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)

A64


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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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mdcu visualizing cells

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