Monochromator, fluorescence spectrum

Fluorescence spectrometers for in vivo diagnostics are commonly based on fibre optic systems [30-33], The excitation light of a lamp or a laser is guided to the tissue (e.g. some specific organ) via glass fibre using appropriate optical filters (instead of an excitation monochromator). Fluorescence spectra are usually measured either via the same fibre or via a second fibre or fibre bundle in close proximity to the excitation fibre. Scanning monochromators or OMA systems as reported above are used for emission spectroscopy. 

Polarization effects The transmission efficiency of a monochromator depends on the polarization of light. This can easily be demonstrated by placing a polarizer between the sample and the emission monochromator it is observed that the position and shape of the fluorescence spectrum may significantly depend on the orientation of the polarizer. Consequently, the observed fluorescence intensity depends on the polarization of the emitted fluorescence, i.e. on the relative contribution of the vertically and horizontally polarized components. This problem can be circumvented in the following way. 

For an excitation wavelength of 457.9 nm, the maximum intensity of the fluorescence spectrum for auramine O in acrylamide was found to occur at / = 505 nm, so the monochromator was set to record the intensity at that wavelength. Figure 26 presents the output of the monochromator/PMT detector as a function of time during polymerization. 

Fig. 17. Delayed fluorescence spectrum of 5 X 10-63/ anthracene in ethanol.84 Half-bandwidth of analyzing monochromator was 0.05 ju-1 at 2.5 n K Intensity of exciting light was approximately 1.4 X 10 einstein cm. a sec.-1 at 2.73m-1 (366 mju). (1) Normal fluorescence spectrum (distorted by self-absorption). (2) Delayed emission spectrum at sensitivity 260 times greater than for curve 1. (3) Spectral sensitivity of instrument (units of quanta and frequency).

In contrast to the preceding set-up, spectrofluorimeters record an entire fluorescence spectrum. Each of the two motorised monochromators can scan a spectral band. It is possible to record the emission spectrum while maintaining a constant excitation wavelength or to record the excitation spectrum while maintaining a constant emission wavelength. Spectra often show small differences when they are obtained using different instruments. 

Fig. 7. Excitation, fluorescence, and synchronously-scanned spectra of oestrone in ethanol, and their second derivatives. A, excitation spectrum monitored at a fluorescence wavelength, Af B, fluorescence spectrum obtained at an excitation wavelength, Aex C, synchronously-scanned spectrum obtained with a constant interval, AA, between the excitation and emission monochromators. (From A. F. Fell, in Proc. 1st Symp. Anal. Steroids, Eger, Hungary, S. Gorog (Ed.), Amsterdam, Elsevier Press, 1982, pp. 495-510.)...

The multi wavelength fluorescence detector consists of two monochromators, the first that selects the wavelength of the excitation light and the second disperses the fluorescent light and provides a fluorescence spectrum or allows the separation to be monitored at a selected fluorescence wavelength,... 

Figure 5. Dispersed fluorescence spectrum and fluorescence decay resulting from excitation of jet-cooled anthracene to S, + 766 cm 1 (122). The fluorescence spectrum was obtained with 1.6 A monochromator resolution (sR). An arrow marks the excitation wavelength. The decay corresponds to detection of the vj = 390 cm 1 band in the spectrum with R — 3.2 A.

Methyl salicylate was placed in a rare gas matrix at 4.2 K. A tuneable dye laser was used to excite the sample, while a monochromator was used to measure the emission spectrum. By tuning the dye laser it was possible to measure the excitation as well as the fluorescence spectrum of methyl salicylate. A partially resolved vibrational progression was observed both in the emission and in the excitation spectra. A fluorescence lifetime of 12 ns was measured at all of the emission wavelengths. As expected deutera-tion changed the vibrational progressions. It also lengthened the fluorescence lifetime. From the vibronic structure it was argued that a double minimum potential does not exist in the excited states. An estimate of... 

A fluorescence spectrum is the plot of the fluorescence intensity as a function of wavelength (Fig. 2.1). Since we have to excite the sample and to record the emitted intensity at different wavelengths, the layout of a fluorometer consists of an excitation source (a lamp or a laser), an excitation monocliromator or a filter (if a lamp is used as the source of excitation), a cuvette holder where we can put the sample, an emission monochromator or a filter (if we do not want to record the whole spectrum but just the fluorescence intensity), a photon detector and a recorder (Fig. 2.2). 

All types of nonresonance fluorescence, particularly direct-line fluorescence, can be analytically useful sometimes it is more intense than resonance fluorescence, and it offers the advantage that scattering of the exciting radiation can be eliminated from the fluorescence spectrum by removing it with a filter or a monochromator. Self-absorption problems (absorption of the emitted radiation by the sample atoms) can also be avoided by measuring fluorescence at a nonresonance line that is not also absorbed. 

Second, it is well suited to gain information on molecular states if the fluorescence spectrum excited by a laser on a selected absorption transition is dispersed by a monochromator. The fluorescence spectrum emitted from a selectively populated rovibronic level (f, / ) consists of all allowed transitions to lower levels (i , 7 ) (Fig. 1.51a). The wavenumber differences of the fluorescence lines immediately yield the term differences of these terminating levels J ). 

The excitation spectrum can be further simplified and its analysis facilitated by recording 3. filtered excitation spectrum (Fig. 4.6b). The monochromator is set to a selected vibrational band of the fluorescence spectrum while the laser is tuned through the absorption spectrum. Then only transitions to those upper levels appear in the excitation spectrum that emit fluorescence into the selected band. These are levels with a certain symmetry, determined by the selected fluorescence band. 

Fig. 4.5 Experimental setup for sub-Doppler spectroscopy in a collimated molecular beam. Photomultiplier PMl monitors the total undispersed fluorescence, while PM2 behind a monochromator measures the dispersed fluorescence spectrum. The mass-specific absorption can be monitored by resonant two-color two-photon ionization in the ion source of a mass spectrometer...

Since the fluorescence spectrum should be relatively simple because of the selective radiative excitation of only the analyte, monochromators with only limited resolution are needed. Detectors that can be used in a photon counting mode (i.e., can detect individual photons) optimize the LODs. For this reason, PMTs are commonplace in AE... 

Most single molecule experiments in solids at low temperatures described in this book have been performed using fluorescence excitation techniques in which the pure electronic absorption spectrum is monitored by detection of the total Stokes shifted fluorescence as a function of the excitation frequency. There is, however, also the possibility to excite the molecule with a fixed frequency in the maximum of its absorption line and disperse the emitted fluorescence light by a monochromator. In this fashion the vibrationally resolved fluorescence spectrum of a single molecule can be recorded. 

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