Detailed protocols for whole brain imaging using LSM
Whole brain and hemisphere images were acquired with the Ultramicroscope II (Lavision Biotec). Samples were mounted to a custom 3D printed holder using RapidClear Mounting Gel (Sunjin lab). For whole brains (TRAP brains), the brain was mounted with the ventral side on top. For hemisphere, the cut surface (midline of the brain) was placed in touch with the holder and with the most lateral part on the top. Samples were securely mounted to the holder after mounting gel solidified (~5 minute at 4°C). Mounted samples were imaged inside an imaging chamber filled with 150ml of Rapidclear (reusable by periodical filtering). Samples were left in imaging chamber for 20-40min before imaging to allow the equilibrium of imaging liquid. Brains were imaged using a 2x/0.5NA objective at 0.6x zoom (whole brain, TRAP) or 0.8x zoom (hemisphere, CAPTURE). Multi-color imaging was enabled by applying filters setting to a supercontinuum white laser (NKT photonics). Samples were with two light sheets (NA=0.144) illuminating from both sides of the sample. Z-step was set to 5.16µm (at 0.6x zoom) or 4µm (at 0.8x zoom). Five horizontal focal points were set to each imaging plane for creating a homogeneous field of view.
All raw images were acquired as 16-bit TIFF files. The raw images were further processed by blind 3D deconvolution using AutoQuantX3 (Media Cybernetics). The parameters of the deconvolution were based on published methods using a similar light sheet microscope with a few modifications50. Briefly, the modality was set to “Multi-photon fluorescence” 3D-blind deconvolution with 20 iterations. Noise was set to zero and using “unfiltered image” as “initial guess”. In the expert settings, montage was turned on XY but off on Z, with 30-pixel overlap in XYZ. Other settings such as NA, spacing and magnitude were set based on the actual experiments. Either deconvolved or raw images can be 3D-rendered and visualized using Imaris (Bitplane, v8.1.2), for taking snapshot images and making movies.