6 Principles and applications of temporal-focusing wide-field two-photon microscopy

Temporal focusing allows for rapid optically sectioned two-photon widefield microscopy. Depth sectioning is provided in a wide-field manner, without spatial focusing, by controlling the temporalwidth of femtosecond laser pulses near the focal plane. This spatial control of the temporal pulse width is achieved by diffracting the light off a grating resulting in spectral component separation and temporal broadening; these spectral components are only recombined at the focal plane to reproduce short, femtosecond pulses. Applications include (i) high speed functional imaging in the brain, (ii) fast FLIM and PLIM, (iii) cell-selective optogenetic excitation, and (iv) temporal focusing photodynamic therapy that may allow selective killing of cancer cells