General Information
Conventional one-photon confocal laser scanning microscopy (CLSM) often provides high resolution but is limited in sensitivity and spatial resolution by background flare noise resulting from "out-of-focus" fluorescence. In CLSM, the repeated scanning of UV light greatly reduces cell or tissue viability. The two- or three-photon excitation laser scanning microscope (nonlinear microscope), however, circumvents this limitation by using two or three red-wavelength photons to obtain both sensitivity and depth resolution without a confocal aperture. The MEFIM technique considerably reduces autofluorescence and photodamage above and below the focal plane, and the volume of the focal plane depends on a diffraction spot created by the objective lens.
Two-photon absorption was theoretically predicted by Goppert-Mayer in 1931, and it was experimentally observed for the first time in 1961 by using a ruby laser as the light source (Kaiser and Garrett, 1961). The original idea of two-photon fluorescence scanning microscopy was proposed by Sheppard et al. (1977) and was experimentally demonstrated for biological imaging by Winfried Denk and Watt Web (1990).
Physics of Two-Photon Excitation
The probablility of two-photon absorption depends on the co-localization of two photons within the absorption cross section of the fluorophore, and the rate of excitation is proportional to the square of the instantaneous intensity. Two-photon excitation is made possible by the very high local instantaneous intensity that is provided by a combination of diffraction-limited focusing of a single laser beam in the specimen plane and the temporal concentration of a femtosecond (fsec) mode-locked laser. The two-photon advantage is roughly proportional to the inverse excitation duty cycle, for example, a 100,000-fold improvement over CW illumination is achieved by using 100-fsec pulses at 76 MHz repetition rate. The use of such short pulses and small duty cycles is, in fact, essential for image acquisition in a reasonable time while using "biologically tolerable" power levels.
Advantages of MEFIM
(i) In one-photon excitation CLSM, photobleaching occurs well above and below the focal (volume) plane; in MEFIM, the photobleaching is considerably reduced, and illumination of laser light occurs only at the focal plane.
(ii) Repeated scanning on the specimen in CLSM, particularly with UV light, induces rapid photoisomerization and high background autofluorescence. MEFIM reduces these complications, providing better penentration at infrared wavelengths and thus prolonging cell viability during image acquisition.
(iii) The CLSM technique requires special UV optics for the UV excitation probe. MEFIM uses conventional microscope optics.
(iv) In CLSM, the emission wavelength is close to the excitation wavelength (about 50-200nm). In MEFIM, the fluorescence emission occurs at a wavelength substantially shorter than the excitation wavelength.