Second Harmonic Generation enables label free imaging of polarized/oriented structures in tissues, e.g. collagen, through excitation with a pulsed IR laser.
image/credits: Mouse, breast tumour section, Nuclei (Cyan), Collagen (Orange). Sample: Raul Peña/Antonio García de Herreros (IMIM).
Multiphoton Excitation is a non-linear process that excites fluorescence only in the focal volume of the laser beam, hence phototoxicity can be better controlled than with laser-scanning 1-photon imaging (i.e. confocal) because the sample is not excited above and below the imaging plane. The collection efficiency is also higher because no pinhole is required, providing higher optical transmission and more sensitivity.
Image/credits: Zebrafish epiboly (EGFP signal) imaged at 300-350um depth, at 3 different time points, by Maria Marsal (Lab of E. Martin Blanco, IBMB-CSIC, 2015)