Wavelength-ratiometric

Abstract The response signal of an immense number of fluorescence reporters with a broad variety of structures and properties can be realized through the observation in changes of a very limited number of fluorescence parameters. They are the variations in intensity, anisotropy (or polarization), lifetime, and the spectral changes that allow wavelength-ratiometric detection. Here, these detection methods are overviewed, and specific demands addressed to fluorescence emitters for optimization of their response are discussed. 

In principle, the problems of intensity-based sensing can be avoided using wavelength-ratiometric probes, i.e., fluorophores that display spectral changes in the absorption or emission spectrum on binding or interaction with the analytes (Figure 1.1). In this case, the analyte concentration can be determined independently of the probe concentration by the ratio of intensities at two excitation or two emission wavelengths. 

Probe development simpler than wavelength ratiometric... 

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15))... 

E. U. Akkaya and J. R. Lakowicz, Styryl-based wavelength ratiometric probes A new class of fluorescent calcium probes with long wavelength emission and a large stokes shift, Anal. Biochem. 213, 285-289 (1993). 

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. 

Figure 13.5. Methods of fluorescence sensing, (a) Single excitation/emission wavelength intensity changes with analyte concentration (b) wavelength-ratiometric A/B changes with analyte concentration (c) liftetime based (time domain) r changes with analyte concentration (d) lifetime-based (phase modulation) Am and A change with analyte concentration.

Badugu R, Lakowicz JR, Geddes CD. A wavelength-ratiometric fluoride-sensitive probe based on the quinolinium nucleus and boronic acid moiety. Sensors and Actuators B 2005, 104, 103-110. 

Langner M, Hui SW. Merocyanine interaction with phosphatidylcholine bilayers. Biochim. Biophys. Acta. 1993 1149 175-179. Ross E, Bedlack RS, Loew EM. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential. Biophys. J. 1994 67 208-216. 

A more recent class of pH probes are the seminaphtofluoresceins (SNAFL) and the seminaphtorhodafluors (SNARF). These probes were reported for use as wavelength-ratiometric probes (86), Their absorption and emission spectra change significantly in response to pH. 

Gross, E, Bedlack, E S., and Loew, L. M., 1994. Dual-wavelength ratiometric fluorescence measurement of the membrane dpote potential, Bi hys, J. 67 208-216. 

For an emission wavelength-ratiometric probe, one can use Eq. 19.11 with the values of Sx(X2) and SsfAj) these values ace tdated to the relative intensities of die fine and bound forms of the probe ... 

Like the Na and K probes, Pura-2 and Indo-1 absorb in the UV. This is disadvantageous because, in addition to problems of cellular autofluorescence, it is difficult to obtun microscope optics with high UV transmission. Hence, it is desirable to develop calcium (Kobes with longer excitation and emission wavelengths. Coumarin- and styiyl-based calcium probes have been developed but have not yet been widely used. Excitation spectra of one such probe are shown in Figure 19.51. These probes allow excitation up to 520 nm, but wavelength-ratiometric measurements require a second excitation wavelength below 430 nm. 

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