Fluorescence excitation spectra, measurement

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Fig. 6.2.5 Fluorescence spectra of pholasin after treatment with 5M guanidine hydrochloride. Left, excitation spectrum measured at 460 nm right, emission spectrum measured with excitation at 360 nm. From Henry et al., 1973, with permission from Elsevier.

Now we will assume in addition that the total measured fluorescence is proportional to the above volume average. This can be accomplished experimentally by suspending the fluorescent particle in an integrating enclosure and monitoring the fluorescence with an optical fiber which is pushed through a small hole in the side of the enclosure. Our interest in what follows is to use Eq. (8.4) to simulate a fluorescence excitation spectrum. 

To measure a fluorescence excitation spectrum, a band of fluorescence from a solute in C was selected by the monochromator M2 and was received by the photomultiplier After amplification the outputs of Pi and P2 were passed to the ratio recorder, R. The frequency drum on Mi was motor-driven and as the frequency was varied the slits were adjusted so as to maintain the output of P2 approximately constant. If the contents of F were so chosen as to make the output of P2 proportional to the quantum output of Mj at all frequencies, then since Pi was proportional to ht (see eq. 2) and P2 was proportional to I0, the recorded ratio was proportional to t, i.e., the true excitation spectrum was recorded. 

Measurements of the intensity of fluorescence at any wavelength vs the wavelength of monochromatic light used to excite the fluorescence give a fluorescence excitation spectrum. The excitation spectrum is an example of an action spectrum, which is a measure of any response to absorbed light. At very low concentrations of pure substances, action spectra tend to be identical to absorption spectra. However, since the observed response (fluorescence in this case) is proportional to light absorbed, action spectra should be compared to plots of 1-T (where T = transmittance, Section B,l) vs wavelength rather than to plots of e vs X. The two plots are proportional at low concentrations. For a discussion of action spectra see Clayton.123... 

The rotational analysis of the 8000 to 9000 A band system, A2B2-X2Al, has been made by Brand et al. (133). The A2B2 state is severely perturbed by the X2/4, state. The 2B2 state has an O N O bond angle of 102 [Gillispie et al. (395)]. Smalley et al. (909) have measured the fluorescence excitation spectrum of the cooled N02 in the region 5708 to 6708 A. The 2B2 state is the only upper state in this region. 

Radiative lifetimes of NS(B II), 6= u = l2 determined using LIF. Collisional quenching by N2 studied Fluorescence excitation spectrum of NH2(A Ai — X Bj) measured in a supersonic free jet. Lifetimes obtained for single rovibronic levels of NHjfA Aj)... 

By measuring the fluorescence intensity of sufficiently dilute solutions as a function of the exciting wavelengths a fluorescence excitation spectrum is obtained. Such a measurement represents a remarkably sensitive method for investigating the absorption spectrum. With and If. being the intensities of the absorbed light and of the light emitted as fluorescence, respectively,... 

Fluorescence and Chemiluminescence Spectroscopy. - The fluorescence excitation spectrum of PF3 at 9-13 eV, using monochromatised synchrotron radiation, has been examined to resolve the pyramidal geometry of the X Ai ground state of the PFs" cation, which was also confirmed by ab initio calculations. Dimethylamino-substituted triphenylphosphines exhibit dual fluorescence in polar solvents, and fluorescence-decay measurements have shown that the photo-induced intramolecular charge-transfer process occurs in a few picoseconds, even in weakly-polar solvents. 

A few years ago, a powerful version of molecular optical spectroscopy with supersonic beams and jets was developed by Smalley, Wharton and Levy . Supersonic expansion of molecules in an inert carrier gas yields an ideal spectroscopic sample. As a result of the expansion, the translational temperature of the carrier gas decreases to extremely low values (below O.I K). The flow is collisionless so that even extremely unstable species survive. Special attention was paid to fluorescence excitation spectroscopy but the technique is by no means limited to this type of spectroscopy. (Because of fundamental difficulties, however, direct measurement of light absorption in molecular beams is not easy.) Cooled molecules in the beam are electronically excited with a tunable dye laser. The emitted fluorescence is detected and plotted against the wavenumber of the exciting radiation. The obtained fluorescence excitation spectrum is generally very similar to the corresponding absorption spectrum. The technique was used for analysis of the spectra of interesting vdW molecules He. .. NOj, He... Ij, X. .. tetrazine and Xj. .. tetrazine (X = He, Ar, H ) complexes . 

Tropolone is easily deuterated and the Sj-Sq fluorescence excitation spectrum of jet-cooled TRN(OD) was reported by Sekiya et al. [40]. The observed doublet separation, DS = 2 cm 1, is near the 2.2 cm value reported by Alves and Hollis [22], and about 10% of the value observed for TRN(OH). Presently the only available experimental estimate for the ZP tunneling splitting of Sq TRN(OD) is Ao <0.17 cm 1 [80] obtained using a chloroform solvent and NMR spectroscopy to measure the deuteron spin-lattice relaxation time. 

Observations analogous to those for carboxylic acid dimers have been reported for 2-pyridone-2-hydroxypyridine (2PY-2HP) [52, 53], illustrated in Fig. 29.9. This dimer, formed from two isomers, is asymmetric, but it has a symmetric double proton transfer potential since the transfer interchanges the isomers. In a collaborative effort, Pratt, Zwier, Leutwyler and their coworkers [52] measured and analyzed the high-resolution fluorescence-excitation spectrum of the origin... 

Further information can be obtained from measurements of the polarization of the photofragment fluorescence excitation spectrum. The directions and magnitudes of the fluorescence polarization, which can be measured following photodissociation at any wavelength in the excitation spectrum, reflect the orientation of the electric dipole transition moment in the parent molecule and its lifetime with respect to pre-dissociation into the observed products . ... 

---