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
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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
2
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)
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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History of cytology and histology
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
Émile DUCLAUX METCHNIKOFF Émile ROUX
Albert CALMETTE Jules BORDET
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Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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Light microscopy
NB: Ernst Abbe developed a mathematical description for the resolution limit of the microscope
1010
Resolving power
Prof. Dr. Nikolai Lazarov
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
Electron microscopy
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1919
Cryo-electron microscopy
Prof. Dr. Nikolai Lazarov
2020
Atomic force microscope
Prof. Dr. Nikolai Lazarov
The first atomic force microscope was invented by IBM scientists in 1982
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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)
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
Prof. Dr. Nikolai Lazarov
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Freeze-Etching and Freeze-Fracture (Cryofacture)
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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
Prof. Dr. Nikolai Lazarov
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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"
Prof. Dr. Nikolai Lazarov
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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)
Prof. Dr. Nikolai Lazarov
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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