Two photon and multiphoton laser scanning microscopy

If a fluorophore absorbs the light of energy, where λ is the wavelength of the absorbed radiation; it is possible to excite the fluorophore with the light of wavelength 2λ if two photons are simultaneously absorbed by the molecule (Figure 16.5).

 

Figure 16.5 A simplified Jablonski diagram showing single-photon and two-photon excitation of a fluorophore.

The probability of simultaneous absorption of two photons is very small; multiphoton microscopes therefore need very intense light sources. Pulsed infrared lasers, however, have realized the multiphoton microscopy. Titanium:sapphire lasers operating at 800 nm can cause excitation of the fluorophores with λ_max ~ 400 nm through two photon absorption. Multiphoton fluorescence microscopy offers following advantages over single photon microscopy:

1. Biological specimens absorb the near-IR radiation very poorly as compared to the UV and blue green radiation, the electromagenetic region commonly used for fluorescence microscopy; this implies that a thicker specimen can be studied using multiphoton microscopy.
   
   
2. As the fluorophores are excited at ~2λ in a two photon fluorescence imaging experiment, the incident and the emitted radiations are well separated; this separation allows detection of the emitted radiation clear of the excitation radiation and the Raman scattering.
   
   
3. The probability of simultaneous absorption of two photons depends on the square of the light intensity. The laser light in a two-photon set up does not excite the fluorophores along its path due to insufficient photon density to cause two-photon absorption. A photon density high enough to cause excitation is achieved only at the focus, thereby exciting the molecules only in the focal plane (Figure 16.6). A multiphoton microscope therefore does not require a pinhole for recording confocal images.