Applications in Confocal Microscopy
Multiphoton Excitation Laser Scanning Microscopy
In multiphoton excitation microscopy, fluorescent dyes are excited by absorbing the energy of two photons simultaneously (see Figure 1). The dyes are excited at twice the wavelength used in ordinary fluorescence observations, since the photon energy is inversely proportional to the wavelength. Because the excitation probability of the fluorescent dye in multiphoton excitation is proportional to the square of excitation light intensity, only the area proximal to the focal point, where the photon density is high, can be excited; tomographic images can then be obtained by scanning the laser beam (Figure 2, in which the non-excited area is grayed). The multiphoton excitation laser scanning microscopy provides similar tomographic images to the confocal LSM through the different principles.
Using probes such as calcium ion indicators, some of which are excited by ultraviolet light, considerations for the aberration and transmittance of ultraviolet light through the microscope optics can be eliminated in multiphoton excitation microscopy. Specimen damage from the ultraviolet light can also be reduced. Multiphoton excitation is expected strongly to photo-activate caged compounds only at a three-dimensionally specific point in specimens.
A confocal laser scanning microscope must be modified and equipped with a specialized non-linear pulsed laser system in order to perform multiphoton imaging.
The following characteristics should be used as a guide when comparing multiphoton excitation with conventional laser scanning confocal microscopy.
Applications of Multiphoton Microscopy
Applications based on the advantages of using near-infrared light:
Applications based on the advantages of local light absorption: