Visualizing the localization of protein isoforms in HeLa cells with laser confocal microscopy
MDCU Visualizing Cells
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Visualizing Cells
VINIDABUNDITM.D., Ph.D.NIGUN WORAPUNPONG M.D.DEPARTMENTOFANATOMY
FACULTYOFMEDICINE
CHULALONGKORNUNIVERSITY 1
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Unit of length in microscopes
m (micrometer)= 10-6 m
nm (nanometer) = 10-9
mA (Angstrom unit) = 10-10 m
Measure sizes of cells & their components
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RESOLUTION (Resolving power)
Two points of discrimination (theability of a microscope to separate
images of closely positionedobjects)
Limit of Resolution (L.R) is a value
of the resolutionHuman eyes, Limit of Resolution =
0.2 mm.3
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Looking at the structure of
cells in the microscope
Limit of resolutionLM = 0.2 mTEM = 0.002 nm
= 1 (at best)SEM = 10 nm
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RESOLVING
POWER
Naked eye
LM
TEM
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Each image is magnified by a factor of tenBegin from a thumb to the skin
NAKED EYE LM
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LM TEM
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Each image is magnified by a factor of tenFrom cells to ribosomes
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TEM BEYOND THE POWER OF TEM
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Each image is magnified by a factor of ten
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MICROSCOPIES () Light (bright-field) M.
Polarizing M.
Phase & Interference &
Differential interference M. Darkfield M.
Fluorescence M.
Confocal laser scanning M.
Transmissionelectron M.
Scanning
electron M. High-voltage
electron M.
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LM (R=0.2 ) TEM (R=0.5 nm)
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A: Light path in compound
microscope
B: A modern
research LM
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Interference between light waves
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Living cells are seen in
A: Bright-field microscope
B: Phase-contrast microscope14
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C: Normarski differential interference-contrast microscope
D: Dark-field microscope15
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Hematoxylin & Eosin
negative positive
charges charges
Molecules Molecules
H&E SECTIONS
H as Basic dye Neg. for the distribution of DNA, RNA,acidic proteinsas purple, E as acid dye Posit. for basicproteins as pink.
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The optical system of a fluorescence microscope
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Fluorescence Microscope
- Locates specific molecules or proteinsin cells
- Fluorescent dyes(fluorescein green
& rhodamine red)
- Technic : couple fluorescent dyesantibody molecules asimmunocytochemistry
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Multiple-
fluorescent probemicroscopy- a cell in mitosis- 3 different
fluorescent probes,spindle MT (green),centromeres (red),DNA of the condensed
chromosomes (blue)
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Conventional & confocalfluorescence microscope
A : unprocessed image: blurred
B : confocal image:out-of-focus informative
is removed
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SCANNING EM (SEM)
Imageof 3-D
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HRSEM VS TEM
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A SEM VS LM
OF STEREOCILIA
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B. DIFFERENTIAL - INTERFERENCE - CONTRAST LM
C. TRANSMISSION EM
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Fixed Tissues & Sectioned
for the LM & TEMFixation: 1 & 2(TEM)
Dehydration & Clearing
Embedding: Paraffin (LM),Rasin (TEM)
Sectioning: Micro or UltramicrotomeStaining: H&E (LM),
lead hydroxide (TEM)( ***Frozen sections)
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Tissue is sectionedfor LM (4-8m):Various kinds of
staining
Tissue is sectioned
for TEM (600 -1000)
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Summary of H&E Staining
NucleusHeterochromatin BlueEuchromatin NegativeNucleolus Blue
CytoplasmEndoplasmic Ret. BlueGeneral cytoplasm PinkCytoplasmic filaments Pink
Extracellular materialsCollagen fibers PinkElastic fibers PinkReticular fibers Pink
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H&E STAINING
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Transmission Electron
Microscope (TEM) resolves the fine structures
requires the special preparation : very thin, quickpreserved, electron stain, etc.
two dimensions & black, white & grey
Electron scattering ability of heavy electrons
Fluorescent screen for micrographs32
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Glut. as a 1 fixative & Osmium tetroxide as a2fixative (stained and fixed) are two common
chemical fixatives used for electron
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Showing a copper grid (TEM)(glass slides for LM)
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EM micrograph VS LM photograph
R of the TEM, 0.2-0.5 nm is about 103 greater than that of LM35
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Various Technic in visualizing cells(LM or EM)
Conventional Tec.: LM(H&E), EM
Histochemistry : LM(PAS or Feulgen or osmicreaction,etc.)
