Why use ratiometric sensors?
There are a few reasons why investigators have largely switched to ratiometric fluorescence sensors (like those offered by Pokegama Technologies) from simple, intensity-based sensors. Consider a classical, intensity-based sensor that exhibits an increase in fluorescence quantum yield when the analyte binds to it. The measured fluorescence intensity F is a product of several factors:
(Iexc x Texc x ε)λexc x QY x [Fl] x (Tem x QYdetect)λem x E = F
[Eq. 1]
where Iexc is the excitation intensity; Texsub> and Temsub> are optical factors expressing the net transmission of the excitation and emission optical trains, respectively; and ε is the extinction coefficient of the fluorophore; all expressed as functions of the excitation wavelength λexc. QY is the quantum yield of the fluorophore, [Fl] is its concentration, QYdetect is the quantum yield of the detector at λem, and E is some electronic amplification factor that expresses the fluorescence emission intensity in some units such as volts or counts.
The issue is that although one might measure a change in fluorescence intensity F and attribute it to a change in the quantum yield of the sensor caused by binding of the analyte metal ion to the sensor, in fact the apparent intensity change might have been caused (wholly, or in part) from changes in almost any of the factors in the above expression. So for instance, a decline in excitation intensity, or a decrease in thickness of different portions of the cell being measured, or washout of the sensor, or bleaching of the sensor/indicator, and/or quenching by an interferent, could all be misinterpreted as declines in the analyte concentration.
Recognizing these issues, Walt, Tsien, Wolfbeis, and their colleagues developed so-called wavelength-ratiometric fluorescence sensors, where the analyte concentration was related to the relative strength of two emission (or excitation) bands, corresponding to analyte-free and -bound forms of the sensor; an example is shown below.
In panel A of the figure are depicted emission spectra (excitation wavelength 338 nm) of the classic calcium indicator Indo-1 (Grynkiewicz, et al., J. Biol. Chem. (1981)) in the presence of four different Ca2+ concentrations. It should be evident that the shapes of the spectra are different, each being the sum of emissions from differing proportions of the calcium-bound form (peaking at roughly 400 nm) and the free form of the Indo-1 (475 nm). However, if we take emission spectra of the Indo-1 at [Ca2+] = 0.23 μM with excitation intensities varying more than three-fold (panel B), the relative intensities of the free and bound forms don’t change, the spectra are scalar multiples of one another, and the ratio of emission at 400 nm to 475 nm (about 85%) doesn’t change for the three spectra. Thus the ratio measurement is largely insensitive to variation in any (or all) of excitation intensity, sample (cell) thickness, indicator concentration due to bleaching or washout, or quenching, unlike simple intensity-based indicators. It should be noted that the shapes of the spectra and thus the absolute values of the ratios corresponding to differing concentrations of the analyte will vary between differing fluorometers, microscopes and plate readers (reflecting variations in the other factors of Equation 1), and thus require calibration measurements: our MetalloBuffersTM are offered for exactly this purpose.