Multiphoton excitation of fluorescence

Not illustrated is the use of multiphoton excitation of fluorescence (12.15). thus far demonstrated in flame systems only for excitation of atoms. It affords the means to excite otherwise inaccessible states and offers other potential advantages in spatial resolution and for optically thick flames, in spite of inherently low signal levels. 

Multiphoton Excitation of Fluorescence near Metallic Particles Enhanced and Localized Excitation. J. Phys. Chem. B 106 2191-2195. 

Gryczynski I, Malicka J, Shen Y, Gryczynski Z, Lakowicz JR (2002) Multiphoton excitation of fluorescence near metallic particles enhanced and locahzed excitation. J Phys Chem B 106 2191-2195... 

To first order one would expect multiphoton excitation of fluorescence markers to decrease the resolution, since the excitation wavelength increases (Sheppard 1996). 

K.H. Drexhage Multiphoton excitation of fluorescence in standing light waves and measurement of picosecond pulses. Appl. Phys. Lett. 

A more sophisticated mode of LIE detection is the multiphoton-excitation (MPE) fluorescence [47], which is based on the simultaneous absorption of more than one photon in the same quantum event and uses special lasers, such as femtosecond mode-locked laser [48] or continuous wave laser [49], This mode of LIE detection allows mass detection limits at zeptomole level (1 zepto-mole=10 mol) due to exceptionally low detection background and extremely small detection volume, whereas detection sensitivity in concentration is comparable to that of traditional LIE detection modes. A further drawback is the poor suitability of MPE-fluorescence detection to the on-column detection configuration, which is frequently employed in conventional LIE detection. 

Xu, C., and Webb, W. W. (1997) Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy, in Topics in Fluorescence Spectroscopy. J. R. Lakowicz (Ed.), Plenum Press, New York, pp. 475-540. 

C. Xu and W. W. Webb, Multiphoton Excitation of Molecular Fluorophores and Nonlinear Laser Microscopy. In Topics in Fluorescence Spectroscopy, Vol. V, J. R. Lako-wicz, Ed., 1997, p. 471. 

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],... 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

Masters, B. R., So, P. T. C., and Gratton, E. 1997. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophysical Journal 72 2405-12. 

Greenhalgh, C., Cisek, R., Prent, N., Major, A., Aus der Au, J., Squier, J., and Barzda, V. 2005. Time and structural image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy. Proc. SPIE 5969 59692F1-F8. 

We demonstrate the Mil method, which couples the sensitivity of multiphoton excitation on the spectral phase of the laser pulses to probe microscopic chemical environment-induced changes in the multiphoton excitation spectrum of sensitive reporter molecules. We carry out the optimization of the required phase functions in solution and provide theoretical simulations. We show experimental images whereby pH-selective two-photon microscopy is achieved and demonstrate how selective excitation can be used to enhance contrast and, consequently, to achieve functional imaging, using fluorescent probes sensitive to changes in their local environment. 

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