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

http://www.colorado.edu/che/ansethgroup/facilities_album/images/Confocal-microscope.gif

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