FIGURE 2.

Simplified photophysical description of cpFP sensors with emphasis on FLIM. (A) Schematic representation of the equilibrium occurring in GPCR sensors (left) and of their spectroscopic properties (right). It can be observed how the absorption spectrum is actually the sum of two bands, one due to the protonated (acid) form of the chromophore and one to the deprotonated (basic) one. The latter is also the only emissive form in most cpFPs used in sensors. This is certainly true if the sensor is excited at longer wavelengths, as shown in these examples, where only the basic form absorbs. (B) Representative spectral changes for the two limit cases described in the text (left), and FLIM images and histograms of RINm5f cells expressing the cpFP sensor TriPer before (right, top) and after (right, bottom) exposure to 0.2 mM H₂O₂ (reproduced without modifications from Melo et al., 2017; under Creative Commons license: http://creativecommons.org/licenses/by/4.0/). Possible candidates for FLIM can be identified by looking at the changes of the absorption and fluorescence spectra of the sensors upon analyte binding. If only the acid/base equilibrium changes, but not the quantum yield of the basic form, then no lifetime change is expected (limit case 1). An easy way to conceptualize this phenomenon is that if the absorbance of the open and closed forms at the excitation wavelength would be the same (i.e., if the two would absorb the same number of photons), then their fluorescence intensity would also be the same (column “absorbance match”). On the other hand, if there are changes in the fluorescence intensity but not in the acid/base equilibrium (limit case 2), then changes in the quantum yield are occurring and will likely (but not necessarily) result in a lifetime change. We note that this is a simplified scheme which takes into account only a subset of the photophysical processes occurring in cpFP sensors, thus: (a) unexpected results may occur by strictly following this simplified scheme as a general rule for all sensors, (b) lifetime changes can occur also as result of other mechanisms.