Multiphoton Fluorescence Imaging

As has been discussed in the previous chapter, the trade-off between image resolution and signal level plays a crucial role in determining the efficiency of optical gating methods under single-photon (1p) fluorescence imaging. Another important development in fluorescence imaging is the two-photon (2p) scanning fluorescence microscopy because of its advantages over 1p scanning fluorescence optical microscopy (Denk et al. Science 248:73–76, 1990; Schilders and Gu Appl Opt 38:720–722, 1999; Guo et al. Appl Opt 36:968–970, 1997). First, 2p excitation is a nonlinear process and therefore the fluorescence intensity is directly proportional to the square of the excitation intensity. Due to this quadratic dependence, the 2p imaging technique provides a pinpoint excitation/detection method at a deep position within thick samples. Second, if an infrared laser beam is employed for 2p excitation, 2p fluorescence microscopy offers access to ultraviolet (UV) excitation without using UV lasers, and reduces Rayleigh scattering appreciably, provided that the size of scatterers in tissue is smaller than the illumination wavelength. According to these properties of 2p excitation, it has been claimed that 2p excitation results in a deeper penetration depth than 1p excitation (Denk et al. Science 248:73–76, 1990; Guo et al. Appl Opt 36:968–970, 1997). The trade-off between image resolution and signal level for 2p fluorescence imaging is discussed in Sect. 8.1, which determines the penetration depth. The effects of various parameters on 2p fluorescence imaging are discussed in Sect. 8.2. Section 8.3 discusses the Monte Carlo simulation of 2p fluorescence imaging through various complex modeling structures. Section 8.4 presents the comparison of 1p and 2p imaging with three-photon (3p) imaging through skin and human cortex media.