Two-photon laser scanning microscopy is particularly well adapted for in-vivo observations of weakly fluorescent scattering samples (ex: fly embryos, neuronal tissues...).
It offers several advantages compared to confocal microscopy:
- Less photo-damage; Photo-damage is restricted to the focal plane. As a result, longer time-lapse imaging can be done.
- Higher fluorescence collection efficiency: With two-photon, it is not necessary to refocus the fluorescent signal through a confocal aperture, therefore even scattered photons contribute to the usable signal.
- Deeper penetration in thick and scattering tissues: The scattering of light by thick and cloudy specimens does not interfere with image formation, allowing much deeper penetration into tissues.
The facility offers several two-photon laser scanning microscopes.
One two photon is built in an upright microscope configuration with large amounts of clearance. A 40x 1.0NA lens and a 25x 1.05 lens are available.
The other two photon microscope is built in an inverted microscope configuration with a single 25x 0.8 multi-immersion lens.
Both two photons have a Coherent Chameleon Ultra II (tunable 680-1080nm) and Spectra Physics High Q laser (fixed wavelength, ~1043nm) for two-color two-photon imaging. Filter sets on the detection size are optimized for GFP/mCherry, RFP.
Both two photons have motorized stages (xy and z) and fast piezo objective positioners that are closed loop with 400 microns of travel for fast and precise acquisition of z stacks.