Table 1. Validation of the four-channel approach.

		Signal contribution in each emission filter (% of total)
		481 nm	535 nm	595 nm	640 nm
GFP	Predicted	19.8	71.5	7.4	1.4
	Experimental	20.4±2.4	68.0±2.0	8.7±1.0	2.9±0.7
Aeq	Predicted	66.4	28.8	4.3	0.5
	Experimental	64.1±2.4	27.1±2.0	6.5±1.4	2.4±0.6

Predicted signal contribution in each channel was calculated from the integral of filter transmittance and the known published emission spectra of GFP and luminescence of Aeq (see Materials and Methods ). The experimental data for GFP were obtained from transfected HeLa cells with off-peak excitation at 420 nm and recording fluorescence signal in the four emission channels, whereas the Aeq data represent bioluminescence contribution during a Ca²⁺ response. Raw counts were corrected for filter bandwidth/transmittance and camera sensitivity (manufactureŕs data); the counts in each channel were divided by the sum of the counts in the four channels, and were expressed as a percentage. The mean±S.E. of 24 cells is shown.