We have developed an open-source software called BIRDS (bi-channel image registration and deep learning segmentation) for the mapping and analysis of 3D microscopy data and applied this to the mouse brain. The BIRDS pipeline includes image pre-processing, bi-channel registration, automatic annotation, creation of a 3D digital frame, high-resolution visualization, and expandable quantitative analysis. This new bi-channel registration algorithm is adaptive to various types of whole-brain data from different microscopy platforms and shows dramatically improved registration accuracy. Additionally, as this platform combines registration with neural networks, its improved function relative to other platforms lies in the fact that the registration procedure can readily provide training data for network construction, while the trained neural network can efficiently segment incomplete/defective brain data that is otherwise difficult to register. Our software is thus optimized to enable either minute-timescale registration-based segmentation of cross-modality, whole-brain datasets or real-time inference-based image segmentation of various brain regions of interest. Jobs can be easily submitted and implemented via a Fiji plugin that can be adapted to most computing environments.

Acquisition of STPT image dataset
Brain 1 and 2 were obtained with STPT and each dataset encompassed ~180 Gigavoxels, e.g., 11980×7540×1075 in Dataset 1, with a voxel size of 1×1×10 μm3. The procedure of sample preparation and imaging acquisition were described in the paper. Briefly, the adult C57BL/6 mouse was anesthetized and craniotomy was performed on top of the right visual cortex. Individuals neuronal axons were labeled with pCAG-eGFP by two-photon microscopy guided single-cell electroporation and the brain was fixed by caridoperfusion of 4% PFA 8 days later. Striatum-projecting neurons were labeled by stereotactically injecting PRV-cre into the right striatum of tdTomato reporter mice (Ai14, JAX) and the brain was fixed cardioperfusion 30 days later. The brains were embedded in 5% oxidized agarose and imaged with a commercial STPT (TissueVision, USA) excited at 940 nm. Coronally, the brain was optically scanned every 10 μm at 1 μm/pixel without averaging and physically sectioned every 50 μm. The power of excitation laser was adjusted to compensate the depth of optical sections.

There are two group of  1×1×10 μm high-resolution mouse brain datasets using serial two-photon tomography (STPT), and there are coronal sections.