2011.11.25.. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 1 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.
Neurobiológia alapjai - Módszerek
BASICS OF NEUROBIOLOGY - Methods
By Imre Kalló
2011.11.25. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 3
METHODS IN NEUROBIOLOGY III.
Histology techniques: electron 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
B
PRINCIPLE OF TRANSMISSION ELECTRON MICROSCOPY
Illumination source
Condensor lens
Objective lens
Projective lens
Ocular (eyepiece)
Specimen
Fluorescent screen
LM TEM
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 5 2011.11.25.
TRANSMISSION ELECTRON MICROSCOPY (TEM)
CHARACTERISTICS OF THE ELECTRON MICROGRAPHS
Electron density
In the electron microscope, electrons are projected onto ultrathin sections of the sample. Electrons, which permeate the sample, generate a lighter area on the screen underneath by exciting its
fluorescent coating. Many of the electrons are, however scattered by the dense
regions of the sample never reaching the screen and resulting in dark areas on it.
Consequently, a 2D image of the sample is appearing with increasingly darker areas on the screen (and the electron micrograph), where the sample is
correspondingly more „electron dense”.
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 7 2011.11.25.
Standard work
I. Chemical fixation at RT (glutaraldehyde, OsO
4) II. Dehydration
III. Embedding in resins - at 60 °C
IV. Ultracutting
V. Contrasting (uranyl and lead compounds)
PREPARATION OF THE SAMPLES FOR TEM
High resolution work
I. Cryo-fixation (rapid freezing by high pressure – up to 0.6 mm) followed by either
- observation on a cryo-stage or - cryo-ultratomy or
- freeze-fracture (FF) or - freeze-substitution (FS)
II. FS – dissolution of ice by an organic solvent + fixative
III. Embedding - at 60 C
- at LowT
USING HIGHLY ELECTRON DENSE MARKERS
I. Preembedding labelling II. Postembedding labelling
Silver-gold intesified DAB
Autoradiography
Colloidal gold Diaminobenzidine
(DAB)
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 9 2011.11.25.
PRINCIPLE OF SCANNING ELECTRON MICROSCOPY (SEM)
Illumination source
Condensor lens 1
Objective lens
Projective lens
Ocular (eyepiece) Specimen
LM SEM
Specimen
Condensor lens 2
Condensor lens 3
Signal detector
SCANNING ELECTRON MICROSCOPY (SEM)
The SEM scans the surface of
the sample with a 2-3 nm
diameter high-energy electron beam, which interacts with the surface atoms of the sample and generates secundary electrons, back-scattered electrons, x-rays, light, reflected electrons,
specimen current and transmitted electrons. Image is created most commonly by detecting
secundary electrons.
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 11 2011.11.25.
CHARACTERISTICS OF SEM MICROGRAPHS
The scanning electron microscope generates
high resolution images (details in a few
nm range) with characteristicly large depth
of field, which supplies the images with
3D appearance.
PREPARATION OF THE SAMPLES FOR SEM
I/a. Cryo-fixation – low-temperature SEM I/b. Chemical fixation
II. Dehydration – Critical point drying (solvents, transitional fluid – carbon dioxid)
III. Conducting samples
IV. Non-conducting samples – Sputter coating
(gold, gold/palladium)
TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 13 2011.11.25.
FREEZE FRACTURE – STUDYING MEMBRANES, MEMBRANE PARTICLES IN REPLICAS
I. Cryo-fixation II. Freeze-fracture
Fracture is splitting the membranes
III. Coating (C-Pt-C) P surface
E surface
IV. Etching
(SDS) V. Immunolabelling
FREEZE-FRACTURE
REPLICA IMMUNOGOLD LABELLING
Nav1.6 –immunoreactivity in CA1 pyramidal cells
E surface
P surface CA3
CA2 CA1
DG
By courtesy of Zoltán Nusser, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine,
