Red Española de Microscopía Óptica Avanzada
Non-Linear Imaging/ Multiphoton fluorescence Imaging

Multiphoton microscopy uses pulsed long-wavelength light to excite fluorophores within the specimen being observed. The fluorophore absorbs the energy from two long-wavelength photons which must arrive simultaneously in order to excite an electron into a higher energy state, from which it can decay, emitting a fluorescence signal. It differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength, as the summed energies of two long-wavelength exciting photons will produce an emission wavelength shorter than the excitation wavelength. Multiphoton fluorescence microscopy has similarities to confocal LSM. Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures, which give confocal microscopes their optical sectioning quality. 

The longer wavelength, low energy (typically infra-red) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than short-wavelength lasers, so cells may be observed for longer periods with fewer toxic effects.

This technique at work
Universitat de Barcelona, CCiTUB- Centres Cientifics i Tecnologics - Advanced Optical Microscopy Unit
ICFO-Instituto de Ciencias Fotonicas, ICFO-Instituto de Ciencias Fotonicas - Servicio de Microscopia de Super-Resolucion y Nanoscopia- ICFO
Consejo Superior de Investigaciones Cientificas (CSIC), Centro de Biología Molecular Severo Ochoa - Servicio de Microscopia Optica y Confocal
Instituto de Salud Carlos III, Centro Nacional de Investigaciones Cardiovasculares (CNIC) - Microscopy and Dynamic Imaging Unit
Centre for Genomic Regulation, Parc de Recerca Biomèdica Barcelona - Advanced Light Microscopy Unit
Universitat de València, SCSIE - Servei Central de Suport a la Investigació Experimental - SCSIE-Secció de Microscòpia
Universidad de Extremadura, Servicios de Apoyo a la Investigación - Servicio de Tecnicas Aplicadas a las Biociencias
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