Three-photon fluorescence

Figure 8.4 (a) Scanning three-photon fluorescence image of pe lene microcrystals obtained by irradiation of the NIR pulse of 1260 nm with power 70 pj pulse scanning step 100nm. (b) Corresponding optical transmission image of the perylene crystals. 

Another early method used to monitor the laser pulse was the three photon fluorescence (3PF) technique. ( 8, 9) The advantage of 3PF over TPF is two-fold the contrast ratio is 10 1 for 3PF as opposed to 3 1 for TPF and in addition to temporal information about the pulses available by TPF, 3PF also provides pulse shape information.(10-13) This additional information is obtained because the third-order correlation function which relates the 3PF intensity to the pulse Intensity includes dependence upon the phase of the pulse frequency components.(11) Again, the resulting fluorescence is photographed, and a densitometer trace is made to determine fluorescence intensities. Azulene is an example of a molecule which has been studied quite extensively.(14-20) Typical data are shown in Figure 2. 

Sheppard, C. (1996). Image formation in three-photon fluorescence microscopy. Bioimaging, 4 124 -128. 

Bhawalkar J D, Swiatkiewicz J, Pan S J, Samarabandu J K, Liou W S, He G S, Berezney R, Cheng P C and Prasad P N 1996 Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore Scanning 18 562-6... 

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. 

Figure 8.2e shows the dependence of the fluorescence intensity on the excitation power of the NIR light for the microcrystals measured with a 20x objective. In this plot, both axes are given in logarithmic scales. The slope of the dependence for the perylene crystal is 2.8, indicating that three-photon absorption is responsible for the florescence. On the other hand, slopes for the perylene and anthracene crystals are 3.9 for anthracene and 4.3 for pyrene, respectively. In these cases, four-photon absorption resulted in the formation of emissive excited states in the crystals. These orders of the multiphoton absorption are consistent with the absorption-band edges for each crystal. The four-photon absorption cross section for the anthracene crystal was estimated to be 4.0 x 10 cm s photons by comparing the four-photon induced fluorescence intensity of the crystal with the two-photon induced fluorescence intensity of the reference system (see ref. [3] for more detailed information). 

Matsuda, H., Fujimoto, Y, Ito, S., Nagasawa, Y, Miyasaka, H., Asahi, T. and Masuhara, H. (2006) Development of near-infrared 35 fs laser microscope and its application to the detection of three- and four-photon fluorescence of organic microcrystals. J. Phys. Chem. B, 110, 1091. 

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) ... 

Fig. 33 Two-photon fluorescent images of photosensitive films developed (via 350-nm broadband exposure, 4.4mW/cm ) using an Air Force resolution target mask, a Image recorded by channel 1, b image recorded by channel 2, and c fluorescence intensity by scanning an xy line across one set of three-membered elements (line across set 5)...

A very simple method of observing the microwave transitions is to detect the fluorescence from only the final state. Gallagher et al.11,12 used this technique to detect one, two, and three photon Na transitions from the optically accessible nd states to the 3 5 states. The scheme used to detect the Na 16d— 16g... 

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]. 

Due to its relevance to the next section, we observed and analyzed the fluorescent emission of Tryptophan in water solution excited by one, two, and three-photon absorption. For that, three different light sources were used a UV (180-375 nm) lamp, the second harmonic of a Q-switched Nd YAG laser (with 8 ns pulse duration at 532 nm) and a Ti-Sapphire laser delivering pulses at 76 MHz, with 150 fs pulse duration and 500 mW average power at 800 nm. 

Figure 18.4 Fluorescence spectrum of the Tryptophan solution excited by a) one, b) two and c) three-photon absorption process. Insets show the excitation intensity dependence of the fluorescence.

When the Tryptophan-Ag colloid is excited by three-photons, an increase of the fluorescence is observed as the NPs ccoicentralicai increase. The presence of nanoparticles introduces a new energy level in the solution which matches with the energy of two photons at 800nm (figure 18.7b), providing an enhancement for absorption of a third photon by the amino acid. Figure 18.8 shows the increase of the... 

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