Massively increasing the photon flux with shorter excitation pulses (15 fs)
Two-Photon Excitation Fluorescence (TPEF) microscopy (also known as two-photon microscopy) is the method of choice for deep three-dimensional imaging of living tissues. Deep imaging is intrinsic to TPEF microscopy since it uses longer excitation wavelengths (near-IR) that scatter less than the shorter visible wavelengths traditionally used in confocal microscopy. This reduces background illumination coming from the scattered light and increases the contrast ratio at higher depths. As an example, in-vivo brain images at depths of 1mm can be achieved with TPEF microscopy.