Two-Photon Excitation Fluorescence Microscopy

Two-photon excitation (TPE) fluorescence microscopy (Denk et al., 1990; Pennisi, 1997; Esposito et al., 2004) can be considered an important example of the continuing growth of interest in optical microscopy (Diaspro, 1996; Koster and Klumperman, 2003). In spite of its low spatial resolution compared to other modern imaging techniques, such as scanning near-field microscopy (Dürig and Pohl, 1986), scanning probe microscopy (Binnig et al., 1986), or electron microscopy (Ruska and Knoll, 1931), light microscopy techniques, including TPE microscopy, have unique capabilities in the investigation of biological structures in a hydrated state, in living specimens, or at least under conditions that are close to physiological states (Pawley, 1995; Periasamy, 2001; Diaspro, 2002). This fact, coupled with advances in fluorescence labeling, permits the study of the complex and delicate relationships existing between structure and function in the four-dimension (x–y– z–t) biological systems domain (Arndt-Jovin et al., 1985; Beltrame et al., 1985; Wang and Herman, 1996; Herman and Tanke, 1998). As well, the advances achieved in the field of biological markers, especially the design of application-suited chromophores, the development of the so-called quantum dots (Jaiswal et al., 2004), visible fluorescent proteins (VFPs) from the green fluorescent protein (GFP) and its natural homologues to specifically engineered variants of these molecules (Patterson and Lippincott-Schwarz, 2002; Wiedenmann et al., 2004), and the improvements in resolution by means of special optical schemes (Egner et al., 2004; Gugel et al., 2004), are enabling TPE to move from micro scopy to nanoscopy (Hell, 2003; Bastiaens and Hell, 2004).