Two-photon fluorescence excitation

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Mertz J, Xu C and Webb W W 1995 Single-molecule detection by two-photon-excited fluorescence Opt Lett. 20 2532-4... 

Nonlinear optical phenomena, as well as near-field optics, provide us with super resolving capability [20]. The probability of nonlinear optical phenomena is proportional to the number of photons which participate in the phenomenon. For example, the intensity distribution of two-photon excited fluorescence corresponds to the square of the excitation light. Thus, we proposed a combination of the field... 

Swift, J. L., Heuff, R. F. and Cramb, D. T. (2006) A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots. Biophys. J., 90, 1396-1410. 

So PT, Dong CY, Masters BR, Berland KM (2000) Two-photon excitation fluorescence microscopy. Annu Rev Biomed Eng 2 399-429... 

LaMorte, Y. J., Zoumi, A. and Tromberg, B. J. (2003). Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level. J. Biomed. Opt. 8, 357-61. 

Volkmer, A., Subramaniam, V., Birch, D. J. and Jovin, T. M. (2000). One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 78, 1589-98. 

Tada, J., Kono, T., Suda, A., Mizuno, H., Miyawaki, A., Midorikawa, K. and Kannari, F. (2007). Adaptively controlled supercontinuum pulse from a micro structure fiber for two-photon excited fluorescence microscopy. Appl. Opt. 46, 3023-30. 

When a single laser is used, the two photons are of identical wavelength, and the technique is called two-photon excitation fluorescence microscopy. When the photons are of different wavelengths X and Xi (so that 1 jX + jXi = 1 j Xv), the technique is called two-color excitation fluorescence microscopy. 

Two-photon excitation fluorescence is currently the most widely nsed nonlinear contrast mechanism for microscopic investigations. The first experimental demonstration of two-photon excitation fluorescence was provided in 1961 (Kaiser and Garrett 1961), even though the first theoretical description of two-photon excitation flnorescence stems back to 1931 (Goppert-Mayer 1931). Three-photon absorption was demonstrated a few years later by Singh and Bradley (1964). Two-photon absorption is a third-order nonlinear effect, whereas three-photon absorption is a fifth-order nonlinear effect. The transition rate for two-photon absorption, R, depends on the square of the intensity, /, as follows (see Boyd 1992) ... 

BariUe, R., Canioni, L., Rivet, S., Sarger, L., Vacher, R, and Ducret, T. 2001. Visualization of intracellular Ca + dynamics with simultaneous two-photon-excited fluorescence and third-harmonic generation microscopes. Appl. Phys. Lett. 79 4045M7. 

Peti-Peterdi, J., Morishima, S., BeU, P. D., and Okada, Y. 2002. Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus. Am. J. Physiol. Renal Physiol. 283(1) F197-F201. 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

Figure 4.8 A schematic representation of NCE and two photon excited fluorescence detection. Excitation light dashed line. Emission light solid line. The dotted line displays light for visualization [59].

Zugel, S.A., Burke, B.J., Regnier, F.E., Lytle, F.E., Electrophoretically mediated microanalysis of leucine aminopeptidase using two-photon excited fluorescence detection on a microchip. Anal. Chem. 2000, 72, 5731-5735. 

Our own interest in 2PA was kindled by the observation by Perry s group of strong blue fluorescence when a toluene solution of ( )-4,4 -bis(di-n-butyl)aminostilbene (1) (Figs. 11.2 and 11.13), was irradiated with 605 nm 5-ns laser pulses, a wavelength at which this species (Amax = 385 nm) shows no linear absorption. Moreover, the fluorescence intensity was proportional to the square of the laser light intensity. This two-photon-excited fluorescence spectrum was essentially identical... 

Fig. 5. Schematic drawing of the microsheath flow apparatus combined with two-photon excitation fluorescence microscopy. DM dichroic mirror BF barrier filter M mirror changing light path and L lens for bearing images.

The two compounds 121 and 122 were studied for applications in two-photon excitation fluorescence microscopy (TPEM). The corresponding Zn complexes showed an enhanced efficiency and a significant increase of the two photon absorption (TPA) cross section <07JA11888>. 

Several articles and reviews on different aspects of multi-photon excitation of biomolecule system are available. For example, Birch [11] consideraticms concentrate mainly on the impact of multi-photon techniques to the time-resolved fluorescence spectroscopy. Lakowicz and Gryczynski [12] have discussed examples of three-photon excited fluorescence. Rehms and Callis studied the two-photon excited fluorescence emission of aromatic amino acids [13]. Kierdasz et al analyzed emission spectra of Tyrosine- and Tryptophan-containing proteins using one-photon (270-3 10 nm) and two-photon (565-6 10 nm) excitation [14]. 

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