exploring synaptic circuits with antibodies and confocal microscopy partii: principles and...
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Exploring synaptic circuits with antibodies and confocal microscopy PartII: Principles and applications of confocal microscopy
Marco SassoèDip. Anatomia, Farmacologia e Medicina LegaleIstituto Nazionale di NeuroscienzeTorino, Italy
Doctoral School of Neuroscience
Turin University
June 2009
3. In a confocal microscope the vertical resolution is:
a. The same as the lateral resolution
b. Better than the lateral resolution
c. Worse than the lateral resolution
2. Compared to a conventional epifluorescence microscope
a confocal microscope affords:
a. A much better resolution
b. A marginally better resolution
c. The same resolution
5. In a confocal microscope the specimen is:
a. Illuminated point-by-point
b. Illuminated line-by-line
c. Completely illuminated
1. The major advantage of a confocal microscope is:
a. The use of laser illumination
b. The elimination of out-of-focus fluorescent light
c. The high resolution
d. The presence of a Z-motor 4. The Airy Unit defines:
a. The size of the pinhole
b. The size of the laser beam
c. The intensity of the laser beam
Development of GABAergic synapses: from synaptic adhesion to activity-dependent synaptogenesis
Synaptic integration of adult-generated neurons
Synapse organization in a mouse model of Rett syndrome
Synaptic plasticity induced by morphine in the mesolimbic pathway
Laboratory of Synapse Development and Plasticity
Federica Briatore
Laura Viltono
Elena Frola
Alessandro Ciccarelli
Marta Pallotto – Patrizia Panzanelli
Elena Boggio
Annarita Patrizi
Giulio Srubek Tomassy
Maurizio Giustetto
Synapses: 1014 (100-500 trillion) 1 mm3 of cerebral cortex: one billion synapses!
Martin Chalfie Osamu Shimomura Roger Y. Tsien
The Nobel Prize in Chemistry 2008
L’eliminazione genetica di MeCP2 induce alterazioni sinaptiche
neurone-specifiche
VI
I strato
IV
V
II-III
100 µm
WT
GFP / NeuN
KO
WT
Strato
1
KO
WT
Strato
5
Elena BoggioMaurizio Giustetto
Marta Pallotto
WT KO
Ruolo dell’inibizione GABAergica nell’integrazione sinaptica dei
neuroni neogenerati
Resolution
R = 0,5 / NA
GABA GABAAR1
PSD95
Fritschy et al., 2006
Panzanelli et al., 2004
Sassoè-Pognetto et al., 2003
Microscopio a epifluorescenza
Fluorescenza
excitation
emission
fluorochromes
fluorophores
autofluorescence
Indirect (secondary) fluorescence
The pinhole is conjugate to the focal point of the lens
Sistema confocale•Sorgente luce puntiforme
•Illuminazione puntiforme del campione (con obiettivo ad elevata AN)
•Pinhole su piano di rilevamento
Vantaggi:
-Eliminazione piani fuori fuoco – immagini più nitide
-Eliminazione di interferenza laterale – aumento contrasto
-Sezioni ottiche (e ricostruzioni 3D)
-Miglior risoluzione (teorico)
TUBE LENS
confocal