Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10 5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy.Three properties TPEM give this method a tremendous advantage over conventional optical sectioning microscopies for the study of thick samples: 1) The excitation is limited to the focal volume because of the intensity-squared dependence of the two-photon absorption. This inherent localization provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. Confinement of excitation to the focal volume also minimizes photobleaching and photo damage - the ultimate limiting factors in fluorescence microscopy of living cells and tissues. 2) The two-photon technique allows imaging of UV fluorophores with conventional visible light optics in both the scanning and imaging systems, because both the red excitation light (~700 nm) and the blue fluorescence (>400 nm) are within the visible spectrum. 3) Red or infrared light is far less damaging to most living cells and tissues than bluer light because fewer biological molecules absorb at the higher wavelengths. Longer wavelength excitation also reduces scattering of the incident light by the specimen, thus allowing more of the input power to reach the focal plane. This relative transparency of biological specimens to 700 nm light permits deeper sectioning, since both absorbance and scattering are reduced.
Two‐Photon Excitation Microscopy for the Study of Living Cells and Tissues
