Two-photon excitation, TPE

Two-photon excitation (TPE) fluorescence microscopy (Section 11.2.1.2) can be applied to the detection of single molecules in solution. By comparison with one-... 

With intense laser pulses, new nonlinear optical phenomena are possible. The prime example is two-photon excitation (TPE). The peak power in a laser pulse from a Ti sapphrre laser (pulse width 100fs) can readily reach 10 W or higher, with a focused intensity of lO W/cm. Under these conditions, excitation can occur with two photons that have half of the energy (twice the wavelength) of the corresponding one-photon transition (see Fig. 2a). The rate of TPE is given by ... 

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.)...

One-photon spectroscopy is due to the linear term, whereas the nonlinear terms lead to the simultaneous absorption of two or more photons. Although the theory was worked out almost 50 years ago, observation of multiphoton absorption was made feasible only after the development of lasers. This chapter deals with the application of two-photon excitation (TPE) to kinetic studies in low-pressure gas-phase samples. For a systematic, extensive discussion of spectroscopic applications, one of the excellent reviews available should be consulted. ... 

Two photon excitation (TPE) and its application to PCS analysis has been described and discussed in detail and shall be briefly reviewed with regard to fluorescent proteins and intracellular usage [6,7,86,87]. 

Selection rules for single-photon and two-photon excitation (TPE) are different [42,107, 108] however, most resins that polymerize under UV (A) exposure can undergo similar reactions when two photons (2A) are absorbed simultaneously (two-photon photopolymerization), provided that the fight intensity is large enough. 

The other important development, based on the fact that the ROS diffusion distance is limited to hundreds of nanometres, utilises the fact that the irradiated volume in PDT can be as small as 1 pm, when femtosecond pulsed lasers are used as an excitation source. Precise localisation of excitation light should, in principle, allow the treatment of tissues without any damage to surrounding structures, which is crucial in the treatment of sensitive tissues such as those found in the eye and the brain of the patient. See Sect. 9.11 for more details of this emerging treatment modality two-photon excited PDT (TPE PDT). 

Of particular interest for us here is the utilisation of multiphoton processes requiring high energy pulses. Such multiphoton processes have caused much excitement in the last decade and offered novel solutions for biological and medical applications, in particular, multiphoton imaging and two-photon excited PDT (TPE PDT) [28, 29]. 

FCS Fluorescence correlation spectroscopy FRET Fluorescence resonance energy transfer RFP Red fluorescent protein TPE Two-photon excitation... 

Fig. 4 TPE of GFP and DsRed. Wavelength dependence of the fluorescence emission yield r was determined for rsGFP and DsRed with two-photon-excitation. Photon yields per molecule were measured under conditions well below photobleaching. The optimal wavelength for joint excitation of both dyes was 940 nm with a maximum intensity of 20 mW...

As noted above, fluorescence emission often follows two-photon absorption. This one-photon process occurs at a higher frequency than that of the exciting laser. Usually the spectral separation between the frequencies is large, minimizing scattered hght problems. The method is highly sensitive, as shown below, and has been used in many TPE applications. Offering excellent time resolution (better than 10 s), it has so far been the only one used in kinetic studies. 

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. 

Figure 11.15 (a) Confocal fluorescence images of human dermal fibroblast cells (HDF), human melanoma cells (C8161), and Chinese hamster ovary cells (CHO), incubated with 3 (100 (iM, 5 min, Aex=488 nm, scale bar = 10 (im). (b) A two-photon (TPE) luminescence image of live CHO cells incubated with 3 (,1 x = 758 nm, 180 fc excitation, scale bar... 

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