Multiphoton Microscopy

In multiphoton microscopy, a fluorophore (or fluorochrome) is excited by multiphoton absorption discussed in Chapter 5, and the resulting up-converted fluorescence (also discussed in Chapter 5) is used to obtain an image. Both two-photon and three-photon absorption-induced up-converted fluorescence have been used for multiphoton microscopy (Denk et al., 1990; Maiti et al., 1997). However, for practical reasons of three-photon absorption requiring extremely high peak power, only two-photon microscopy has emerged as a powerful technique for bioimaging. Two-photon laser scanning microscopy (TPLSM) can use a red- and near-infrared-wavelength short pulse (picoseconds and femtoseconds) laser as the excitation source and produce fluorescence in the visible range. Therefore, two-photon microscopy extends the range of dynamic processes by opening the entire visible spectral range for simultaneous multicolor imaging (Bhawalkar et al., 1997). Two-photon excitation, as discussed in Chapter 5, involves a simultaneous absorption of two laser photons from a pulsed laser source, to achieve fluorescence at the desired wavelength. The transition probability for simultaneous two-photon absorption is proportional to the square of the instantaneous light intensity, hence necessitating the use of intense laser pulses. It is preferable to use ultra-short laser pulses (picosecond or femtosecond pulses from mode-locked lasers), whereby the average power can be kept very low to minimize any thermal damage of the cell or biological specimen. A Ti: Sapphire laser (See Chapter 5), which produces very short (~100 fs) pulses of light around 800 nm (at a rate of ~80-MHz repetition rate), with a very large peak power (50kW), has been a popular choice for two-photon microscopy.

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