INTRODUCTION TO CELL BIOLOGY - Lazarovnikolai.lazarov.pro/.../Introduction_to_Cell_Biology.pdf ·...

Introduction to Cell Biology

1.Aims and scopes of cytology, histology and embryology

2.Brief historical review

3.Microscopy and microscope types

4.Methods for microscopic observations

5.General principles of cytological and histological techniques

6.Techniques of cell and molecular biology

Prof. Dr. Nikolai Lazarov

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Cytology – now Cell Biology:(Gr. κύτος, kytos, a hollow + logos, study)

Objective of cytology, histology and embryology

Histology: (Gr. ἱστός, histos, web or tissue + logos)

general histology

special histology = microscopic anatomy of organs

Embryology: (Gr. έμβρυον, embryon + logos)

general embryology (embryogenesis)

special embryology (organogenesis)


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History of cytology and histology

Period of observations (1590-1839)

Robert Hooke, 1665

Antony van Leeuwenhoek, 1678

Period of generalization (1839-)

Cell theory: Schleiden and Schwann (1838-1839)

Omnis cellula e cellula: Rudolf Virchow(1852)

Omnis nucleusе nucleo:Walther Flemming (1860)

Drawing of the structure of cork by Robert Hook

Period of the study of the cell structure

Period of cell and molecular biology –

20th century

biochemical cytology

cytogenetics

cytophysiology

molecular biology

cellular ecology


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History of cytology and histology


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History of light microscopy

NB: the invention of the compound microscope is credited to the Dutch spectacle maker, Zacharias Janssen, around the year 1590


Émile DUCLAUX METCHNIKOFF Émile ROUX

Albert CALMETTE Jules BORDET

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Microscopy

Microscope types:

light (optical) microscope

phase contrast microscope

interference microscope

differential interference microscopy (DIC)

fluorescence microscope

polarizing microscope

confocal laser scanning microscope

electron microscope (EM)

transmission EM (TEM)

scanning EM (SEM)

scanning tunneling microscope (STM)

atomic force microscope


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Light microscope

Mechanical part:

stand (arm&base)

objective holder (stage)

tube

Optical part:

ocular lens (eyepiece)

objective lens – types

condenser

Illumination part:

illumination source (mirror or light)

filters

Ernst Abbe(1840-1905)

Carl Zeiss(1816-1888)

NB: Zeiss made contributions to lens manufacturing that have aided the modern production of lenses while Abbe created the mathematical foundation of microscope design


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Light microscopy

NB: Ernst Abbe developed a mathematical description for the resolution limit of the microscope

1010

Resolving power



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Frederik Zernicke(1888 – 1966)Nobel Prize in Physics, 1953

does not require staining to view the slide

possible to study living cells and the cell cycle

Phase contrast microscope


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А. Lebedeff, 1930 –designed and built the first interference microscope useful for assessingsurface properties of cellsand other biologic objects

Interference microscope


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Polarizing microscope

designed to observe specimens that are visible primarily due to their optically anisotropic character allows tissue structures containing oriented molecules (such as cellulose, collagen, microtubules, and microfilaments) to be recognized

two filters – polarizer and analyzer


Albert H. Coons

(1912 – 1978)

Fluorescence microscope

used to display naturally occurring fluorescent (autofluorescent) molecules – neurotransmitters, vitamin A

the immunohistochemical techniques for labeling antibodies were developed in the early 1940s by Albert H. Coons

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Confocal laser scanning microscope

a technique for obtaining high-resolution optical images with depthselectivity and reconstruct them into a three-dimensional image

Marvin Minsky(1926 – 2016)

The first confocal scanning microscope was built by Marvin Minsky in 1955


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1939, first commercial TEM(Ruska, von Borries)

Max Knoll and Ernst Ruska 1931, the first transmission electron microscope (TEM)

Electron microscope


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1965, Charles Oatleythe first scanning EM (Stereoscan)

Electron microscope


Electron microscopy

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1919

Cryo-electron microscopy


2020

Atomic force microscope


The first atomic force microscope was invented by IBM scientists in 1982


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Vital observations

Ross Granville Harrison

(1870-1959)

Alexis Carrel(1873-1944)

The Nobel Prize in Physiology or

Medicine 1912

Supravital microscopy

Cell, tissue and organ cultures: in vitro and in vivo

primary cell cultures:

dissociated (cell cultures)

explant (tissue cultures)

secondary: cell lines

Medical applications:

Study of the metabolism of normal and cancerous cells

Development of new drugs

Study of parasites that grow only within cells, such as viruses, mycoplasma and some protozoa

Vaccine creation

Cytogenetic research: chromosome analysis

determination of human karyotype

genetic disorders

gene and cell engineering

Observation – inverted microscope


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Methods for observation of fixing cells and tissues

preparation of histological sections

– the paraffin technique steps:

removal of tissue: biopsy, necropsy

fixation: types of fixatives

rinsing

dehydration

clearing

infiltration

embedding: paraffin – Klebs (1869)

sectioning: microtome – Oschatz (1843)

staining: types of stains

mounting

specimen preparation for TEM

Johannes Purkinje

(1787-1869)


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Methods for observation of fixing cells and tissues

Johannes Purkinje

(1787-1869)

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Interpretation of structures in tissue sections


