Orca fusion digital camera

For single-cell gliding on glass, cells were grown in CYE at 28°C to an A_{600 nm} ≈ 0.7. Cells were diluted to an A_{600 nm} ≈ 0.05 and 100 μL were spotted into μ-Slide chambers with glass coverslip bottom (Ibidi). After 5-minute incubation, floating cells were washed out with fresh CYE medium and gliding of adherent cells was monitored by phase contrast microscopy on a Nikon Eclipse TE-2000 microscope equipped with a 100× NA 1.3 Ph3 objective, a perfect focus system to maintain the plane in focus, and an Orcaflash 4.0 LT digital camera (Hamamatsu Photonics, Shizuoka, Japan). GldL-alfa/NBalfa-sfGFP localization was observed by Hilo microscopy. Cells were grown in CYE overnight without shaking at 28°C. NBalfa-sfGFP expression was induced with 1 mM IPTG for 1 hour prior to observation. Cells were spotted on a 2% low-melting agarose pad for immediate observation. Hilo fluorescence microscopy and FRAP experiments were performed with a Nikon Eclipse Ti2 microscope equipped with a 100x NA 1.45 Ph3 objective, an Orca-Fusion digital camera (Hamamatsu Photonics), a perfect focus system, and an Ilas2 TIRF/FRAP module (Gataca Systems, Massy, France).

For live-cell imaging, 5–10 µL S. rosetta cultures were pipetted directly onto superfrost plus glass slides (Fisherbrand) between two thin streaks of petroleum jelly (applied using a 22-gauge needle with syringe), over which an 18 mm circle glass coverslip (Fisherbrand) was gently suspended to create vertical space for the cells to move freely. Brightfield fast time-lapse series (100 FPS) were acquired on a Nikon Eclipse Lvdia-N microscope equipped with an Andor Zyla 4.2 PLUS sCMOS camera, using a 40x0.75 NA Plan Fluor objective and the Nikon Elements software. Videos were later converted to the.mov format using Fiji. For culture images, S. rosetta cells were fixed with 2% glutaraldehyde (Sigma-Aldrich, Germany) for 10 min, and 5–10 µL were transferred to glass slides, covered with a glass coverslip, and sealed with clear nail polish. DIC images were collected on an inverted Nikon Eclipse Ti microscope using a 60x1.4 NA Plan Apochromat oil objective, an Orca-Fusion digital camera (Hamamatsu), and the Nikon Elements software.

Ex vivo coronal brain slices (300 μm) encompassing the medial-caudal portion of the third ventricle were cut from B6;129S-Slc17a6^{tm1.1(flpo)Hze/J} mice bilaterally injected with AAV1-CAG-FLEXFRT-ChR2(H134R)-mCherry in the PBN to test possible monosynaptic inputs onto tanycytes. Brain slices were superfused with oxygenated ACSF containing 1 µM TTX (Tocris, 1069) and 100 µM 4-aminopyridine (Sigma Aldrich, 275875) with a peristaltic pump (Multichannel systems, PPS2) at a flow rate of 3 ml min⁻¹ at 25 °C throughout. A BX51WI microscope (Olympus) equipped with a DIC prism (Olympus, WI-DICHTRA2), and LUMPlanFI/IR 60X/0.90W and Plan N4×/0.10 objectives (Olympus) was used. channelrhodopsin-2(ChR2)-mCherry⁺ axons in close apposition to the third ventricle were excited with a CoolLED (pE-100) light source at 535 nm and imaged on an ORCA-Fusion digital camera (Hamamatsu, C14440). Tanycytes were clamped at a holding potential of −70 mV, and data acquired on an EPC10 USB Quadro patch-clamp amplifier (HEKA) were sampled at 20 KHz, and filtered at 2 KHz. ChR2–mCherry⁺ terminals were excited with 50-ms light pulses at 470 nm (CoolLED, pE-100) synchronized to the recording of possible optically induced EPSCs in tanycytes. The time response (in ms) and amplitude (in pA) of EPSCs were analysed in PatchMaster Next (HEKA).