Single-photon excited fluorescence

Figure 3.47. Normalized single-photon excitation (SPE) and two-photon excitation (TPE) spectra (left) and single-photon excited fluorescence (SPEF) and two-photon excited fluorescence (TPEF) spectra (right) of 85 in THE In the TPE spectrum, the wavelength data are divided by 2, considering the TPA characteristic. (From Ref. [447] with permission of the American Chemical Society.)...

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... 

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. 

D. J. S. Birch, G. Hungerford, B. Nadolski, R. E. Imhof and A. D. Dutch, Time-correlated single-photon counting fluorescence decay studies at 930nm using spark source excitation, /. Phys. E Sci. Instrum. 21, 857-862(1988). 

Figure 1. Fluorescence intensity versus incident laser light intensity at 320 nm. The straight line was fit to the low-intensity data and is drawn with the assumption that the fluorescence intensity is proportional to the intensity of the incident laser light. The curved solid line is the result predicted by the phenomenological equation for single-photon excitation with y = 4.2 X 10 cm ls. The area of the exciting laser beam was about 0.1 cm. ...

Figure 11.6 Steady-state fluorescence spectra of fluorescein (a standard single- and two-photon fluorophore) obtained using single-photon excitation at 500 nm and two-photon excitation at 800 nm. Although the two-photon transition energy is higher, 3.1 vs 2.48 eV, fast relaxation within the excited state manifold leads to the rapid population of the same low-lying vibrational levels in Si as evidenced by the equivalence of the emission spectra...

Perhaps the most significant contrihution that two-photon excited fluorescence has made in biological research to date is in the area of fluorescent imaging. Prior to the invention of two-photon microscopy in 1990, depth resolution in fluorescence microscopy was achieved using the confocal microscope, the principles of which are illustrated in Figure 11.9. Fluorescence is excited via single-photon excitation... 

Figure 11.11 A direct comparison between the spatial dependence of two- and single-photon absorption from a fluorescent probe in an isotropic solution contained in a cuvette and viewed at 90° to the excitation direction. Single-photon excitation at 488 nm produces fluorescence throughout the sample, whereas the restriction of two-photon fluorescence (excited at 800 nm) to the Rayleigh range around the laser focus is evident. The photographs were provided courtesy of Dr Mireille Blanchard-Desce (CNRS UMR 6510 Rennes)...

Figure 11.17 Single and two photon excited fluorescence anisotropy decays recorded for rhodamine 6G in ethylene glycol, the initial anisotropies for both processes are close to the theoretical maxima (dashed lines) for excitation from an isotropic ground state and (in the case of the two-photon excited population) a diagonal transition tensor dominated by Sxx...

This relationship is modified by two constants the molecular shape factor/ (a function of the molecular dimensions) and the boundary coefficient C, which takes into account the interaction between the solvent and the solute. In principle, two-photon fluorescence anisotropy decays in isotropic media should yield the same diffusion times as for single photon excitation, but with significantly increased initial fluorescence anisotropy this can be seen in Figure 11.17, which compares single- and two-photon anisotropy decays for the fluorescent probe rhodamine 6G in ethylene glycol. Rotational drflusion times for small molecular probes vary from nanoseconds to hundreds of picoseconds for isotropic rotational drflusion in low viscosity solvents. 

Figure 11.20 Structures of the quadrupolar two-photon polyenes 0M62 (a) and LP79 (b) are engineered to undergo a quadrupolar intramolecular charge redistribution upon two-photon excitation and are based around a fluorene core with electron donating end groups, (c) A standard single-photon visible fluorescent probe rhodamine 6G...

Figure 11.21 StimuLated emission depletion (STED) microscopy. The sample is excited using single-photon excitation (PUMP pulse) in a confocal microscope arrangement. A time-delayed DUMP pulse selectively depletes close to 100% of the exdted state population in a region around the focus of the PUMP pulse. Using this approach. Hell and co-workers were able to obtain a 5-fold reduction in the fluorescent spot size in the vertical (Z-direction) and a greater than a 2-fold reduction in the horizontal Y/X) direction, leading to a final image size of 97 by 104 nm...

Two-Photon Confocal Microscopy. Confocal microscopy provides the possibilities of optical sectioning of a sample by the use of a spatial filter to improve axial resolution of the optical microscope (20). These optical sections can be used to reconstruct the 3D structure of a polymeric specimen. Confocal microscopy using fluorescence is usually performed by a single-photon excitation (linear absorption) of a fluorophore which may be chemically an integral part of the system (polymer) being investigated or just... 

---