introduction to cell biology -...

IntroductionIntroduction toto Cell BiologyCell Biology

1.1.Aims and scopes of cytology, histology Aims and scopes of cytology, histology and embryologyand embryology

2.2.Brief historical reviewBrief historical review

3.3.Microscopy and microscope typesMicroscopy and microscope types

4.4.Methods for microscopic observationsMethods for microscopic observations

5.5.General principles of cytological and General principles of cytological and histological techniqueshistological techniques

6.6.TechniquesTechniques ofof cellcell andand molecularmolecular biologybiology

Prof. Dr. Nikolai LazarovProf. Dr. Nikolai Lazarov

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

Objective of Objective of cytology, histology and embryologycytology, 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 History of cytology and histologyof 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 nucleus е

nucleo: Walther

Flemming (1860)

Drawing of the structure of cork by Robert Hook


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� Period of the study of the cell structure

� Period of cell and molecular biology –

20th century

�biochemical cytology

�cytogenetics

�cytophysiology

�molecular biology

�cellular ecology

History History of cytology and histologyof cytology and histology


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History History of light microscopyof light microscopy

NB:NB:NB:NB:NB:NB:NB:NB: the invention of the compound microscope the invention of the compound microscope is credited is credited to the Dutch spectacle maker, Zacharias Janssen, to the Dutch spectacle maker, Zacharias Janssen, aroundaround the year the year 15901590159015901590159015901590


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Émile DUCLAUX METCHNIKOFF Émile ROUX

Albert CALMETTE Jules BORDET


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MicroscopyMicroscopy

�� Microscope typesMicroscope 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 microscopeLight microscope

� Mechanical part:Mechanical part:

� stand (arm&base)

� objective holder (stage)

� tube

� Optical partOptical part::

� ocularocular lens (eyepieceeyepiece)

� objectiveobjective lens – types

� condensercondenser

�� IlluminationIllumination partpart::

� illumination source (mirror or light)

� filters

Ernst AbbeErnst Abbe

Carl ZeissCarl Zeiss

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


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�� resolving powerresolving power D = 0.2 D = 0.2 µµmm

�� depth of fielddepth of field

Light microscopyLight microscopy

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


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Frederik Zernicke Frederik Zernicke (1(1888888 –– 1966)1966)Nobel Prize in Nobel Prize in PhysicsPhysics, 1953, 1953

� does not require staining to view the slide

� possible to study living living cells and the cell cyclecells and the cell cycle

Phase contrast microscopePhase contrast microscope


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

Interference microscopeInterference microscope


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

� two filters – polarizer and analyzer

Polarizing microscopePolarizing microscope


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Albert H. CoonsAlbert H. Coons

1941 –

Fluorescence microscopeFluorescence microscope

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


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Confocal laser scanning microscopeConfocal laser scanning microscope

�� a technique for obtaining higha technique for obtaining high--resolution resolution optical images with depth optical images with depth selectivity selectivity and reconstruct them into a three-dimensional image

Marvin MinskyMarvin Minsky1941 –


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1939, first commercial TEM 1939, first commercial TEM

(Ruska, von Borries)(Ruska, von Borries)

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

Electron microscopeElectron microscope


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1965, Charles Oatleythe first scanning EM (Stereoscan)

Electron microscopeElectron microscope


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Electron microscopyElectron microscopy


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Vital observationsVital observations

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

�� primary cell culturesprimary cell cultures::

�� dissociateddissociated (cell cultures(cell cultures))

�� explantexplant ((tissue culturestissue cultures))

�� secondarysecondary: : cell linescell lines

RossRoss Granville Granville HarrisonHarrison

(1870(1870--1959)1959)

Alexis CarrelAlexis Carrel

(1873(1873--1944)1944)

The Nobel Prize The Nobel Prize in Physiology or in Physiology or

Medicine 1912Medicine 1912

�� Medical applicationsMedical 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

�� Supravital microscopySupravital microscopy


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Methods for observation Methods for observation of fixing cells and tissuesof fixing cells and tissues

�� preparation of histological sectionspreparation of histological sections–– thethe pparaffin araffin ttechniqueechnique stepssteps::� 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 TEMspecimen preparation for TEM

Johannes Johannes

PurkinjePurkinje(1787-1869)


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FreezeFreeze--Etching and Etching and FreezeFreeze--Fracture (Cryofacture)Fracture (Cryofacture)


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�� uultracentrifugeltracentrifuge – T. Svedberg

�� ccell fractionationell fractionation – A. Claude

� allows the isolation of cell constituents

by differential centrifugation

�� density gradient centrifugationdensity gradient centrifugation

Theodor Theodor

SvedbergSvedberg

(1884(1884--1971)1971)

Nobel PrizeNobel Prize, , 19261926

Albert ClaudeAlbert Claude

(1899(1899--1983)1983)

Nobel Prize,Nobel Prize,19741974

Cell fractionationCell fractionation


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Autoradiography Autoradiography of tissue sectionsof tissue sections

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

BelangerBelanger andand LeblondLeblond,,1946 1946 –– begin of modernbegin of modern ARGARG�� electron microscope ARGelectron microscope ARG�� light microscope ARGlight microscope ARG

