Two-color excitation

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. 

The advantage of two-color excitation over two-photon excitation is not an improvement in imaging resolution, but the easier observation of microscopic objects through highly scattering media. In fact, in two-color excitation, scattering decreases the in-focus fluorescence but only minimally increases the unwanted fluorescence background, in contrast to two-photon excitation. 

Spiro-oxazine (NOSH) photo-induced ring closure reactions were first described by Bohne et al., who used two-laser two-color excitation in the UV and visible regions [71,72]. In this work, they found that photoexciting the merocyanine in cyclohexane leads to a bleached product which recovers quantitatively to the merocyanine form over 30 xsec. The transient bleach state had an absorbance... 

The first direct time-resolved evidence for energy transfer from an upper excited triplet state in solution at room temperature was published in 1987 [50]. This study made use of the two-color technique to photoexcite the 7, state of benzophenone, 70, in benzene solvent. As the extensive (almost quantitative) triplet depletion was not accompanied by any product formation, it was concluded that the excitation energy was transferred to the triplet manifold of the benzene solvent. The energetics of this donor-acceptor system are certainly conductive to this process. The benzophenone 7, and Tn energies (69 kcal/mol and ca. 120 kcal/ mol, respectively—the second photon in the two-color excitation provides roughly 50 additional kcal/mol to the 71 state) bracket the benzene 7j energy (85 kcal/mol) and therefore benzene acts in the same way toward benzophenone as 1,3-cyclohexadiene acts toward anthracene, i.e., as an exclusive upper triplet energy accepter. 

Two-color excitation can thus provide orders of magnitude intensity gains for GSA/ESA upconversion processes through the optimization of both Oq and o. Eor practical applications such as lasing or imaging, this can lead to superior performance [6]. Two-color upconversion excitation methods have been applied in other cases to generate efficient upconversion where little or none was observed under one-color conditions, or to advance mechanistic studies. 

In view of the advantages inherent in the use of two-color excitation schemes, and the experimental difficulties associated with their implementation, it would... 

While the enzymes were again labeled with PDI, the bilayer was doped with 3, 3 -dioctadecyloxacarbocyanine perchlorate (DiO). Two-color excitation was employed to excite both the PDI and the DiO label efEciently. By choosing an appropriate optical filter before the detection apparatus, it was possible to detect the majority of PDI emission and block most of the DiO emission (Fig. 25.5). Thus, emission from the enzymes and the substrate was discriminated based on their relative brightness, and single enzymes appear clearly as bright spots against the background of the low intensity bilayer (Fig. 25.6). 

When excited at 266 nm, all four compounds exhibited a broad blue fluorescence centered at a400 nm with lifetimes ranging from 100 to 3500 ps. Excitation with a second 355-nm laser pulse which was delayed relative to the first 266-nm pulse produced a fluorescence centered at V600 nm with a lifetime of vlO ns. Since the emission spectra (Figure 7) obtained by excitation of l-(chloromethyl)naphthalene and l-(bromomethyl)-naphthalene are superimposable in the region from 550 to 750 nm, this red fluorescence was attributed to the 1-naphthylmethyl radical. In a similar manner, the red fluorescence resulting from two-color excitation of 2-(chloromethyl)naphthalene and 2-(bromomethyl)naphthalene was attributed to the 2-naphthylmethyl radical. This two-color fluorescence technique used in conjunction with OMCDs has proven to be a powerful tool in the study of photodissociation of haloaromatic compounds. 

At the same time Kaldor, et al. (25) reported the observation of infrared multiphoton photodissociation of UFg using a one color CF4 laser irradiation source (16 pm) coincident with V3. A dissociation threshold and yield were estimated to be in the range of those found for SFg. The preliminary report was recently followed by a more extensive paper from the Exxon group (26) in which infrared multiphoton excitation and dissociation in the V3 mode was further described. In addition, two color excitation (16 ym) and dissociation (10.6 ym) experiments similar to those described by Wittig were described. 

A circumvention of this problem is through the use of two-color excitation, or double resonance. An infrared laser can excite a selected rotational state (with a greatly reduced Doppler width because v is much less). As discussed in a following section on overtone excitation, the absorption of the second (visible) photon leads to an excited state in a much more precisely defined energy. 

Xue, G. and Yeung, E.S., Two-color excitation system for fluorescence detection in DNA sequencing by capillary array electrophoresis, Electmphoresis, 23, 1490, 2002. 

G. Jung, J. Wiehler, B. Steipe, C. Brauchle, A. Zumbusch, Single-molecule microscopy of the green fluorescent protein using two-color excitation. Chem. Phys. Chem. 2,392 (2001)... 

A recent extension of the experimental study of Ag by the NeNePo technique, utilizing two-color excitation, is given by Boo et al. [420]. 

Jung, G., Wiehler, J., Steipe, B., Brauchle, C., and Zumbush, A., Single-molecuele microscopy of the Green Fluorescent Protein using simultaneous two-color excitation, Chem. Phys. Chem., 6, 392-396, 2001. 

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