Light source requirements for confocal and multiphoton microscopy
Lasers for confocal micrsocopy
Lasers for multiphoton microscopy (MP)
Multiphoton microscopy uses the nonlinear process of exciting a molecule by two quanta half the energy required to step the electronic system from ground state to the excited state. As the probability of this 2-particle process depends on the photon concentration in a square mode, the excitation inherently occurs only in a layer around the focus which inherently leads to optical sectioning without pinholes. The required high photon density is created by pulsing the laser energy. Mid-range fluorochromes are excited in the near IR, which also improves the penetration into thick biological samples.
As the 2-photon effect requires a very high photon density, the method became applicable only when short pulsed lasers at sufficient output power were available. The laser of choice is a titanium-sapphire laser with pulse lengths of about a hundred femtoseconds. These lasers emit up to 10 W light and are tunable in the range of 650 … 1,100 nm.
A modern type of laser is based on optical fibers. These fiber lasers have a very good beam profile, can reach a high output power and are comparably compact and maintenance-free. In the future, fiber lasers will more and more invade the area of confocal and multiphoton microscopy. One example is the all-fiber white light laser, already used as a tunable source for confocal illumination.