2-photon imaging and photostimulation

Studying neuronal activity needs to keep the most integrated sample to conserve neuronal connections and physiological conditions. To respect these conditions, thick tissue slices or alive animals have to be used and specific technique allowing to observe fluorescence in the depth has to be used: the 2-photon microscopy.

This technique allows to observe standard fluorochromes in depth by exciting them with infra-red light thanks to a non linear optical effect called 2-photon absorption. This effect happens only by using a special femtosecond pulsed laser and specific detectors placed close to the sample.

Figure 1: A) Scheme showing the penetration of the light into tissues depending on the wavelength ; B) Scheme with the Jablonski diagram of 1P and 2P excitation ; C) Optical scheme of the Femtonics system with the 2 femtosecond lasers, the 2 scanheads and the different detectors.

Two 2-photon systems are available on the BIC: The SP5-MP and the Femtonics microscopes.

The first one, based on a confocal head, allows 2-photon imaging on fixed or live samples, coupled with galvo or resonant scanners.

The second one is equipped with 2 scanheads (galvo and resonant) usable simultaneously to couple imaging and photostimulation in 2-photon.

Contact: Sébastien Marais

Figure 2: A) Maximum projection of neurons expressing GCaMP6 in an organotypic brain slice of 350 µm ; B) Movie of the calcium activity in a dendrite reported by the fluorescent protein GCaMP6f