Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**
Consortium leader
PETER PAZMANY CATHOLIC UNIVERSITY
Consortium members
SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER
The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***
**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben
***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg.
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Neurobiológia alapjai - Módszerek
BASICS OF NEUROBIOLOGY - Methods
By Imre Kalló
Methods in Neurobiology I.
Histology techniques: light microscopic studies
Imre Kalló
Pázmány Péter Catholic University, Faculty of Information Technology
I. Histology techniques: light microscopic studies II. Applications using fluorescent dyes
III. Histology techniques: electron microscopic studies IV. Techniques to map neuronal connections
V. Molecular biological techniques VI. Living experimental models VII. Electrophysiological approaches VIII. Behavioral studies
IX. Dissection, virtual dissection, imaging techniques
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Techniques based on invivo or post mortem sampling - HISTOLOGY
I. PRESERVATION OF THE SAMPLE 1. Aim of this procedure
a. Advantages b. Disadvantages 2. Preservation techniques
a. Heat, microwaves
b. Chemical preservation (e.g. 4% PFA, 0.1-10%
GA, Acrolein etc.)
- Perfusion of the fixation solution - Immersion of sample in the fixation
solution
II. CUTTING SECTIONS 1. Aim of this procedure
2. Prerequisites of precise cutting Embedding
Sharp knives 3. Cutting tools
Scalpel, razor blade – free hand cutting 200-500 μm
Microtome 5- 20 μm
Freezing microtome 10- 50 μm
Vibratome, vibroslicer 20-300 μm
Cryostat 10-300 μm
Ultratome 0.5-2 μm 40- 80 nm
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III. STAINING TECHNIQUES (in order to localise and identify tissue components, cells, cellular organelles or molecules)
Histochemistry:
Dyes – binding to molecules according to their physico-chemical properties Proteins
Lipids
Carbohydrites Nucleic acides
Enzim histochemistry - demonstration of endogenous enzymes Acidic and alkaline phosphatases
Dehidrogenases etc.
Autoradiography
Binding or incorporation of radioactively labelled ligands, molecules
Immunohistochemistry – detection of molecules with antigenic properties direct labelling
indirect labelling enzyme - labels fluorescent - labels
intensification techniques
In situ hybridization histochemistry - detection of DNA, heteronuclear and messenger RNA
riboprobes oligoprobes
radioactively labelled probes
non-radioactively labelled probes
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IV. OBSERVATION POSSIBILITIES
Transmission Electron Microscopy
Light Microscopy
Unaided Human
Eye Scanning
Tunneling Microscopy
0.2 nm 0.2 μm 0.2 mm
Atoms
Macro molecules
Viruses
Bacteria
Cells
1 m
100 mm
10 mm
1 mm
100 μm
10 μm
1 μm
100 nm
10 nm
1 nm
0.1 nm
Human ovum
LIGHT MICROSCOPE
XVII. century
XXI. century
Current microscopes use multiple, variously filtered and targeted illuminations, corrected lens systems, automated, motorized units, digital recording
techniques, computerized image processing and analyzing support.
Illumination
Condensor lens
Objective lens
Projective lens
Ocular (eyepiece) Specimen
Principle
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NATIVE PREPARATIONS ARE SUFFICIENTLY VISIBLE AT SPECIAL ILLUMINATON AND CONTRAST MODES
Bright field
Dark field
Phase contrast
Differential interference contrast
COLOURED PREPARATIONS AT BRIGHT FIELD CONDITIONS
Toluidin blue Nickel (Ni) enhanced DAB Ni-DAB/DAB
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FLUORESCENCE MICROSCOPY
Arc lamp
Excitation Diaphragm
Excitation Filter
Emission Filter Objective
Dichroic Beam Splitter
Fluorophores in the tissue
CONFOCAL LASER MICROSCOPY
Laser
Excitation Pinhole
Emission Filter Objective
Emission Pinhole
PMT Dichroic Beam
Splitter
Fluorophores in the tissue
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Principle :
Dyes in the tissue absorb two, small energy (infrared ) photons almost concurrently.
Advantages:
- no fading
- no phototoxicity
- increased focal depth
TWO-PHOTON MICROSCOPY
Infrared
Laser
Objective
Scanning Mirror
Green PMT Dichroic Beam
Splitters
Red PMT Green Filter
Red Filter
Fluorophores in the tissue S
ingle-Photon Exc. Two-Photon Exc. Fluorescent Decay
