of glycogen, abundant free ribosomes, occasional bile canaliculi, and peroxisome-like dense bodies. Embryonal fibroblast-like cells had pleomorphic and folded nuclei with prominent perinuclear chromatin and dispersed cytoplasmic organelles. Fibroblast-like cells were surrounded by bundles of collagen fibrils. Intermediate or transitional types of cells were seen. No tumor cells were immunoreactive for mouse alpha-fetoprotein (AFP) antibody, unlike those in hepatocellular adenomas or carcinomas. Epithelial and mesenchymal tumor cells contained intermediate filaments throughout the cytoplasm; some of these cells stained for keratin but not for vimentin. These findings suggest that mouse hepatoblastomas are derived from bipotential liver blastema cells and are composed of a mixture of several cell populations.

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TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages

Scientific Reports ◽

10.1038/s41598-020-79637-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Viola Oorschot ◽

Benjamin W. Lindsey ◽

Jan Kaslin ◽

Georg Ramm

Keyword(s):

Electron Microscopy ◽

Progenitor Cell ◽

Light And Electron Microscopy ◽

Cell Populations ◽

Human Tissues ◽

Scanning Em ◽

Stem Cell Populations ◽

Heterogeneous Tissues ◽

Cytochemical Markers

AbstractIdentifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.

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Immuno-correlative light and electron microscopy (iCLEM) using TEM v1

10.17504/protocols.io.btmrnk56 ◽

2021 ◽

Author(s):

Viola Oorschot ◽

Jillian C Danne ◽

Benjamin Lindsey ◽

Jan Kaslin ◽

Georg Ramm

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Light And Electron Microscopy ◽

High Resolution Electron Microscopy ◽

Cell Populations ◽

Tissue Sections ◽

Resolution Electron ◽

Stem And Progenitor Cells ◽

Scanning Em ◽

Nerve System

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy with the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-TEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-TEM provides high resolution ultrastructural detail of cells, and consecutive ultrathin (62-70 nm) tissue sections can be examined using different labelling techniques involving the use of immunofluorescent and immunogold markers.

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Cell populations in skeletal muscle after regeneration

Development ◽

10.1242/dev.23.2.531 ◽

1970 ◽

Vol 23 (2) ◽

pp. 531-537

Author(s):

J. C. T. Church

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Muscle Regeneration ◽

Light And Electron Microscopy ◽

Cell Populations ◽

Fruit Bat ◽

General Terms ◽

Measuring Techniques ◽

Eidolon Helvum ◽

The Web

Much is known of the processes involved in the regeneration of skeletal muscle after injury. Yet in most accounts, the results are expressed in general or relative terms. Thus Adams, Denny-Brown & Pearson (1962) state that provided the architecture of the muscle survives, the reconstruction of ‘considerable lengths’ of muscle fibre is feasible. Wright (1963) makes a plea for the application of quantitation, with the choice of ‘some muscle that is sufficiently small for counting and measuring techniques to be reasonably applicable…’ The web of the fruit bat Eidolon helvum (Kerr) contains a number of small muscles which have proved very suitable for the detailed study, in light and electron microscopy, of muscle regeneration (Church & Noronha, 1965; Church, Noronha & Allbrook, 1966) (Fig. 1 A–D), and for the quantitation of recovering lesions (Church, 1968). Satellite cells (Mauro, 1961), which are possibly the main source of myoblasts in regenerating muscle, have often been observed, yet again their numbers have only been expressed in general terms.

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The dissociation of transplantable tumors.

Journal of Histochemistry & Cytochemistry ◽

10.1177/25.7.197162 ◽

1977 ◽

Vol 25 (7) ◽

pp. 544-553 ◽

Cited By ~ 9

Author(s):

J S Noel ◽

R M Zucker ◽

N C Wu ◽

S Y Demaray

Keyword(s):

Electron Microscopy ◽

Single Cell ◽

Solid Tumors ◽

Surface Area ◽

Light And Electron Microscopy ◽

Enzymatic Digestion ◽

Cell Populations ◽

Tissue Surface ◽

Transplantable Tumors ◽

Enzymatic Methods

Four animal transplantable solid tumors, composed of varying morphologic architecture and intercellular specializations, were studied by light and electron microscopy. These tumors were dissociated into viable single cell populations using a combination of mechanical and enzymatic methods. The conditions necessary for optimal dissociation consisted of (a) preparation of the tumor to maximize the tissue surface area, (b) enzymatic digestion with continuous agitation and (c) additional agitation to release loosely attached cells. Other factors that influenced the dissociation were optimized and discussed.

