The two-photon effect is a process in which a dye molecule is excited by simultaneously absorbing two photons. In the microscope available in the Nano-Bio-Imaging Core Facilitity, the intensity of light at the focal point of the infrared laser pulse, focused on the sample is high enough that our absorbing fluorescent dye can be excited by the simultaneous absorption of two infrared photons. Due to the 2-photon effect, the dye is excited only in a small volume around the focal point (approximately 1 µm3). The photon density is not high enough for the two-photon excitation outside the focal point, leaving the sample intact there. Consequently, this method minimize the photo-degradation of sample which often limits in vivo studies with laser beams. It is also excellent for Ca2 + imaging and high-volume fluorescent morphometric studies in both in vivo and in vitro samples, as well as for photo-elimination and photo-activation methods using two photon lasers.
Advantages: it can be measured at depths of up to 800-1200 µm; focal excitation; a wide detection angle and a more freely adjustable lens under which any kit can be easily mounted; the excitation laser wavelength is tunable; kHz sampling is available and up to three-dimensional images can be captured using a scanning procedure.
- Microscope: Femto2D-SMART 2-photon microscope
- Laser: Mai Tai HP (690-1040 nm)