Fluoview fvmpe rs

A two-photon imaging experiment was performed before and after conditioning. The mice were anesthetized with isoflurane, and the head was fixed in place under a two-photon microscope using a titanium head bar. V1 calcium signal was recorded using Olympus FluoView FVMPE-RS upright two-photon laser-scanning system (Olympus, Tokyo, Japan). The stimulation light was delivered by an LED light source. The LED was positioned 8 cm from the left eye of the mouse, with a light intensity of approximately 5 μW/mm². Blue light and green light were presented six times each in a randomized order, each for a duration of 1 s, followed by a 10-s inter-trial interval. The entire imaging device was enclosed by a blackout fabric to prevent light leakage into the imaging photomultiplier (PMT). The calcium indicator (GCaMP6s) was excited using a laser at a wavelength of 920 nm. Images were acquired at 30 Hz. During the recording, mice were anesthetized with isoflurane (2% for induction and 0.5–1% for maintenance) and placed on a heating pad to maintain body temperature.
For the second recording on the day after conditioning, the location of such target region was attempted to align with the previous area for each mouse. The procedure of the second recording was identical to the first recording.

After the 5-day conditioning, the head-fixed awake mice were immobilized in homemade tubes to prevent wild free movement, and imaged under the Olympus FluoView FVMPE-RS upright multiphoton laser-scanning system with an Olympus XL Plan N 25×/1.05 WMP ∞/0–0.23/FN/18 water immersion objective lens (Japan). Both GCamp6s and SR101 were excited at 920nm with an INSIGHT X3-OL laser (Spectra-Physics, USA). Laser power was kept below 30 mW to avoid phototoxicity. Emitted fluorescence was detected through 495 to 540 nm and 575 to 645 nm bandpass filters. For 3D astrocyte calcium signal imaging, the calcium transients were recorded in cortical layer 2/3 100 to 300 µm below the pial surface. Appropriate cells were selected for imaging based on SR101 by adjusting zoom factor and z-axis. Image volume was scanned at 512×512 pixels for 20 to 30 layers in 2 μm z-axis step, resulting 0.121 to 0.144 µm lateral resolution and 2 µm axial resolution. Each plane was scanned twice and averaged for noise cancelling consideration. The volumetric sampling rate was 0.25 to 0.33 Hz. Typically, acquisitions of 2 to 3 minutes were made for each field of view. Since all mice received aseptic chronic cranial window surgery, imaging multiple trials in weeks was feasible.

Mice were perfused with 4% PFA at 45 days after implantation of hG008 cells. For preparation of PASSIVE CLARITY-processed mouse brains, brain tissues were fixed with 4% PFA at 4 °C overnight and then incubated in hydrogel solution (4% PFA, 4% acrylamide, 0.25% VA044 in PBS) at 4 °C for 3 days [24 (link)]. Brain tissues were degassed and polymerized in the same hydrogel solution at 37 °C for 3 h. Four-mm thick coronal sections, except cerebellum and olfactory bulb, were cut. Hydrogel-embedded tissue sections were washed with clearing solution (200 mM sodium borate buffer (pH 8.5) containing 4% SDS) at 37 °C with shaking for 2 h. Sections were then incubated in fresh clearing solution at 48 °C for 5 days. Imaging was performed by multi-photon microscopy (FLUOVIEW FVMPE-RS; Olympus) and 3D images were reconstructed using FV31S-SW software with a maximum intensity projection algorithm (Olympus).