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Immuno-correlative light and electron microscopy (iCLEM) using STEM v1

10.17504/protocols.io.bv89n9z6 ◽

2021 ◽

Author(s):

Viola Oorschot ◽

Jillian C Danne ◽

Benjamin Lindsey ◽

Jan Kaslin ◽

Georg Ramm

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Light And Electron Microscopy ◽

High Resolution Electron Microscopy ◽

Cell Populations ◽

Resolution Electron ◽

Stem And Progenitor Cells ◽

Immunofluorescence Labelling ◽

Morphological Detail ◽

Scanning Em

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy and the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-STEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-STEM involves the examination of ultrathin tissue sections (62-70 nm) using immunofluorescence labelling and subsequent SEM imaging to obtain a high resolution overview of these sections with greater morphological detail compared to iCLEM-SEM. This protocol should be of particular interest to EM facilities with SEM, but not TEM access.

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Electron microscopy of endocardial cell populations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083291 ◽

1978 ◽

Vol 36 (2) ◽

pp. 486-487

Author(s):

T. G. Sarphie ◽

C. R. Comer ◽

D. J. Allen

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Cellular Transformation ◽

Cell Populations ◽

Cell Surfaces ◽

Kb Cells ◽

Dna Viruses ◽

Cell Cycles ◽

Ultrastructural Studies ◽

Endocardial Cell

Previous ultrastructural studies have characterized surface morphology during norma cell cycles in an attempt to associate specific changes with specific metabolic processes occurring within the cell. It is now known that during the synthetic ("S") stage of the cycle, when DNA and other nuclear components are synthesized, a cel undergoes a doubling in volume that is accompanied by an increase in surface area whereby its plasma membrane is elaborated into a variety of processes originally referred to as microvilli. In addition, changes in the normal distribution of glycoproteins and polysaccharides derived from cell surfaces have been reported as depreciating after cellular transformation by RNA or DNA viruses and have been associated with the state of growth, irregardless of the rate of proliferation. More specifically, examination of the surface carbohydrate content of synchronous KB cells were shown to be markedly reduced as the cell population approached division Comparison of hamster kidney fibroblasts inhibited by vinblastin sulfate while in metaphase with those not in metaphase demonstrated an appreciable decrease in surface carbohydrate in the former.

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The Morphology of Vacuolated Cells in the Liver Following Administration of Vitamin A.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072484 ◽

1973 ◽

Vol 31 ◽

pp. 408-409

Author(s):

Odell T. Minick ◽

Hidejiro Yokoo ◽

Fawzia Batti

Keyword(s):

Electron Microscopy ◽

Body Weight ◽

Vitamin A ◽

Light And Electron Microscopy ◽

Liver Cells ◽

Prominent Feature ◽

Vascular Space ◽

Vacuolated Cell ◽

Vacuolated Cells ◽

Vacuolated Cytoplasm

Vacuolated cells in the liver of young rats were studied by light and electron microscopy following the administration of vitamin A (200 units per gram of body weight). Their characteristics were compared with similar cells found in untreated animals.In rats given vitamin A, cells with vacuolated cytoplasm were a prominent feature. These cells were found mostly in a perisinusoidal location, although some appeared to be in between liver cells (Fig. 1). Electron microscopy confirmed their location in Disse's space adjacent to the sinusoid and in recesses between liver cells. Some appeared to be bordering the lumen of the sinusoid, but careful observation usually revealed a tenuous endothelial process separating the vacuolated cell from the vascular space. In appropriate sections, fenestrations in the thin endothelial processes were noted (Fig. 2, arrow).

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Control of Autogenous Vein Grafts Prior to Reimplantation, By Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009378x ◽

1972 ◽

Vol 30 ◽

pp. 106-107

Author(s):

John H. L. Watson ◽

John L. Swedo ◽

M. Vrandecic

Keyword(s):

Electron Microscopy ◽

Light And Electron Microscopy ◽

Physiological Saline ◽

Experimental Conditions ◽

Vein Grafts ◽

Arterial Blood ◽

Saphenous Veins ◽

Autogenous Vein ◽

Control Of Parameters ◽

Post Implantation

The ambient temperature and the nature of the storage fluids may well have significant effects upon the post-implantation behavior of venus autografts. A first step in the investigation of such effects is reported here. Experimental conditions have been set which approximate actual operating room procedures. Saphenous veins from dogs have been used as models in the experiments. After removal from the dogs the veins were kept for two hours under four different experimental conditions, viz at either 4°C or 23°C in either physiological saline or whole canine arterial blood. At the end of the two hours they were prepared for light and electron microscopy. Since no obvious changes or damage could be seen in the veins by light microscopy, even with the advantage of tissue specific stains, it was essential that the control of parameters for successful grafts be set by electron microscopy.

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