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Virtual microscopy



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Freeze-Etching and Freeze-Fracture (Cryofacture)


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ultracentrifuge – T. Svedberg

cell fractionation – A. Claude

allows the isolation of cell constituents

by differential centrifugation

density gradient centrifugation

Theodor Svedberg

(1884-1971)Nobel Prize,

1926

Albert Claude(1899-1983)Nobel Prize,

1974

Cell fractionation


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Autoradiography of tissue sections

Antoine Lacassagne(1884-1971) 1924, developed the firstautoradiographic method

Belanger and Leblond,1946 – begin of modern ARG electron microscope ARG light microscope ARG

a technique that permits the localization ofradioactive substances in tissues by meansof emitted radiation effects on photographicemulsions


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Sir William Henry Bragg(1862-1942)

Sir William Lawrence Bragg(1890-1971)Nobel Prize in Physics, 1915

X-ray crystallography

a method of determining the arrangement of atoms within a crystal to solve the crystal structure of:

proteins cholesterol and vitamin B12

hemoglobin and myoglobin etc.

"for their services in the analysis of crystal structure by means of X-rays"


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Histochemistry and cytochemistry

Histochemistry = LM results

Cytochemistry = ЕМ results

Quantitative analysis: principles

to preserve structure of cells and tissues

localizations on the original sites in the cell: to avoid translocation

specificity of the reaction: positive and negative controls

Qualitative analysis: microspectrophotometry

Founder of the method: Francois-Vincent Raspail (1794-1878)


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Enzyme histochemistry: principles and applications

Enzyme, substrate, product

Principles:

fresh, unfixed material – cryostat

short-term fixation at lower temperature

pH optimum of the detected enzyme: buffers

Basic requirements:

demonstration of final product, not the enzyme

insoluble product: true localization in the cell

color product: easily visible on the background

Enzyme + Substrate = unstained reaction product

Product + Dye = insoluble colored final reaction product


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Catalytic enzyme histochemistry

Hydrolases: method of Gomori (1950, 1952)

acid phosphatase – lysosomes and Golgi complex

Rat kidney

oxidoreductases:method of Nachlas (1957)

succinate dehydrogenase –mitochondria

catalyzes the oxidation of succinate to fumarate

succinate + acceptor = fumarate + reducted acceptor

Rat kidney


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Demonstration of proteins

the histochemical methods usually do not permit identification of specific proteins in cells and tissues

elastic fibers: orcein

Weigert's resorcin-fuchsin

amino acids: immunocytochemistry

chemical groups: paraldehyde-fuchsin – neurosecrete, insulin

solubility and isoelectric point


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Demonstration of oligosaccharides & polysaccharides

PAS-reaction (Periodic Acid-Schiff)

demonstration of glycogen in tissues

demonstration of glycoproteins

demonstration of glycosaminoglycans


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Demonstration of lipids

Sudan IV(red)

Sudan ІІІ (orange)

Sudan В(black)

best revealed with dyes that are soluble in lipids:

http://www.botany.hawaii.edu/bot606/606Photo/BasicAnatomy/StainCSlip/CoverSlip_files/SudanBazOilSeed.jpg

http://www.botany.hawaii.edu/bot606/606Photo/BasicAnatomy/StainCSlip/CoverSlip_files/SudanBazOilSeed.jpg


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Demonstration of nucleic acids

based on basophilia of nuclei acids

RNA: method of Brachet

(1940-1941)

methyl green-pyronin

DNA: method of Feulgen

and Rossenbeck (1924)

(Feulgen reaction)


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coupling with an enzyme:

PAP method(Sternberger et al., 1970)

ABC technique (Hsu, 1981)

coupling with gold particles

coupling with a fluorescent compound: immunofluorescence method – Coons, 1941

Methods of labeling antibodies:

based on an antigen-antibody reaction

Immunohistochemistry


Immunohistochemistry

Methods of localizing of antigens:

direct method – detection with the fluorescence microscope

indirect method – more sensitive but requires more steps

based on an antigen-antibody reaction

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Immunohistochemical technique


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Sоuthern blotting –detection of a specific DNA

sequence, Edwin M. Southern

Western blotting –detection of specific proteins

Northern blotting –

detection of RNA fragments

(or isolated mRNA)

Hybridization techniques


In situ hybridization

Radioactive in situ hybridization (ISH):

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Nonradioactive in situ hybridization:originally developed by Pardue and Gall (1969),

and (independently) by John et al. (1969)

Flow diagram for ISH procedure

Microscopy

Immunocytochemical visualization


Denaturation of in situ target DNA(probe and target)

Preparation of slides and fixation of material

Choice of the probe and its labeling


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Medical applications

gene mapping

localization of gene expression

systematization of nuclear DNA and RNA

replication

cell sorting

In fundamental research:

In clinical research:

cytogenetics

prenatal diagnostics

gene disorders

diagnostics of infectious and malignant diseases

biological dosimetry

Lichtman et al.: Nature 2007, 450:56-62 44


Human brain connectivity

Connectome Brainbow

transgenic painting in the brain


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Thank you…

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INTRODUCTION TO CELL BIOLOGY - Lazarovnikolai.lazarov.pro/.../Introduction_to_Cell_Biology.pdf ·...

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