Visualizing Cells

VINIDA BUNDIT M.D., Ph.D. NIGUN WORAPUNPONG M.D. DEPARTMENT OF ANATOMY FACULTY OF MEDICINE CHULALONGKORN UNIVERSITY

Unit of length in microscopes m (micrometer)= nm (nanometer) 10-6 m = 10-9 m

A (Angstrom unit) = 10-10 m

Measure sizes of cells & their components
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RESOLUTION (Resolving power)

Two points of discrimination (the ability of a microscope to separate images of closely positioned objects) Limit of Resolution (L.R) is a value of the resolution Human eyes, Limit of Resolution = 0.2 mm.

 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

Each image is magnified by a factor of ten From cells to ribosomes

LM TEM

Each image is magnified by a factor of ten

TEM BEYOND THE POWER OF TEM

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 )

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: B: Bright-field microscope Phase-contrast microscope

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C: D:

Normarski differential interferencecontrast microscope Dark-field microscope

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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

The optical system of a fluorescence microscope

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Fluorescence Microscope
- Locates specific molecules or proteins in cells - Fluorescent dyes
(fluorescein & rhodamine green red)

- Technic : couple fluorescent dyes antibody molecules as immunocytochemistry

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Multiplefluorescent probe microscopy

- a cell in mitosis - 3 different fluorescent probes,
spindle MT (green), centromeres (red), DNA of the condensed chromosomes (blue)

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A : unprocessed image : blurred B : confocal image:outof-focus informative is removed

Conventional & confocal fluorescence microscope

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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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B. DIFFERENTIAL - INTERFERENCE - CONTRAST LM C. TRANSMISSION EM

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Fixed Tissues & Sectioned for the LM & TEM

Fixation: 1 & 2(TEM) Dehydration & Clearing Embedding: Paraffin (LM), Rasin (TEM) Sectioning: Micro or Ultramicrotome Staining: H&E (LM), lead hydroxide (TEM)
( ***Frozen sections)
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Tissue is sectioned for LM (4-8m) :Various kinds of staining

Tissue is sectioned for TEM (600 -1000)

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Summary of H&E Staining

Nucleus Heterochromatin Euchromatin Nucleolus Cytoplasm Endoplasmic Ret. General cytoplasm Cytoplasmic filaments Extracellular materials Collagen fibers Elastic fibers Reticular fibers Blue Negative Blue Blue Pink Pink Pink Pink 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, quick preserved, electron stain, etc.

two dimensions & black, white & grey

Electron scattering ability of heavy electrons Fluorescent screen for micrographs
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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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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 LM

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Various Technic in visualizing cells (LM or EM)

Conventional Tec.: LM(H&E), EM Histochemistry : LM(PAS or Feulgen or osmic reaction,etc.) Cytochemistry: immunocytochemistry (reaction bet. Ag. & Ab.), LM & EM Cell fractionation: EM Radio-autography: LM & EM Negative staining : EM
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What are artifacts?

1. 2. 3. 4. 5. 6. Postmortem degeneration Shrinkage Precipitation Wrinkles & Folds Nicks 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 fluorescein

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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, dissolved tissue, replica : frozen, crack, sublimate the ice (etch), etc.

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E-Face

P-Face

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EM & Freeze-fracture EM replica 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 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

Insulin as secretory protein for the secretion

A : 10 min chase, labeled protein has moved from RER Golgi stacks B : 45 min chase, labeled protein at the secretory granules 52

VISUALIZING MOLECULES IN LIVING CELLS

Some of the methods are used to study these dynamic processes Most of current methods use optical microscopy, are based on the use of fluorescent tags and indicator The molecules that can be specifically imaged, such as Ca+2 or H+, specific proteins, RNAs, DNA sequences
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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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VIEWING THE CELL & TISSUES in LM slides

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THE CELLS are the major components of basic tissues

THE CYTOLOGY: MAJOR CELLULAR COMPONENTS

@ 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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Levels of Anatomic Organization

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4 Basic tissues as functional constitution of organs controlling the body system: 4

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 epithelial tissue, connective tissue , muscular tissue & nervous tissue.

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1. THE EPITHELIAL TISSUE

A & B as covering type, C = glandular type

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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2. THE CONNECTIVE TISSUE

Connective tissue cells + Extracellular matrix in large amount (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 &

SPECIAL, Cells and 3 types of fibers

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Connective tissue cells: tissue, leucocytes, fibroblast,

plasma cell, etc.

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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)

A
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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, coordinated responses in various effector organs
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NEURONS: vesicular N. & Nissl bodies

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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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