Fluorescence relative positions

A fluorophore in the proximity of the NP senses the altered EM-field and its fluorescence properties are consequently modified. There are (at least) two enhancement effects an increase in the excitation of the fluorophore and an increase in its quantum efficiency (QE). The increased excitation of the fluorophore is directly proportional to the to the square of the strength of the E-field and is a function of the wavelength and relative position of the NP. The maximum enhancement of this type is achieved if /.res equals the peak absorption wavelength of the dye. 

Fig. 3.1. Perrin-Jablonski diagram and illustration of the relative positions of absorption, fluorescence and phosphorescence spectra.

FIGURE 7. Relative positions of absorption, fluorescence, and phosphorescence The 0-0 peak is common to both absorption and fluorescence spectra (see Fig. 6). ISC, Intersystem crossing. (Modified from Ref. 2.)... 

Standards may be used in two different manners. First, the standard may be an external standard. It is this method that was described above. The experimental samples and the standard samples are kept separate during the isolation, staining, and flow analysis. Each day the fluorescence intensity of the experimental lines are calculated relative to the standard. The standard is defined as 100% or 100 AU. Second, the standard may be used as an internal standard. In an internal standard, the standard and experimental lines are mixed at the isolation, staining, or analysis stage. This results in two G, peaks in the resulting histogram (Fig. 3). The relative position of the experimental Gt peak with respect to the G, peak of the... 

FIGURE 1. Jablonski diagram for the relative positions of the electronic energy levels of a molecule, where F = fluorescence rate, P = phosphorescence rate and VR = vihrational relaxation... 

To demonstrate that STORM can indeed resolve nearby fluorescent molecules with sub-diffraction-limit resolution, we first engineered samples with known relative positions of the fluorescent labels - double-stranded DNA labeled with two Cy3-Cy5 pairs separated by a well-defined number (135) of base pairs, corresponding to an inter-CyS distance of 46 nm along the contour of DNA [4]. The DNA strands were immobilized in a flat configuration to a quartz slide through multiple biotin-streptavidin linkages. The two Cy5 dyes were turned on and off, repetitively, and the image sequence was analyzed to determine the positions of individual activated Cy5 dye. We then constructed... 

Fluorescent screens are used only for the detection of x-ray beams, while photographic film and the various kinds of counters permit both detection and measurement of intensity. Photographic film has the advantage of being able to record a number of diffracted beams at one time and their relative positions in space, and the film can be used as a basis for intensity measurements if desired. Intensities can be measured much more rapidly with counters, and these instruments are more popular for quantitative work. However, most counters record only one diffracted beam at a time. 

The relative position of the excited states of benzophenone (10) is shown in Figure 5.13a. Intersystem crossing leads from a (n, r ) state to a state this type of transition is favored by spin-orbit coupling to such an extent that st and no fluorescence is observed. If, however, S and T, are (n,7T ) states and are disposed as in Figure 5.13b, d>sr is so small that practically no phosphorescence can be observed although the molecule has n- jr transitions. The relative disposition of (n,Ji ) and states may... 

Fig. 4. Relative position of lower excited singlet state (S,), triplet state (Tt) and metal ions (Mi), electronic levels, and transitions between them in metal ion complexes with organic ligands, (A) luminescence (fluorescence and phosphorescence) with levels of metal ion located above Sr levels, (B) phosphorescence with metal ion levels located between S,- and Trlevels, (C) sensitized (native) luminescence with metal ion levels located below Ti-levels...

Figure 7. Measurement of the critical breakdown potential of a myeloma cell membrane using an ac field. The chemical formula of propidium iodide is shown in the upper figure. The middle figure shows a typical myeloma cell under the microscope (bright field). The relative positions of the platinum electrodes are indicated. The lower figure gives some photographs taken at different times after a cell was electroporated by an ac field Ecrit. Within 1-3 s, two narrow, fluorescent bands appeared at the two loci facing the electrodes (the leftmost photo). The next three photos, from left to right, were taken at 20 s, 1 min, and 3 min, respectively, after the application of a 200-ms ac field of 1 kV/cm at 100 kHz. (Reproduced with permission from reference 12. Copyright 1990.)...

In order to put into evidence the contribution of eacli Tip residue to the global fluorescence of the protein, construction of mutants was performed. In each mutant, one Trp residue was replaced by a phei Ialanlne. Figure 7.7a shows the fluorescence emission spectra of the wild type and tlic diflerence spectra of the tiyptophan mutants calculated by subtraction of their respective TuoTesccnce emission from that of the wild type enzyme. Therefore, the results described in the figure give the fluorescence emission spectra of the specific tiyptophan re ue missing in each of the mutants. The spectra obtained indicate that l ip 186 contribute the most to the fluorescence intensity of the wild type. The position of the emission maximum indicates the relative position in the protein of each Trp residue In fact, the maximum of the fluorescence spectrum of Trp 186 - 332 run) indicates that the Trp residue is buried m the protein core... 

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