Anan Li

Continuously tracing brain-wide long-distance axonal projections in mice at a one-micron voxel resolution.

Revealing neural circuit mechanisms is critical for understanding brain functions. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge. Here, we describe a whole-brain approach to systematically obtaining continuous neuronal pathways in a fluorescent protein transgenic mouse at a one-micron voxel resolution. This goal is achieved by combining a novel resin-embedding method for maintaining fluorescence, an automated fluorescence micro-optical sectioning tomography system for long-term stable imaging, and a digital reconstruction-registration-annotation pipeline for tracing the axonal pathways in the mouse brain. With the unprecedented ability to image a whole mouse brain at a one-micron voxel resolution, the long-distance pathways were traced minutely and without interruption for the first time. With advancing labeling techniques, our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.

3D BrainCV: Simultaneous visualization and analysis of cells and capillaries in a whole mouse brain with one-micron voxel resolution

Systematic cellular and vascular configurations are essential for understanding fundamental brain anatomy and metabolism. We demonstrated a 3D brainwide cellular and vascular (called 3D BrainCV) visualization and quantitative protocol for a whole mouse brain. We developed a modified Nissl staining method that quickly labeled the cells and blood vessels simultaneously in an entire mouse brain. Terabytes 3D datasets of the whole mouse brains, with unprecedented details of both individual cells and blood vessels, including capillaries, were simultaneously imaged at 1-μm voxel resolution using micro-optical sectioning tomography (MOST). For quantitative analysis, we proposed an automatic image-processing pipeline to perform brainwide vectorization and analysis of cells and blood vessels. Six representative brain regions from the cortex to the deep, including FrA, M1, PMBSF, V1, striatum, and amygdala, and six parameters, including cell number density, vascular length density, fractional vascular volume, distance from the cells to the nearest microvessel, microvascular length density, and fractional microvascular volume, had been quantitatively analyzed. The results showed that the proximity of cells to blood vessels was linearly correlated with vascular length density, rather than the cell number density. The 3D BrainCV made overall snapshots of the detailed picture of the whole brain architecture, which could be beneficial for the state comparison of the developing and diseased brain.

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding.

Visualization of brain circuits using two-photon fluorescence micro-optical sectioning tomography

Neural circuits are fundamental for brain functions. However, obtaining long range continuous projections of neurons in the entire brain is still challenging. Here a two-photon fluorescence micro-optical sectioning tomography (2p-fMOST) method is developed for high-throughput, high-resolution visualization of the brain circuits. Two-photon imaging technology is used to obtain high resolution, and acoustical optical deflector (AOD), an inertia-free beam scanner is used to realize fast and prolonged stable imaging. The combination of these techniques with imaging and then sectioning method of a plastic-embedded mouse brain facilitated the acquisition of a three-dimensional data set of a fluorescent mouse brain with a resolution adequate to resolve the spines. In addition, the brain circuit tracing ability is showed by several neurons projecting across different brain regions. Besides brain imaging, 2p-fMOST could be used in many studies that requires sub-micro resolution or micro resolution imaging of a large sample.

High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level.

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei.

Fast optical sectioning obtained by structured illumination microscopy using a digital mirror device

Abstract. High-throughput optical imaging is critical to obtain large-scale neural connectivity information of brain in neuroscience. Using a digital mirror device and a scientific complementary metal-oxide semiconductor camera, we report a significant speed improvement of structured illumination microscopy (SIM), which produces a maximum SIM net frame rate of 133 Hz. We perform three-dimensional (3-D) imaging of mouse brain slices at diffraction-limited resolution and demonstrate the fast 3-D imaging capability to a large sample with an imaging rate of 6.9×107  pixel/s of our system, an order of magnitude faster than previously reported.

Rapid Reconstruction of 3D Neuronal Morphology from Light Microscopy Images with Augmented Rayburst Sampling

Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.

Modified Golgi-Cox method for micrometer scale sectioning of the whole mouse brain

One of the major challenges of connectomics is obtaining a physical map of the neurons that comprise a circuit and the sites within the whole mouse brain. However, there is no report that addresses the preparation of whole mouse brain tissue for microsectioning. In this paper, such tissue is prepared by a modified Golgi-Cox method in which the staining time is prolonged to half a year, the darkening solution is changed to 1% LiOH, and the brain is embedded in resin. Projections of several coronal sections are reconstructed by serial 1-μm sectioning and simultaneous imaging of the specimen. This approach ensures that the stained neurons are present throughout the whole mouse brain from superficial to deep layers and that the neuronal soma and traces of the processes can be distinguished in local magnification.

A high-resolution anatomical rat atlas.

This paper reports the availability of a high‐resolution atlas of the adult rat. The atlas is composed of 9475 cryosectional images captured in 4600 × 2580 × 24‐bit TIFF format, constructed using serial cryosection‐milling techniques. Cryosection images were segmented, labelled and reconstructed into three‐dimensional (3D) computerized models. These images, 3D models, technical details, relevant software and further information are available at our website, http://vchibp.vicp.net/vch/mice/.

NeuroGPS-Tree: automatic reconstruction of large-scale neuronal populations with dense neurites

The reconstruction of neuronal populations, a key step in understanding neural circuits, remains a challenge in the presence of densely packed neurites. Here we achieved automatic reconstruction of neuronal populations by partially mimicking human strategies to separate individual neurons. For populations not resolvable by other methods, we obtained recall and precision rates of approximately 80%. We also demonstrate the reconstruction of 960 neurons within 3 h.