A wireless miniScope for deep brain imaging in freely moving mice.

New Method: Here we developed a miniature wireless fluorescence microscope (miniScope) that allows recording of brain neural activities at single cell resolution. The wireless miniScope has onboard field-programmable gate array (FPGA) and Micro SD Card storage, and is powered by a battery backpack.

Comparison with Existing Methods: Existing miniaturized microscope systems have connecting cables between the microscope sensor and the data acquisition system, consequently limiting the recording to one animal at a time. The wireless miniScope allows simultaneous recording of multiple mice in a group, and could also be applied to freely behaving small primates in the future.

2.

Methods 2.1 Microscope design

The wireless miniScope imaging system comprised a 3D printed miniature epifluorescence microscope body ( Barbera et al., 2016 ), an image sensor board hosting the CMOS image sensor (MT9V022, ON Semiconductor), a daughter board hosting the field-programmable gate array (FPGA, AGL400V5, Microsemi Corporation), a LED driver, a Micro SD Card and a communication interface with host computer ( Fig. 1a and 1b ). Frames from the image sensor (10 fps, 200 × 200 pixels, corresponding to a 500 μm × 500 μm field of view) were saved directly to the Micro SD Card as raw data, including a header with timestamp and frame number to facilitate image extraction. The daughter board also hosted an IR sensor that was used as a trigger for initiation of recording and for synchronization with other wireless imaging microscopes. Two operation modes were available: a streaming mode and a recording mode. In the streaming mode, the system was tethered and streamed live images to the host computer through a custom data acquisition board and a USB cable. The streaming mode was useful for positioning of the microscope and focus adjustments. The recording mode was used during the experiments, and the wireless system stores all frames into the Micro SD Card. The entire system was tested using a single 3.8 g rechargeable LiPo battery (180 mAh), which could provide 40 minutes of continuous recording. A brief summary of the miniScope’s main optical parameters was listed below: Number Name Weight Specs and vendor 1 Miniscope Housing 1.7g Custom design, 3D printed with SLArmor Nickel-NanoTool, Protolabs 2 LED XLamp XP-E blue LED, Cree 3 Collimating lens 4-mm diameter, 2.73-mm focal length aspherical lens, #83–605, Edmund optics 4 Excitation filter 3 mm × 3 mm×1mm, ET470/40×, Chroma Technology 5 Dichroic mirror 5 mm × 5 mm×1mm, FF495-Di02, Semrock 6 Emission filter 3 mm × 3 mm×1mm, ET525/50m, Chroma Technology 7 Objective lens 4-mm diameter, 2.73-mm focal length aspherical lens, #83–605, Edmund optics 8 Imaging lens 4-mm diameter 6-mm focal length achromatic doublet lens, #63–690, Edmund optics 9 Imaging sensor PCB including SD card 2.2g Custom design 10 Polymer Lithium Ion Battery See below See below Total weight of wireless miniScope without battery: 3.9 grams Dimension of wireless miniScope: 16 mm × 18 mm × 30 mm Below are three different models of battery that we tested with wireless miniScope. Type Model Size Weight Expected runtime 100 mAh GM261534 2.5mm × 15mm × 34 mm 2.2 g 16 min 180 mAh GM052025 5mm × 20mm × 25mm 3.8 g (Used in exp.) 28 min 240 mAh GM502030 -PCB 5mm × 20mm × 30mm 5.0 g 38 min All the design files and codes can be found at the following GitHub link: https://github.com/giovannibarbera/wireless_v1.0 2.2 Animal surgery All surgical procedures were performed in accordance with the guidelines of Institutional Animal Care and Use Committee, the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health. Transgenic mice (minimum 30 g of body weight) expressing Cre recombinase under the control of the dopamine D1 receptor promoter (D1-Cre, FK150 line, C57BL/6J congenic, Gensat, RRID: MMRRC_036916-UCD) or dopamine D2 receptor promoter (D2-Cre, ER44 line, C57BL/6J congenic, Gensat, RRID: MMRRC_032108-UCD) were used in the experiment. Adeno-Associated Virus (AAV) expressing calcium indicator Gcamp6s, AAV1.CAG.Flex.GCaMP6s.WPRE.SV40 (University of Pennsylvania Vector Core), was injected into the dorsal striatum of mice using the stereotactic coordinates (A/P: −0.93 mm, M/L: +1.8 mm, D/V: −3.46 mm, with 30° angle shift to caudate). Ten days after AAV injection, a 1mm diameter GRIN lens was implanted into the mouse brain right above the dorsal striatum under ketamine/xylazine anesthesia (ketamine:100 mg/kg, xylazine:15 mg/kg), as previously described ( Barbera et al., 2016 ). Three weeks after GRIN lens implantation, miniScope base was mounted on the mouse head. Data acquisition was performed 4 weeks after GRIN lens implantation.

Data acquisition and analysis

The open field behavior tests were performed during the light cycle. Mouse were under light isoflurane anesthesia when the miniScope was mounted on the mouse head. Mouse was then allowed to recover from anesthesia in home cage for 30 minutes. We conducted 3 sessions (5 min per session) open field test for the two mice simultaneously in a 34 cm × 40 cm × 20 cm (length × width × height) chamber with 5 min interval between imaging sessions. The GCaMP6s fluorescent signal and the video of mice behavior were recorded simultaneously. The calcium images and behavior video were processed and analyzed using custom scripts in MATLAB.