�� a technique a technique that permits the localization of radioactive substances in tissues by means of emitted radiation effects on photographic emulsions


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Sir William Henry Bragg(1862-1942)

Sir William Lawrence Bragg(1890-1971)Nobel Prize in Nobel Prize in PhysicsPhysics,, 19151915

XX--ray crystallographyray crystallography

�� a method of determining the a method of determining the arrangement of arrangement of atomsatoms within a within a crystalcrystal�� to to solvsolvee the the crystal crystal structure ofstructure 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 Histochemistry and cytochemistrycytochemistry

�Histochemistry = LM results

�Cytochemistry = ЕМ results

Quantitative analysisQuantitative 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 analysisQualitative analysis: microspectrophotometry

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


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Enzyme histochemistryEnzyme histochemistry: : principles andprinciples and applicationsapplications

�� EnzymeEnzyme, , substratesubstrate, , productproduct

�� PrinciplesPrinciples::

� fresh, unfixed material – cryostat

� short-term fixation at lower temperature

� pH optimum of the detected enzyme: buffers

�� Basic requirementsBasic requirements::� demonstration of final product, not the enzyme

� insoluble product: true localization in the cell

� color product: easily visible on the background

EnzymeEnzyme + + SubstrateSubstrate = unstained reaction product= unstained reaction product

ProductProduct + + DyeDye = = insoluble coloredinsoluble colored final reaction productfinal reaction product


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Catalytic enzyme histochemistryCatalytic enzyme histochemistry

�� HydrolasesHydrolases:: method of Gomori (1950, 1952)�� acid phosphataseacid phosphatase – lysosomes and Golgi complex

Rat Rat kidneykidney

�� oxidoreductases:oxidoreductases:method of Nachlas (1957)�� succinate dehydrogenasesuccinate dehydrogenase –

mitochondria� catalyzes the oxidation of

succinate to fumaratesuccinate + acceptor = fumarate + reducted acceptor

Rat Rat kidneykidney


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Demonstration of proteinsDemonstration 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 Demonstration of oligosaccharides & polysaccharidesoligosaccharides & polysaccharides

�� PASPAS--reaction (reaction (PPeriodic eriodic AAcidcid--SSchiff)chiff)�demonstration of glycogenglycogen in tissues

�demonstration of gglycoproteinslycoproteins�demonstration of glycosaminoglycansglycosaminoglycans


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Demonstration of lipidsDemonstration of lipids

�� Sudan IVSudan IV((redred))

�� SudanSudan ІІІІІІ((orangeorange)

�� SudanSudan ВВ((blackblack))

�� best revealed with dyes best revealed with dyes that are soluble in lipids:that are soluble in lipids:


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Demonstration of nucleic acidsDemonstration of nucleic acids

�� based on basophilia based on basophilia of nuclei acids of nuclei acids

�� RNARNA: : method ofmethod of BrachetBrachet(1940(1940--1941)1941)

methyl greenmethyl green--pyroninpyronin

�� DNADNA:: method ofmethod of FeulgenFeulgen

andand RossenbeckRossenbeck (1924)(1924)

((FeulgenFeulgen reaction)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 labelingMethods of labeling antibodiesantibodies::

�� based on an antigenbased on an antigen--antibody reactionantibody reaction

ImmunohistochemistryImmunohistochemistry


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ImmunohistochemistryImmunohistochemistry

�� Methods of localizing ofMethods of localizing of antigensantigens::�� directdirect method – detection with the fluorescence microscope

�� indirectindirect method – more sensitive but requires more steps

�� based on an antigenbased on an antigen--antibody reactionantibody reaction


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Immunohistochemical techniqueImmunohistochemical technique


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SSооuthernuthern blotting –detection of a specific DNA

sequence, Edwin M. Southern

WesternWestern blotting –detection of specific proteins

NorthernNorthern blotting –

detection of RNA fragments

(or isolated mRNA)

Hybridization techniquesHybridization techniques


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In situIn situ hybridizationhybridization

�� Radioactive Radioactive in situin situ hybridization (ISH):hybridization (ISH):


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�� NonradioactiveNonradioactive in situin situ hybridization:hybridization:��originally developed byoriginally developed by ParduePardue andand GallGall (1969), (1969),

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

Flow diagram for ISH procedure

Microscopy

Immunocytochemical visualization

In situ hybridization

Denaturation of in situ target DNA(probe and target)

Preparation of slides and fixation of materialPreparation of slides and fixation of material

Choice of the probe and its labelingChoice of the probe and its labeling

In situIn situ hybridizationhybridization


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Medical applicationsMedical applications

�� gene mappinggene mapping

�� localization of gene localization of gene expressionexpression

�� systematization of systematization of nuclearnuclear DNA and RNADNA and RNA

�� replicationreplication

�� cell sortingcell sorting

�� In In fundamental researchfundamental research::

�� In In clinical researchclinical research::

�� cytogeneticscytogenetics

�� prenatal diagnosticsprenatal diagnostics

�� gene disordersgene disorders

�� diagnostics of infectious diagnostics of infectious and malignant diseasesand malignant diseases

�� biological dosimetrybiological dosimetry


38 Thank youThank you……

introduction to cell biology -...

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