I noticed that in the development of many new biosensors of substances, researchers always measure the Ex and Em wavelength during the absence and present of the substance (Examples shown in the following pictures, from left to right: Calcium sensor, NE sensor). Why is it always necessary to measure excitation and emission spectra in calcium-free and calcium-bond conditions during the construction of Calcium ion sensors? I am confused with the reason why they measure the spectra of sensor without the substance. Do they want to see if the fluoresence intensity of sensor expression in the cells without the combination of the substance?