Filtered excitation spectrum

Often excitation spectra of polyatomic molecules are rather complex and difficult to analyze, in particular if several absorption lines overlap with the laser line profile. In such cases filtered excitation spectra are helpful where instead of the total fluorescence from all excited levels only fluorescence lines selected through a spectrometer are detected which are emitted from a single upper level. Although this decreases the detected fluorescence intensity, it simplifies the excitation spectrum and makes the assignment much easier. 

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. 4.6 Section of the spectrum of NO2 excited at Aex = 488 nm in a collimated NO2 beam with a collimation ratio of sine = 1/80 (a) total fluorescence monitored and (b) filtered excitation spectrum, where instead of the total fluorescence only the fluorescence band at X = 535.6 nm for the lower vibrational level (0,10) was monitored by PM2 behind a monochromator [393]...

Fiq.l0.4a-c. Section of the excitation spectrum of NO2 obtained with a single-mode argon laser, tunable around x = 488 nm, (a) In an-N02 cell (p = 0.01 torr), (b) in a collimated NO2 beam with a collimation ratio of 1 80, (c) filtered excitation spectrum. Instead of the total fluorescence as in (b) only the (0,1,0) fluorescence band was monitored... 

Fig. 5.2 Immunofluorescent demonstration of smooth muscle actin (FITC, green channel) in the blood vessel wall of the human kidney. Red autofluorescence of erythrocytes, elastic lamellae and kidney tubules was captured with a filter exciting the autofluorescence in red spectrum under a longer exposure than with the filter exciting specific fluorescence in the green spectrum. Nuclei are counterstained with DAPI (blue channel)...

Fig. 9.1 Immunolocalization of Adenosine Receptor A (FITC, green) in mouse myocardium using mouse monoclonal primary antibody after blocking mouse endogenous immunoglobulins by preincubation with unconjugated Fab fragment Goat Antimouse IgG. Red color accounts for cardiomyocytes and erythrocytes autofluorescence captured under illumination with a filter excit ing the autofluorescence in red spectrum. Nuclei are counterstained with DAPI (blue). Courtesy of Stephanie Grote...

Fig. B5.2.1. Corrected excitation spectrum (broken line) and excitation polarization spectrum of indole in propylene glycol at -58 °C. The fluorescence is observed through a cut-off filter (Corning 7-39 filter) (reproduced with permission from Valeur and Weber3 ).

In multicomponent systems A"0 can be written as a sum of the individual absorption coefficients A ot = 2TA , where each AT,(A ) depends in a different way on the wavelength. If one or more of the components are fluorescent, their excitation spectra are mutually attenuated by absorption filters of the other compounds. This effect is included in Eqs. (8.27) and (8.28) so that examples like that of Figure 8.4 can be quantified. The two fluorescent components are monomeric an aggregated pyrene, Mi and Mn. The fluorescence spectra of these species are clearly different from each other but the absorption spectra overlap strongly. Thus the excitation spectrum of the minority component M is totally distorted by the Mi filter (absorption maxima of Mi appear as a minima in the excitation spectrum ofM see Figure 8.4, top). In transparent samples this effect can be reduced by dilution. However, this method is not very efficient in scattering media as can be seen by solving Eqs. (8.27 and 8.28) for bSd — 0. Only the limit d 0 will produce the desired relation where fluorescence intensity and absorption coefficient of the fluorophore are linearly proportional to each other in a multicomponent system. 

The integrated fluorescence signal //was collected with a g-in. glass light pipe and detected through a combination of dielectric and colored glass filters with a photomultiplier tube. Fluorescence excitation and elastic scattering spectra were recorded simultaneously, in order to identify the type (TM or TE) of resonance responsible for the peaks seen in the excitation spectrum. 

It can easily be shown that for white noise with E[k] 2 = const, and a random binary (-l,l)-sequence, whose total power is independent of its spectrum, Eq. (77) is maximized if the excitation spectrum is proportional to the power spectrum of the filter C[k] °c [F[k] 2. 

In general, when one wants to determine if global and/or local structural modifications have occurred within a protein, circular dichroism experiments are performed. Also, one can record the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from one state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

Figure 9. Fluorescence and REMPl excitation spectra of the styrene-TMA adduct in a supersonic jet. The top panel shows the fluorescence excitation spectrum of the total emission bands marked S are due to bare styrene. The middle panel shows the fluorescence excitation spectrum observed through a filter transmitting at 385 + 3 nm and the bottom panel the excitation spectrum of the trimethylamine ion (a fragment of the cluster ion). Two vibronic band systems are clearly observed, one being assigned to an R-isomer and the other to an E-isomer, as shown at the top of the figure. Adapted from Ref. [27].

Figure 13. Kalman filter resolution of Mg excited spectrum of powder of K2Cr207+Cr03+Cr (40 45 15) mixture in the region of the overlapped Cr 2p doublets.

In a spectrofluorometer, the filters are replaced with scanning monochromators. Either the excitation spectrum (similar to the absorbance spectram) or the emission spectrum may be recorded. 

Figure 23. The high-resolution laser excitation spectrum of the T2n3/2- 2Z+ transition of SrN3 recorded using a monochromator as a narrow band filter. [Reprinted with permission from ref. 96. Copyright 1988 American Institute of Physics.]...

Most of the experiments on optically induced ESR in a-Si H have been performed with white light, sometimes with a filter to block the infrared below-gap light. One study has reported an excitation spectrum for the optically induced ESR (Pawlik and Paul, 1977), but these experiments were performed at high microwave powers and high light intensities that complicate the interpretation. The ESR intensity is, of course, not an absolute measure of the number of centers because the observed signal represents a dynamical balance between the excitation and decay mechanisms. One can also observe an artificial increase in the ESR during optical excitation because the excited electrons and holes allow the equilibrium ESR to be relaxed... 

X-ray tube and for excitation with the filtered Bremsstrahlung spectrum from a tungsten X-ray tube. The data shown were obtained from diluted aqueous solutions which can be considered to be virtually free from any matrix effects. A detection limit of 10 pg for a 10 pi sample corresponds to a concentration of 1 pg/1. A linear dynamic range of four orders of magnitude is obtained for most elements for example, lead at concentrations of 2-20,000 pg/1 using cobalt as an internal standard at 2000 pg/1. 

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