The University of Toledo

Multi-Photon Microscopy

The Advanced Microscopy & Imaging core facility houses a TCS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems) equipped with both conventional and high-speed resonance scanners. The Multi-photon instrument is attached to an inverted microscope and allows deeper penetration into tissue with less photo-bleaching/damage. Eight laser plus multi-photon excitations are available, producing the following laser excitation lines: 458, 476, 488, 496, 514, 561, 594, 633, and 710-990nm (from the tunable Ti-Sapphire MP laser). The system is capable of collecting up to 5 colors simultaneously for quantitative confocal image analysis, 3D reconstruction, FRAP and FRET, animation, stereo imaging, single layer projection, time lapse collection, and co-localization analysis.

Multi-photon Microscopy using pulsed Infra-Red Laser:

Two photons are better than one! A direct comparison of confocal (left) and multi-photon excitation (right)

 

Advantages No attenuation of exciting light by out of focus fluorescence. Longer wavelengths, less scattering and photodamage, deeper penetration No scatter of fluorescence light away from focal plane (signal not degraded)