Cytochemistry: immunocytochemistry (reactionbet. Ag. & Ab.), LM & EM
Cell fractionation: EM Radio-autography: LM & EM
Negative staining : EM36
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What are artifacts?1. Postmortem degeneration2. Shrinkage
3. Precipitation4. Wrinkles & Folds5. Nicks
6. Rough handling
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Immuno-cytochemistry- Antibodies detect specific molecules- LM & TEM- A specific target molecule (Antigen)
Antibodies- Using 1or/and 2Ab coupled
with fluorescein41
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FREEZE-FRACTURE &
FREEZE-ETCH TEM View surfaces, inside the cell Study membranous particles Processes : frozen, crack (middle of lipid bilayer),
shadowed with platinum, dissolvedtissue, replica
: frozen, crack, sublimate the ice (etch), etc.
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E-FaceP-Face
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EM & Freeze-fracture EMreplica of the nuclear
envelope
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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 particlesActin 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 orchromotography
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The radiolabeledprecursor,(H3,thymidine was injected intoan experimentalanimal, which wassacrificed 24 hr later.
Histologic sections &were coated with aphotographic
emulsion & exposedin the dark for 24 hr.
Development of thephotographicemulsion followed by
staining the sectionreveals thelocalization of silvergrain (black dots) onsome nuclei
LM TEM
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EM-AUTORADIOGRAPHY
Pancreatic B cells +3 H-leucine5 min as pulse & chase
+3H Insulin assecretory protein for the
secretionA : 10 min chase, labeled
protein has moved fromRER Golgi stacks
B : 45 min chase, labeledprotein at the secretorygranules
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VISUALIZING MOLECULES
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VISUALIZING MOLECULESIN LIVING CELLS
Some of the methods are used to study thesedynamic processes
Most of current methods use optical
microscopy, are based on the use offluorescent tags and indicator
The molecules that can be specificallyimaged, such as Ca+2 or H+, specific proteins,RNAs, DNA sequences
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MEASURE THE INTRACELLULAR
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MEASURE THE INTRACELLULAR
CONCENTRATION OF FREE Ca+2
Fluorescent indicator (Aequorin,
a luminescent protein)
light
Egg of the medaka fish
Microelectrode withaequorin & Fertilization
Show a wave of release of freeCa+2 from internal stores justbeneath the plasma membrane
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Ca+2
VIEWING THE CELL & TISSUES
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VIEWING THE CELL & TISSUES
in LM slides
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THE CELLS th j t f
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THE CELLS are the major components ofbasic tissues
THE CYTOLOGY: MAJOR CELLULAR COMPONENTS
@ The plasma membrane (plasmalemma): closing thecellular contents & separating them from the external
environment
@ The cytoplasm: cytosol,various membrane-boundorganelles, inclusions & cytoskeletal elements
@ The nucleus: genetic material(DNA) & is surroundedby a double membrane
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Levels of Anatomic Organization
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4 Basic tissues as functional constitution oforgans controlling the body system:
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EPITHELIAL T.: COVERING & GLANDULAR Epithelial tissue CONNECTIVE T.: GENERAL & SPECIAL Connective tissue
(i.e., blood, cartilage & bone)
MUSCULAR T.: SMOOTH, SKELETAL & CARDIAC MUSCLE
NERVOUS T.: NEURONS & SUPPORTING TISSUE
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This long, tube-like organ is
constructed from epithelialtissue, connective tissue ,muscular tissue & nervoustissue. 59
1 THE EPITHELIAL TISSUE
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EPITHELIAL TISSUES ARE COMPOSED OF GROUPS OF CELLSwith no free intercellular substances: MOSTLY KNOWN AS
THE PARENCHYMAL TISSUE of the Organ-System.
1. THE EPITHELIAL TISSUE
A & B as covering type, C = glandular type
A B c
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2. THE CONNECTIVE TISSUE
Connective tissue cells + Extracellular matrix in largeamount (ECM: collagen, elastic, reticular fibers& proteoglycans) in large amount
Functions : Supportive & connecting frame network(or stroma)
Is directed supplies by blood & lymphatic vvs.
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CONNECTIVE TISSUE GENERAL &
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CONNECTIVE TISSUE : GENERAL &SPECIAL, Cells and 3 types of fibers
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Connective tissue cells: tissue, leucocytes, fibroblast,
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, y , ,plasma cell, etc.
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3 THE MUSCULAR TISSUE
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3. THE MUSCULAR TISSUE
Three types: skeletal, smooth and cardiac muscle
Muscle fibers or cells function for contraction
(microfilaments: actin & myosin)
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B C65
4 THE NERVOUS TISSUE
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4. THE NERVOUS TISSUE
Nervous system: CNS (brain & spinal cord) and PNS(peripheral ganglia, nerves, nerve endings)
Nervous tissue of CNS consists of neurons & glia, but of
PNS consists of neurons (of ganglia) & supporting cells(satellites & Schwann cells)
Functions as receives stimuli from both the internal &
external environments appropriate, coordinatedresponses in various effector organs
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NEURONS: vesicular N. &
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Nissl bodies
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Reference
Molecular Biology of THE CELL by Alberts,
et. al., Fourth edition, Chapter 9, page 579-615, 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 Publishercompany.
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