ACR in Ratiometric Mode
Ratiometry offers ease of calibration and enables quantitative measurement by eliminating variables such as indicator concentration, photobleaching, indicator leakage, and cell thickness. Importantly, a red-emitting indicator significantly reduces interference from cellular autofluorescence, which typically occurs at shorter wavelengths (e.g., flavin nucleotide cofactors, FMN and FAD, emit green fluorescence peaking at ~540 nm).
ACR in Non-Ratiometric Mode
When excited at 540 nm (Figure. 4.1), ACR emits maximally at 650 nm; it shows a 50-fold increase in intensity on going from the Ca2+-free to the Ca2+-bound form. The red fluorescence of ACR is not as intense as the green fluorescence of dyes, but is useful for many applications.