Extended Data Fig. 1: Multipurpose photometry system for FLiP, fibre photometry and optogenetics.

The system consists of two independent multicolour photometry units. The top photometry unit consists of three subcomponents used for: (1) red channel fluorescence photometry, (2) Chrimson optogenetic laser activation and (3) green channel fluorescence lifetime and intensity photometry. For (1), red channel photometry was accomplished using a fibre-coupled 565-nm LED (M565F3, Thorlabs) for excitation with output collimated in free-space by L2 and filtered by F1 (554/23, Semrock). Red fluorescence was separated from the excitation light by dichroic D1 (573LP, Semrock), filtered by F2 (630/60, Semrock) and focused onto a PMT (H10770(P)A-40, Hamamatsu) by L3. For (2), Chrimson optogenetic light was provided by a fibre-coupled 593.5-nm laser (SKU: YL-593-00100-CWM-SD-03-LED-F, Optoengine) with output collimated by L1 and combined with the red photometry path via M2, a mirror that can be inserted or removed, respectively, for Chrimson optogenetic stimulation or red channel photometry. For the green channel fluorescence lifetime measurement mode of (3), a 50-MHz 473-nm pulsed laser (BDS-473-SM-FBE, Becker and Hickl) was fed through a rotating neutral density filter for power adjustment, reflected by D3 (488LP dichroic, Semrock) and focused onto a patch cable by L6. Emission light was passed through D3, reflected by D2 (532LP dichroic, Semrock), filtered by F4 (517/22, Semrock) and focused by L5 to a high-speed hybrid PMT (HPM-100-07-Cooled, Becker and Hickl). The hybrid PMT was connected to a time-correlated single-photon counting board (SPC-830, Becker and Hickl) for fluorescence lifetime measurements. For the green channel fluorescence intensity measurement mode of (3), a fibre-coupled 470-nm LED (M470F3, Thorlabs) was collimated by L4, filtered by F3 (482/18, Semrock) and reflected by a removable mirror (M4); emission light was detected by a PMT (H7422-40, Hamamatsu). Alternatively, when fluorescence lifetime measurements were not needed, the bottom photometry unit was used. This simple ‘dual-colour fluorescence intensity photometry’ unit consists of 470-nm and 565-nm LEDs (Thorlabs), two PMTs (H10770(P)A-40, Hamamatsu) and a Doric Minicube (FMC5_E1(465-480)_F1(500-540)_E2(555-570)_F2(580-680)_S, Doric Lenses) that are connected by patch cables. For both photometry units, LEDs were driven by a digital signal processor system (RX8-5-12, Tucker-Davis Technology) for frequency modulation to carry out locked-in amplification of sensor signals detected by PMTs. In addition to the two main photometry units, a 593.5-nm laser (SKU: YL-593-00100-CWM-SD-03-LED-F, Optoengine) and a 473-nm laser (MBL-III-473, Optoengine) with independent patch cable connections were installed for Chrimson optogenetics and stGtACR2 optogenetics, respectively, for VTA DAN activity manipulation while monitoring the NAc.