Two-photon Fluorescence Microscopy for Biological Imaging

The 2PA effect has been widely involved in numerous appheations as reviewed by Prasad et al. [25], such as frequency-up conversion lasing [26], optical power limiting [27,28], microfabrication [29,30], optical data storage and processing [31 ] and biological or medical applications [32]. As this chapter, which addresses 2PA of lanthanide complexes, will concern mainly biological applications, we will focus in this part on two-photon excited fluorescence microcopy in biology. 

The use of a Ti sapphire laser, which produces a light in the range between 700 and 1200 nm, in the so-caUed transparency window of biological tissues [33], leads to decreased linear absorption (Fig. 5.2a) and scattering along the beam [34] (Fig. 5.2b). This allows a deeper penetration of the beam within tissues up to 600-800 mm in the neocortex for instance [35], and is illustrated by in vivo two-photon microscopy blood vessels in the mouse brain [36] (Fig. 5.3a). 

A reduced photobleaching is obtained, sinee only the region at the focal point is excited, as 2PA varies quadratieally with the ineident laser intensity [38] (Equation 5.1). 

The 2PA nonlinear absorption process can be deseribed from the semi-classical theory, which leads to express the nonlinear electric polarisation P of an optical medium and the electronic dipole moment p induced at molecular scale following Equations 5.6 and 5.7, respectively, as a function of odd-order susceptibilities and hyperpolarisabilities, respectively [39]  

In these equations, eq and a are respectively the vacuum permittivity and the linear absorption coefficient. E a ) represents the apphed electric field at the frequeney co [ ( w) is the complex conjugate of ( )], whilea , —m) and/( , , — ), which are fourth-order tensors, are respectively the third-order susceptibUity and hyperpolarisabUity. The real part of these tensors represents the induced refractive index change and is a function of the laser intensity (m) oc ( u) ( ), while the imaginary part describes the 2PA process [40]. 

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