One-photon fluorescence

In TPA induced fluorescence spectroscopy the excited state population for fluorescence is created by the simultaneous absorption of two photons instead of the one-photon absorption in standard fluorescence experiments. The principles of this technique are described in Ref. [23]. It allows an experimental determination of two-photon absorption cross-sections provided the material is fluorescent and that its two-photon fluorescence quantum efficiency is known (which is usually assumed to be equal to the one-photon fluorescence quantum efficiency if the same excited state is reached). In this case the method generally provides high sensitivity. 

In some favorable cases TPE was efficient enough to form measurable amounts of photodecomposition products. Examples are the dissociation of iodoform by a ruby laser s and of water by a doubled dye laser. In the first, the reaction was followed by titrating the liberated iodine, while in the second, OH radicals were monitored by laser-induced fluorescence, using the same laser frequency for both two-photon dissociation of HjO and one-photon fluorescence excitation of OH. 

As discussed in Section 14.2.1, normal (one-photon) fluorescence spectroscopy (IPE) has been the method of choice in capillary electrophoresis (CE) and gel electrophoresis (GE) with visible and NIR fluorescent dyes. MPE fluorescence, however, is also quite suitable for detection in CE and GE. For instance. Song et al. fractionated coumarine dyes with capillary electrophoresis, and detected the dyes at at-tomole concentrations by 2PE fluorescence [189]. Generally, detection Hmits are comparable to those attainable by normal fluorescence. However, MPE is particu-... 

The anti-Stokes signal is blue-shifted from the pump and Stokes frequencies, and therefore it is not affected by one-photon fluorescence. 

The ns (n + 2)s transition is two-photon allowed. Detection of the npper level is through its one-photon fluorescence to the np state. The object of the control is either to populate or not to populate the upper state using the interference between different two-photon transitions. The principle is very much the same as in Section 7.4 bnt the implementation is different. Here only one pulse is used but this pulse is ultrashort so it is a superposition of many pulses each with its own well-defined ffeqnency. It is the interference between these components that allows us to direct the outcome. The shape of the pulse, meaning the amplitude... 

To check the impact of silk fibroin on the fluorescence emission of the fluorescent molecules, the one-photon fluorescence spectra of the powder of 4NF, 8NF, and of their functionalized silks were acquired under the same conditions. The results showed that 4NF fibers had strong fluorescent emission, while the emission from the 4NF powder was hardly detectable (Figure 7.6b). The fluorescence plots of the 8NF solution, 8NF fibers, and the powders exhibit a mono tonic bathochromic shift in the order of 8NF solution < 8NF fibers < 8NF powder. The emission spectrum... 

In this method the TPA ctoss section of a chromophore is calculated by comparing its TPA against a standard whose TPA cross sections as well as one-photon fluorescence qirantum yields are known." "" The TPA cross section is calculated by the equation... 

CAHRS and CSHRS) [145, 146 and 147]. These 6WM spectroscopies depend on (Im for HRS) and obey the tlnee-photon selection rules. Their signals are always to the blue of the incident beam(s), thus avoiding fluorescence problems. The selection ndes allow one to probe, with optical frequencies, the usual IR spectrum (one photon), not the conventional Raman active vibrations (two photon), but also new vibrations that are synnnetry forbidden in both IR and conventional Raman methods. 

B1.18.5.5 CONTRAST ENHANCEMENT AND PRACTICAL LIMITS TO CONFOCAL ONE-PHOTON-EXCITATION FLUORESCENCE MICROSCOPY... 

The vast majority of single-molecule optical experiments employ one-photon excited spontaneous fluorescence as the spectroscopic observable because of its relative simplicity and inlierently high sensitivity. Many molecules fluoresce with quantum yields near unity, and spontaneous fluorescence lifetimes for chromophores with large oscillator strengths are a few nanoseconds, implying that with a sufficiently intense excitation source a single... 

A dye molecule has one or more absorption bands in the visible region of the electromagnetic spectrum (approximately 350-700 nm). After absorbing photons, the electronically excited molecules transfer to a more stable (triplet) state, which eventually emits photons (fluoresces) at a longer wavelength (composing three-level system.) The delay allows an inverted population to build up. Sometimes there are more than three levels. For example, the europium complex (Figure 18.15) has a four-level system. 

The example we consider is the two-photon fluorescence excitation specfrum of 1,4-difluorobenzene, shown in Figure 9.29 and belonging to the >2 point group. The transition between the ground and first singlet excited state is Table A. 3 2 in Appendix A shows that 82 = r(T ) and, therefore, according to Equation (7.122), the electronic transition is allowed as a one-photon process polarized along the y axis which is in-plane and... 

Nevertheless, 1,4-difluorobenzene has a rich two-photon fluorescence excitation spectrum, shown in Figure 9.29. The position of the forbidden Og (labelled 0-0) band is shown. All the vibronic transitions observed in the band system are induced by non-totally symmetric vibrations, rather like the one-photon case of benzene discussed in Section 7.3.4.2(b). The two-photon transition moment may become non-zero when certain vibrations are excited. 

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

Fig. 5 Linear absorption (1, 2) and one-photon-excited fluorescence (1, 2 ) for the quantum yield standard Cresyl Violet (1, 1 ) and the proposed standard PD 2631 (2, 2 ) for NIR wavelengths. Molecular structures are shown to the left...

Fisz, J. J. (2007). Fluorescence polarization spectroscopy at combined high-aperture excitation and detection Application to one-photon-excitation fluorescence microscopy. J. Phys. Chem. A 111, 8606-21. 

In conventional fluorescence spectroscopy, a fluorophore is excited by absorption of one photon whose energy corresponds to the energy difference between the ground state and the excited state. Excitation is also possible by the simultaneous absorption of two photons of lower energy (i.e. of longer wavelength) via a short-lived virtual state (Figure 11.4)3). For instance, absorption of two photons in the red can... 

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. 

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