Intensity-based methods

The term filterFRET here refers to intensity-based methods for calculating fluorescence resonance energy transfer (FRET) from sets of images of the preparation collected at different excitation and/or emission wavelength. The term is not intended to imply that interference filters are actually present in the setup very similar considerations apply when donor- and acceptor fluorophores are spectrally resolved by other means, such as monochromators or spectral detectors. 

The various possible schemes for fluorescence sensing are summarized in Figure 1.1. At present, most fluorescence assays are based on the standard intensity-based methods, in which the intensity of the probe molecule changes in response to the analyte of interest. However, there has been the realization that lifetime-based methods possess intrinsic advantages for chemical sensing. (A more detailed description of... 

Continuous wave techniques do not offer the optimum use of luminescence for sensing applications. CW methods, also known as intensity-based techniques, have many inherent limitations. These limitations will be discussed later in the chapter. Many of the limitations of intensity-based methods can be overcome by using steady-state modulated excitation of the form... 

At present, most fluorescence sensors or assays are based on intensity measurements, i.e., intensity-based sensing, in which the intensity of the probe molecules change in response to the analyte of interest. Intensity-based methods are initially the easiest to implement because many fluorescent probes change intensity in response to analytes. These intensity changes can be due to changes in extinction coefficient due to probe ionization, changes in quantum yield of the probe on analyte binding, or due... 

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

Intensity-based methods. 1H— H RDCs have been obtained by analyzing the intensity ratios of the diagonal and cross-peaks in a series of 2D CT COSY spectra.177 This method can only be applied to resolved resonances, for example, those of anomeric protons.149 Similar limitations apply to /-modulated ID directed COSY,153,178 which uses selective 180° pulses to produce a series of ID spectra for each pair of coupled spins. This approach has recently been extended to include additional selection blocks yielding a versatile method for the measurement of coupling constants in compounds with severely overlapping proton resonances such as those found in carbohydrates.154 The problem of overlapping resonances can also be resolved by involving 13C nuclei, as demonstrated on natural abundance (13C COSMO HSQC)32 or uniformly 13C isotopically enriched carbohydrates (2D-HSQC-(sel C, sel H)-CT COSY experiment).73,158... 

Intensity-based methods. Intensity-based methods detecting 13C—13C pairs are practical only for 13C isotopically enriched oligosaccharides. 13C—13C CT-COSY was used to measure one-bond 13C—13C RDCs in uniformly 13C isotopically enriched lactose.73... 

In comparison to intensity-based methods, techniques that rely on FRET provide large changes in emission profiles and open the opportunity for ratio-metric fluorescence measurements [59]. Assays of this type are less prone to false positives from nonspecific binding events. 

Fluorescent probes that give rise to ratiometric fluorescence responses are valuable in the design of chemosensors. They are particularly attractive when the concentration of the analytes is limited since many of the problems associated with direct intensity-based methods, such as dye bleaching, medium-induced fluctuations in absorbance or emission intensity, and interference from the medium are avoided [31]. This class of chemosensors also offers the advantages of being less likely to be biased by non-specific interactions or impurities, while the signal-to-noise ratio is improved [31]. 

The proton NMR of the cobalt monomer and polymers also confirms the presence of diastereomers due to the S-leucine moiety. Peaks due to the diastereomers is most evident in the gamma proton of the P-diketones in the monomer, for which four resonances are observed in pairs of different relative intensities based on the method of preparation. 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

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