A modified Olympus FV300 and IX71 allows multiphoton imaging.
Bovine Ligament simulataneously imaged with SHG and MPE.

Multiphoton imaging and spectroscopy laboratory

Multiphoton microscopy (MPM) is a type of laser scanning microscopy that derives its contrast from non- linear optical properties of a sample and is currently attracting a great deal of interest across all fields of biophysics. Non-linear imaging has many advantages over standard fluorescence confocal laser scanning microscopy, with the principle advantages being an increase in depth penetration and stain-free molecular contrast.

The earliest form of multiphoton microscopy, multiphoton fluorescence excitation (MPE), was discovered in 1990. This technique uses two photons of half the excitation wavelength to excite molecular fluorescence. As well as extraneous fluorophores the technique can also be used to excite indigenous fluorophores, such as elastin.

The next non-linear phenomenon to be applied to biological imaging was second harmonic generation (SHG). SHG derives image contrast from the non-linear optical properties of specific molecules possessing the correct symmetry. One such tissue component known to exhibit the correct symmetry is collagen.

The most recent non-linear imaging modality is Coherent Anti-Stokes Raman Scattering (CARS). CARS derives image contrast from molecular vibrations within the sample and is particularly sensitive to lipid-enriched structures such as lipid membranes.