Excitation intensity

It should be noted that this technique is not without some disadvantages. The blackbody emission background in the near IR limits the upper temperature of the sample to about 200°C [43]. Then there is the dependence of the Raman cross-section ( equation (B 1.3.16) and equation ( B1.3.20)-equation ( B 1.3.21)) which calls for an order of magnitude greater excitation intensity when exciting in the near-IR rather than in the visible to produce the same signal intensity [39]. 

Equations (C3.4.5) and (C3.4.6) cover the common case when all molecules are initially in their ground electronic state and able to accept excitation. The system is also assumed to be impinged upon by sources F. The latter are usually expressible as tlie product crfjo, where cr is an absorjition cross section, is tlie photon flux and ftois tlie population in tlie ground state. The common assumption is tliat Jo= q, i.e. practically all molecules are in tlie ground state because n n. This is tlie assumption of linear excitation, where tlie system exhibits a linear response to tlie excitation intensity. This assumption does not hold when tlie extent of excitation is significant, i.e. 

Valkunas L, Liuolia V and Freiberg A 1991 Picosecond processes in chromatophores at various excitation intensities Photosynthesis Res. 27 83-95... 

Figure 8-14. (a) (—versus pump-probe delay al 1.91 eV lor VW.= -16 V and pump excitation intensities 1.2 mJ/ein2 (solid line) and 0.24 niJ /cm" (dashed line). The inset shows (-A7/T)Mr at 1.91 eV and 20 ps versus excitation intensity, (b) same as (a) Tor pump excitation intensity 1.2 nij/eni2 with V[1M =-16 V (solid line) and th, is=-8 V (dashed line). The inset shows ( A7//)/h/. at 1.91 eV and 20 ps versus field open squares=posilive bias filled eirclcs=negative bias (adapted from Ref. (40J). 

Figure 5 shows the dependence of the total emission intensity on the excitation intensity and its spectral width obtained from DCM-encapsulated dendrimers. A nitrogen laser (wavelength of 337 run, pulse duration of 4 ns, and repetition rate of 10 Hz) was used as the excitation source. A cylindrical lens focused the excitation beam onto a stripe 200 pm wide on a quartz cuvette... 

Fig. 5. a Total emission intensity, b Linewidth, both as functions of excitation intensity for DCM/dendrimer solution in cuvette. DCM concentration was 4.0 mmol/1. Inset in a shows plot in logarithmic scale at moderate excitation intensity... 

Fig. 8a-f. Evolution of emission spectra from waveguides with increasing excitation intensity for films with a-c 2 wt% d-f 10 wt% Rd/dendrimer content. The intensity increased for each spectrum from the bottom as follows 1.7,2.1, and 4.0 mj/cm ... 

Emission spectra at these points are shown in Figure 8.2d. The band shapes were independent of the excitation intensity from 0.1 to 2.0 nJ pulse . The spectrum of the anthracene crystal with vibronic structures is ascribed to the fluorescence originating from the free exdton in the crystalline phase [1, 2], while the broad emission spectra of the pyrene microcrystal centered at 470 nm and that of the perylene microcrystal centered at 605 nm are, respectively, ascribed to the self-trapped exciton in the crystalline phase of pyrene and that of the a-type perylene crystal. These spectra clearly show that the femtosecond NIR pulse can produce excited singlet states in these microcrystals. 

Figure9.6 Typical luminescenceintensityversustimetrajectories of a single CdTe QD (4.6 nm) embedded in a PVA (a) and a trehalose (b) matrix dispersed on a cover glass surface at room temperature. The excitation intensity was 1.7 kW cm and the integration time was 200 ms bin ...

Figure 9.7 On-time histogram of single CdTe QDs in PVA matrix at 0.28 kWcrn and 7.1 kWcrn . All histograms were built from time traces recorded under identical experimental conditions except the excitation intensity.

Levels of carotenoids are much lower in the skin relative to the macula of the human eye, but higher light excitation intensities and longer acquisition times can be used in Raman detection approaches to compensate for this drawback. Since the bulk of the skin carotenoids are in the superficial layers of the dermis, and since the concentrations are relatively low, the thin-film Raman equation given above, Equation 6.1, should still be a good approximation. 

Fluorescence and phosphorescence spectra corrected for the instrumental sensitivity were measured with a spectrometer described previously (()). Corrected excitation spectra were obtained with constant excitation intensity controlled by a rhodamine B quantum counter. For phosphorescence polarization measurements the apparatus was set up in an "In Line" arrangement (j ) and equipped with a Glan-Thomson polarizer and a sheet polarizer (analyser) (10). 

Even if we forget, for a moment, the overlap problem and assume that we obtained a pure sensitized emission image, interpretation of this image is still ambiguous. That is because first, the intensity of S varies linearly with the excitation intensity and with the detector sensitivity. The exact same preparation will, when measured on a different microscope, yield different s.e. intensities. In fact, as much as renewing the arc lamp would impede comparison of results obtained on the same microscope. Second, the interpretation... 

Ratio imaging nicely cancels out some of the main complications in the interpretation of wide-field images in that it normalizes fluorescence intensity differences caused by for example, cell height (Fig. 7.T1) as well as possible slow drift in excitation intensity. Light sources invariably are much less stable than detectors. Incidentally, for these reasons emission ratio imaging has been applied for over 3 decades by the Ca2+ imaging community. 

Note that parameters ft and 5 depend on signal amplifications in the utilized detectors and on the elements in the optical path (optical filter, spectral detection bands) only, while a and y are additionally influenced by relative excitation intensity. This is usually a fixed constant in wide-field microscopy but in confocal imaging laser line intensities are adjusted independently. Furthermore, note that the a factor equals 5 multiplied by y (see Appendix for further detail). 

Note that G as derived here relates the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel and that it is identical to the correction factor y/ [2] or G [6, 14]. Note however, that if the correction factors or 5 change, G and

correction factor C [3] is a constant that depends only on fluorophore properties and filter settings, and therefore it does not change with excitation intensity or detector gain. This is a clear advantage for confocal filterFRET. C (Eq. (7.14)) and G (Eq. (7.19)) are related as ... 

What is the donor/acceptor ratio in a given cell Again, this ratio cannot be directly derived because it concerns two quantities that stem from fluorophores with different properties (absorption coefficient, quantum yield, spectra) and that emit into two channels differing in gain, filters, and excitation intensity. Thus, the (overlap corrected) intensity of acceptors in channel A will be a factor k times that of donors in D, at equimolar concentrations,3 or ... 

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