Chameleon ultra 2

5 to 6 dpf ^ITNGal4, UAS:GCaMP3 larvae were individually mounted in 2% low-melting-point agarose in a 35 mm petri dish and the eyes and tail subsequently freed from the agarose with a scalpel. Visual stimuli consisted of small moving spots (subtending 5° visual angle) back-projected onto a curved screen in front of the animal, appeared at 76° to the left or right of the midline and then moving 152° right or left across the frontal region of visual field (at an average angular velocity with respect to the fish of 30°/s). At each imaging plane, 12–18 repetitions of each of the visual stimuli (L > R, R > L) were presented in pseudo-random order.
2-photon calcium imaging was simultaneously performed using a custom-built microscope equipped with a 20 × /1.00 NA Olympus objective and a Ti:Sapphire ultra-fast laser (Chameleon Ultra II, Coherent Inc) tuned to 920 nm. Average laser power at the sample was 5–10 mW. Images were acquired by frame scanning at 3.6 Hz with 1 µs dwell time per pixel. For each larva, 1–3 focal planes through the ITN nuclei of the larva were imaged. Image acquisition and visual stimulus presentation were controlled using software written in LabView and MATLAB.

The fluorescence lifetime of iLOV in the absence and presence of mBeRFP moiety was measured using a home-built fluorescence lifetime setup (Harp300, Picoquant) coupled with the time-correlated single-photon counting (TCSPC) module (time resolution < 4 ps) and a broadband tunable pulse femtosecond Ti:Sappihire laser (Chameleon Ultra II, Coherent Inc.). The 457 nm femtosecond excitation source was obtained from the secondary harmonic generation with BBO nonlinear crystal to excite the iLOV protein, and the repetition rate was regulated down to 40 MHz with a pulse selection system (Conoptics, model 305). The emission passed through a band-pass filter (ET540/30m, Chroma) and was detected by a micro photodevice (Picoquant) with the time resolution of less than 40 ps. The emission lifetime was determined by fitting the emission trajectory with single exponential decay function.

Mice were imaged using customized two-photon microscopy (2P PLUS, Bruker Corporation) coupled with a femtosecond mode-locked Ti: Sapphire laser (80 MHz, 140 fs, Chameleon Ultra II, Coherent Inc.) with 920 nm (for GCaMP6s only) or 965 nm (for GCaMP6s and jRGECO1a). A Pockels Cell (EO-PC, Thorlabs Corporation) was used to regulate laser power. For neuron imaging, power after the 4× objective (N4X-PF, 0.13 NA, Nikon) and 16× objective (N16XLWD-PF, 0.8 NA, Nikon) was limited to a maximum of 40 mW, dependent on depth. Emission light was filtered using a bandpass 525/70 filter for GCaMP6s and a bandpass 595/50 filter for jRGECO1a, detected by two GaAsP PMTs (model H10770, Hamamatsu Photonics). Images were collected at $\sim 30\mathrm{Hz}$ using a resonant-galvo scanner with $512\times 512\text{pixel}$ resolution for functional imaging. Images were collected using a galvo-galvo scanner with $1024\times 1024\text{pixel}$ resolution for structure imaging. For two-photon imaging in anesthetized experiments, isoflurane (0.6%, mixed with fresh air, $0.5\mathrm{L}/\min$ ) was used to anesthetize mice during imaging. For two-photon imaging in awake experiments, 3 days of routine handling of the mice to acclimate them to the imaging system and immobilization device, which greatly helped to reduce animal motion.

The fluorescence lifetimes were recorded by time-correlated single-photon-counting (TCSPC) measurement using a home-built fluorescence lifetime setup (Harp300, Picoquant, Berlin, Germany). The excitation light source was a broadband tunable pulse femtosecond Ti:sapphire laser (Chameleon Ultra II, Coherent Inc., Santa Clara, CA, USA) equipped with a pulse-selection system (Model 305, Conoptics, Danbury, CT, USA) to modulate the repetition rate. A pulsed excitation laser of 450 nm was generated from the second harmonic generation with a BBO nonlinear crystal. Two long-pass filters (AT470lp, Chroma, Bellows Falls, VT, USA) were utilized to filter out the excitation light. Sample fluorescence was collected by a single-photon-counting avalanche photo diode detector (MPD, Picoquant, Berlin, Germany) and delivered into the TCSPC system for analysis. The typical full width at half-maximum (FWHM) of the system response was 0.15 ns. The recorded data were plotted as a fluorescence lifetime histogram, and the dynamic parameters were obtained from single or double exponential fitting.

After treatment, cells cultured in 35 mm dishes were observed using a two-photon microscope (LSM 710, Carl Zeiss, Jena, Germany) equipped with a Chameleon Ultra II pulsed infrared laser (Coherent Inc., Santa Clara CA, USA), and a W-Apochromat 63x/1.0 NA objective was used. NADH was excited at 730 nm and detected at 460 nm. The maximum projection of each image was obtained from a 5 μm-thick z-stack and analyzed with the CellProfiler program. Cells were delineated, and NADH fluorescent signal per cell was measured.