Supporting Information Figure S1. Expression pattern of GFP and Met-lead in adult Drosophila brains. Different types of neurons expressing GFP (upper part, Elav-gal4 > UAS-GFP; R13F02-gal4 > UAS-GFP; Cha-gal4 > UAS-GFP) or expressing Met-lead (lower part, Elav-gal4 > Met-lead; R13F02-gal4 > UAS-Met-lead; Cha-gal4 > UAS-Met-lead) were examined under a confocal microscope for 3D imaging. The scale bar is 50 μm. Figure S2. Practical application of Met-lead to adult Drosophila brains at a lower magnification (4x, compared with Figure 4 at 20x). (a) The in vivo biosensing of lead in Drosophila (Cha-gal4 > UAS-Met-lead) was carried out using an upright FRET imaging platform. During a 30-s recording, a buffer containing lead ions (10 μM) and ionomycin (5 μM) was added. Representative images of fly brains (cholinergic neurons) in the YFP channel (12 graphs numbered with the time points in minutes, upper), in the CFP channel (middle), and in a ratio (YFP/CFP, YC bottom) are shown. The rainbow color images were combined through a ratio imaging process. (b) and (c) The time-lapse plots from selected regions are displayed as fluorescent intensities (FIs) (YFP in red lines and CFP in blue lines (b) and emission ratios (c)). In (a), the scale bar is 50 μm and the rainbow color ratio bar is 1–3. Figure S3. YC montage images of Met-lead within the larval CNS of Drosophila. Control (upper) and lead-containing (lower) CNSs are shown in different optical sections in an RG merged manner or in a ratio color manner (the ratio color bar runs from –0.5 to 2.8). Supplementary Video S1: Biosensing of intracellular lead ions by Met-lead through a stereo microscope (Case 1). Supplementary Video S2: Biosensing of intracellular lead ions by Met-lead through a stereo microscope (Case2). Supplementary Video S3: Ratio color images of the control larval CNS in each optical section. Supplementary Video S4: Ratio color images of the Pb-treated larval CNS in each optical section. Supplementary Video S5: YC merged images of the control larval CNS in a 3D stack rotated on one axis. Supplementary Video S6: YC merged images of the control larval CNS in a 3D stack rotated on another axis. Supplementary Video S7: YC merged images of the Pb-treated larval CNS in a 3D stack rotated in one axis. Supplementary Video S8: YC merged images of the Pb-treated larval CNS in a 3D stack rotated on another axis. Supplementary Video S9: YC merged images of the control larval CNS ex vivo in a 3D stack rotated on one axis. Supplementary Video S10: YC merged images of the control larval CNS ex vivo in a 3D stack rotated on another axis.