Mf525 39

A fiber photometry system (ThinkerTech, Nanjing, China) was used to record either GRAB_DA signals from genetically identified neurons^{46 (link)} or the SNc-projecting SC neurons that expressed GCaMP7. To induce fluorescence signals, a laser beam from a laser tube (488 nm) was reflected by a dichroic mirror, focused by a ×10 lens (NA 0.3) and coupled to an optical commutator. A 2-m optical fiber (230 μm in diameter, NA 0.37) guided the light between the commutator and implanted optical fiber. To minimize photo bleaching, the power intensity at the fiber tip was adjusted to 0.02 mW. The fluorescence of GRAB-DA or GCaMP was band-pass filtered (MF525-39, Thorlabs) and collected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) was used to convert the photomultiplier tube current output to voltage signals, which were further filtered through a low-pass filter (40 Hz cut-off; Brownlee 440). The analog voltage signals digitalized at 100 Hz were recorded by a Power 1401 digitizer and Spike2 software (CED, Cambridge, UK).

To demonstrate the ultrasonic virtual relay imaging in transmission mode, we built a custom experimental setup consisting of a top microscope assembly and the piezoelectric transducer immersed in a water tank. The cylindrical transducer surrounds the target medium and launches standing pressure waves into it. The top microscope is used in transmission mode for imaging the relayed image through the ultrasonically sculpted virtual GRIN lens. The top microscope is composed of a zoom imaging lens (VZM 600i, Edmund Optics, Inc., USA), a CMOS camera (BFS-U3-51S5C-BD2, FLIR Integrated Imaging Solutions, Inc., Richmond, British Columbia, Canada), and a fluorescent emission filter (MF525-39, Thorlabs, Inc., Newton, New Jersey, USA). A clear glass optical window (WG11050-A, Thorlabs, Inc., Newton, New Jersey, USA) is immersed in the medium and acts as the interface between the microscope and the imaged medium. We designed a fluorescent target object consisting of a negative transparency mask (CAD/Art Services, Inc., Bandon, Oregon, USA) of the word “CMU” (overall size: 374 × 110 µm, minimum feature size: 22 µm) overlaid on a layer of solid agar (A5306, Sigma–Aldrich, Inc., St. Louis, Missouri, USA) homogeneously mixed with Fluorescein dye. the sample is illuminated by a blue laser (λ_light = 473 nm) from the bottom.

The setup schematically shown in Figure 6A was used to measure the emission diagrams of flat-cleaved optical fibers in tissue. Fibers were inserted into a 300 μm thick fluorescently stained brain slice, 473 nm light was coupled into the fiber through an objective lens (Olympus Plan N 40x), and the primary dichroic of the 2P microscope was removed from the system. Light emission from the tissue was collected through the microscope objective, descanned by the scan-head, focused into a pinhole (Thorlabs MPH-16), and detected by a PMT (Hamamatsu H7422P-40, the “pinhole PMT”). A BPF (Thorlabs MF525/39) isolated the wavelength band of interest. The pinhole size was set to 100 μm.

Imaging of the mounted larvae was performed using a custom-made upright fluorescence microscope. We imaged either the entire zebrafish larva (using EC Plan-NEOFLUAR 2.5x/0.085 M27, Carl Zeiss Microscopy) or only the encephalic region (with EC Plan-NEOFLUAR 5x/0.16 M27, Carl Zeiss Microscopy). Fluorescence was excited in wide-field configuration using an LED with emission centred at 470 nm (M470L3, Thorlabs), followed by an excitation band-pass filter (469/35 nm, FF01–469/35, Semrock). The epifluorescence detection was implemented with a dichroic mirror (DC FF495-DI02, Semrock) and an emission band-pass filter (525/39 nm, MF525-39, Thorlabs). The fluorescence signal was recorded with a sCMOS camera (OrcaFLASH 4.0, Hamamatsu Photonics) with 16-bit dynamic range.
To perform high-throughput measurements the excitation LED was equipped with a diffuser to produce an even illumination over an area of about 50 cm², covering all the multi-well plate. In order to image such a large area, a wide-angle lens (MVL8M23, Thorlabs) with 8 mm focal length was mounted directly on the camera.

Fiber photometry was performed using a previously described system (Zhou et al., 2017 (link); Sun et al., 2019 ; Wang et al., 2019 (link), 2020 (link); Wu et al., 2020 (link)). To record fluorescent signals, the beam from a 488 nm laser (OBIS 488LS, Coherent) was reflected by a dichroic mirror (MD498, Thorlabs), focused by an objective lens (10×, NA: 0.3; Olympus), and then coupled to an optical commutator (Doric Lenses). An optical fiber (200 mm o.d., NA: 0.37, 1.5 m long) coupled the light between the commutator and the implanted optical fiber. GCaMP6s fluorescence was collected by the same fiber and objective, then bandpass-filtered (MF525–39, Thorlabs) and detected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) converted the photomultiplier tube current output to a voltage signal, which was further filtered through a low-pass filter (35 Hz cut-off; Brownlee, 440). The analog voltage signals were digitized at 500 Hz and recorded by fiber photometry software (Thinkerbiotech, Nanjing, China) for the duration of each behavioral session.

For fluorescence Ca²⁺ recordings, light from a 473-nm LED was reflected by a dichroic mirror (MD498, Thorlabs). The emission signals collected through the implanted optical fiber in V1 were filtered by a bandpass filter (MF525-39, Thorlabs) and detected by a photomultiplier tube (PMT, R3896, Hamamatsu). The light at the tip of the optical fiber was adjusted to 10–30 μW to minimize bleaching. An amplifier converted the output of the PMT to voltage signals, which were digitized using a data acquisition card (USB6009, National Instrument) at 200 Hz with custom-written programs.

Mice were housed individually for two weeks after AAV injection and optical fiber (200 μm diameter, 0.33 numerical aperture (NA), AniLab) implantation. Fluorescence signals were output to an amplifier (C7319, Hamamatsu) with a 0.01 s moving average filter (MF525-39, Thorlabs). The signals were then digitized (Power 1401) and recorded (Spike2 Software) at a 300 Hz sampling rate. The laser power at the tip of optical fiber was 15 μW for RtTg. For monitoring the neuronal activity of RtTg during startle, we recorded white noise stimuli as well as airpuffs induced startle reflexes and Ca²⁺ transients or EGFP signals simultaneously. The raw data was then analyzed using MATLAB. Ca²⁺ fluorescence changes (ΔF/F) during startle reflex and conditioned fear were aligned to individual bouts and were calculated as (F−F₀)/F₀, where F₀ was the average fluorescence signals from 1 s preceding stimuli (95 dB stimuli or the CS) to stimuli onset. (ΔF/F) values were presented as heatmaps or plotted with shaded area indicating s.e.m.