Escolar Documentos
Profissional Documentos
Cultura Documentos
VINIDA BUNDIT M.D., Ph.D. NIGUN WORAPUNPONG M.D. DEPARTMENT OF ANATOMY FACULTY OF MEDICINE CHULALONGKORN UNIVERSITY
Looking
at the structure of cells in the microscope of resolution LM = 0.2 m TEM = 0.002 nm = 1 (at best) SEM = 10 nm
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Limit
RESOLVING POWER
Naked eye
LM TEM
Each image is magnified by a factor of ten Begin from a thumb to the skin
NAKED EYE LM
MICROSCOPIES ()
Light (bright-field) M. Polarizing M. Phase & Interference & Differential interference M. Darkfield M. Fluorescence M. Confocal laser scanning M. Transmission electron M. Scanning electron M. High-voltage electron M.
LM (R=0.2 )
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B: A modern research LM
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C: D:
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H&E SECTIONS
Hematoxylin negative & Eosin positive
charges
Molecules
charges
Molecules
H as Basic dye Neg. for the distribution of DNA, RNA, acidic proteins as purple, E as acid dye Posit. for basic proteins as pink. 16
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Fluorescence Microscope
- Locates specific molecules or proteins in cells - Fluorescent dyes
(fluorescein & rhodamine green red)
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SCANNING EM (SEM)
Image of 3-D
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HRSEM VS TEM
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A SEM VS LM
OF STEREOCILIA
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H&E STAINING
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Glut. as a 1 fixative & Osmium tetroxide as a 2fixative (stained and fixed) are two common chemical fixatives used for electron
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EM micrograph VS LM photograph
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Immuno-cytochemistry
- Antibodies detect specific molecules - LM & TEM - A specific target molecule (Antigen) Antibodies - Using 1or/and 2Ab coupled with fluorescein
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E-Face
P-Face
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NEGATIVE STAINING
PROCESSES : Isolate macromolecules, e.g., DNA or large proteins, virus : Heavy metal medium to provide contrast in a grid : View in the TEM
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NEGATIVE STAINING
Virus particles
Actin filaments
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AUTORADIOGRAPHY
A typical pulse-chase experiment using radioisotopes (A, B, C, D, as different compartmets in the cell) Detected by autoradiograph or cell-fractionation or chromotography
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The radiolabeled precursor,(H3,thymidi ne was injected into an experimental animal, which was sacrificed 24 hr later. Histologic sections & were coated with a photographic emulsion & exposed in the dark for 24 hr. Development of the photographic emulsion followed by staining the section reveals the localization of silver grain (black dots) on some nuclei
LM
TEM
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EM-AUTORADIOGRAPHY
Pancreatic B cells +3 H-leucine 5 min as pulse & chase
+3H
A : 10 min chase, labeled protein has moved from RER Golgi stacks B : 45 min chase, labeled protein at the secretory granules 52
MEASURE THE INTRACELLULAR CONCENTRATION OF FREE Ca+2 Fluorescent indicator (Aequorin, a luminescent protein) Ca+2 light Egg of the medaka fish Microelectrode with aequorin & Fertilization Show a wave of release of free Ca+2 from internal stores just beneath the plasma membrane
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@ The plasma membrane (plasmalemma): closing the cellular contents & separating them from the external environment @ The cytoplasm: cytosol,various membrane-bound organelles, inclusions & cytoskeletal elements @ The nucleus: genetic material(DNA) & is surrounded by a double membrane
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This long, tube-like organ is constructed from epithelial tissue, connective tissue , muscular tissue & nervous tissue.
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EPITHELIAL TISSUES ARE COMPOSED OF GROUPS OF CELLS with no free intercellular substances: MOSTLY KNOWN AS THE PARENCHYMAL TISSUE of the Organ-System.
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A
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Reference
Molecular Biology of THE CELL by Alberts, et. al., Fourth edition, Chapter 9, page 579615, 2008, Garland Science, Taylor & Francis Group,NY. Histology 1: The cell and basic tissues, by V. Bundit, et. al., Third edition, Chapter 1, page1-30, 2545, Chulalongkorn Publisher company.
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