Ft200emt

Optical stimulation and electrophysiological recordings were performed with a 2 m (bench experiment) or 8 m (fMRI experiment) optic fiber (FT200-EMT, NA = 0.39, 200 μm, Thorlabs). The coating of both ends of the optical fiber was stripped off. One end was glued into an FC/PC connector (Thorlabs), and the other end was carefully polished by using polishing sand papers with appropriately selected grit size (LF1P/3P/5P, Thorlabs). Optical quality of the polishing interface was confirmed by using a fiber inspection microscope (FS200, Thorlabs). For simultaneous Ca²⁺ recording and fMRI, two fibers (one for optical stimulation, the other for Ca²⁺ recording) were closely glued (454, Loctite) together. For simultaneous Ca²⁺ and electrophysiological recording, a Tungsten electrode (1 MΩ, ~100 μm, FHC) was closely glued to the optical fiber tips. The dura was carefully removed, the optical fibers with the electrode were slowly inserted into either the BC or the hippocampus. The reference and ground were placed on the screws, which were fixed above the cerebellum. After implantation, the fibers with the electrode were glued to the skull for acute terminal experiments.

The light path was built based on a previous report (Fig. 1a)^{21 (link),22 (link)}. The light source comes from a 488 nm laser (MBL-III, CNI). Light beams were first reflected through a dichroic mirror (F48-487, reflection 471–491 nm, >94%, transmission 500–1200 nm, >93%, AHF). Then, by using an objective lens fixed on the fiber launch (MBT613D/M, Thorlabs), the light beam was focused on the optical fiber (FT200-EMT, NA = 0.39, 200 μm, Thorlabs). The laser intensity was measured at the optical fiber tip (5 µW for neuronal calcium recording) by an optical power meter (PM20A, Thorlabs). The same optical fiber guided the emitted fluorescent signal back to the light path. The light beam was successively passed through a dichroic mirror and an optical filter (F37-516, 500–550 nm bandpass, AHF). By using a tube lens (AC254-030-A1-ML, Thorlabs), the GCaMP6-mediated fluorescent signal was coupled to a Peltier-cooled SiPM with a transimpedance preamplifier (MiniSM-10035-X08, SensL). Before being recorded by the analog input module of the Biopac 150 system, the signal from the photomultiplier was amplified by a voltage amplifier (DHPVA-100, Femto).

Chlamydomonas reinhardtii wild type strain CC125 and mutant CC2905 (which lacks flagella) were grown axenically at 24 °C in Tris-Acetate-Phosphate medium (TAP)³⁴ under fluorescent light illumination (OSRAM Fluora, 100 μmol/m²s PAR) following a 14 h/10 h light/dark diurnal cycle. Exponentially growing cells at ~2 × 10⁶ cells/ml were resuspended in fresh TAP at the required concentration, loaded in the 7 mm diameter circular observation chamber cored out of a 1 mm thick agar pad sandwiched between coverslips. A CCD camera (Pike, AVT) hosted on a continuously focusable objective (InfiniVar CFM-2S, Infinity USA) recorded at 12.2 fps the phototactic motility of cells within the horizontal sample, visualised through darkfield illumination at 635 nm (FLDR-i70A-R24, Falcon Lighting). Actinic light was provided by a 470 nm LED (Thorlabs M470L2) through a 200 μm-diameter multimode optical fibre (FT200EMT, Thorlabs). Approximation of the fibre output I(x) by a Gaussian (σ_I = 667 μm, peak intensity 260 μmol/m²s) is excellent and will be used throughout the paper. An inverted microscope (TE2000-U, Nikon) fitted with a 10× Plan Apo objective (NA 0.45) and a EMCCD (Evolve, Photometrics) was used to record the chlorophyll fluorescence of CC2905, excited by the epiport-coupled blue LED.

Before pre-training for skilled reaching, rats were anesthetized with isoflurane (5% induction and 2–3% maintenance) and bilaterally injected in the SNc (M-L ±1.8 mm; A-P −5.2 mm, –6.2 mm; D-V –7.0 mm, −8.0 mm) with AAV-EF1α-DIO-hChR2(H134R)-EFYP, AAV-EF1α-DIO-eArch3.0-EYFP, or AAV-EF1α-DIO-EYFP (UNC vector core). 1 μl of virus (titer: 3.4–4.2 × 10¹² vg/ml) was injected per site (4 µl total per hemisphere) at a rate of 0.1 μl/min. After reaching stable performance on the skilled reaching task, optical fibers (multimode 200 μm core, 0.39 NA, Thor Labs FT200EMT) embedded in stainless steel ferrules (2.5 mm outer diameter, 230 μm bore size, Thor Labs #SF230-10) were implanted above SNc contralateral to the rat’s preferred reaching paw (M-L ±2.4 mm, A-P −5.3 mm, D-V −7.0 mm). Optical fibers were calibrated before implantation to determine optical power at the fiber tip as a function of laser output power, which was continuously monitored during experiments by ‘picking off’ 10% of the laser output with a beamsplitter. Rats recovered for at least 7 days after surgical procedures before beginning behavioral training or testing.

Optical fiber was made as previously reported⁸¹ (link) A 0.39 NA, Ø200 μm core multimode optical fibers (FT200EMT, Thorlabs) were assembled with a stainless-steel ferrule terminals (SF230, Thorlabs) and then polished on one side; exposed the silica core on 1 cm, removed TECS cladding and shaped with hydrofluoric acid on the another side. Only optoprobes with ∼9 mW maximal current output power at the tip were used, as confirmed by a photodiode power sensor (S130C, Thorlabs) and a power meter (PM200, Thorlabs). After that, each optical fiber was glued to a single tungsten wire by a UV-curing optical adhesive (NOA61, Thorlabs), the tip of wire was 0.5 mm longer than the tip of optical fiber.