High-definition imaging using line-illumination modulation microscopy.

The microscopic visualization of large-scale three-dimensional (3D) samples by optical microscopy requires overcoming challenges in imaging quality and speed and in big data acquisition and management. We report a line-illumination modulation (LiMo) technique for imaging thick tissues with high throughput and low background. Combining LiMo with thin tissue sectioning, we further develop a high-definition fluorescent micro-optical sectioning tomography (HD-fMOST) method that features an average signal-to-noise ratio of 110, leading to substantial improvement in neuronal morphology reconstruction. We achieve a >30-fold lossless data compression at a voxel resolution of 0.32 × 0.32 × 1.00 µm3, enabling online data storage to a USB drive or in the cloud, and high-precision (95% accuracy) brain-wide 3D cell counting in real time. These results highlight the potential of HD-fMOST to facilitate large-scale acquisition and analysis of whole-brain high-resolution datasets.

Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

BACKGROUND: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. METHODS: To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. RESULTS: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. CONCLUSIONS: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

Skeleton optimization of neuronal morphology based on three-dimensional shape restrictions.

BACKGROUND: Neurons are the basic structural unit of the brain, and their morphology is a key determinant of their classification. The morphology of a neuronal circuit is a fundamental component in neuron modeling. Recently, single-neuron morphologies of the whole brain have been used in many studies. The correctness and completeness of semimanually traced neuronal morphology are credible. However, there are some inaccuracies in semimanual tracing results. The distance between consecutive nodes marked by humans is very long, spanning multiple voxels. On the other hand, the nodes are marked around the centerline of the neuronal fiber, not on the centerline. Although these inaccuracies do not seriously affect the projection patterns that these studies focus on, they reduce the accuracy of the traced neuronal skeletons. These small inaccuracies will introduce deviations into subsequent studies that are based on neuronal morphology files. RESULTS: We propose a neuronal digital skeleton optimization method to evaluate and make fine adjustments to a digital skeleton after neuron tracing. Provided that the neuronal fiber shape is smooth and continuous, we describe its physical properties according to two shape restrictions. One restriction is designed based on the grayscale image, and the other is designed based on geometry. These two restrictions are designed to finely adjust the digital skeleton points to the neuronal fiber centerline. With this method, we design the three-dimensional shape restriction workflow of neuronal skeleton adjustment computation. The performance of the proposed method has been quantitatively evaluated using synthetic and real neuronal image data. The results show that our method can reduce the difference between the traced neuronal skeleton and the centerline of the neuronal fiber. Furthermore, morphology metrics such as the neuronal fiber length and radius become more precise. CONCLUSIONS: This method can improve the accuracy of a neuronal digital skeleton based on traced results. The greater the accuracy of the digital skeletons that are acquired, the more precise the neuronal morphologies that are analyzed will be.

Multicolor high-resolution whole-brain imaging for acquiring and comparing the brain-wide distributions of type-specific and projection-specific neurons with anatomical annotation in the same brain.

Visualizing the relationships and interactions among different biological components in the whole brain is crucial to our understanding of brain structures and functions. However, an automatic multicolor whole-brain imaging technique is still lacking. Here, we developed a multicolor wide-field large-volume tomography (multicolor WVT) to simultaneously acquire fluorescent signals in blue, green, and red channels in the whole brain. To facilitate the segmentation of brain regions and anatomical annotation, we used 4', 6-diamidino-2-phenylindole (DAPI) to provide cytoarchitecture through real-time counterstaining. We optimized the imaging planes and modes of three channels to overcome the axial chromatic aberration of the illumination path and avoid the crosstalk from DAPI to the green channel without the modification of system configuration. We also developed an automatic contour recognition algorithm based on DAPI-staining cytoarchitecture to shorten data acquisition time and reduce data redundancy. To demonstrate the potential of our system in deciphering the relationship of the multiple components of neural circuits, we acquired and quantified the brain-wide distributions of cholinergic neurons and input of ventral Caudoputamen (CP) with the anatomical annotation in the same brain. We further identified the cholinergic type of upstream neurons projecting to CP through the triple-color collocated analysis and quantified its proportions in the two brain-wide distributions. Both accounted for 0.22%, implying CP might be modulated by non-cholinergic neurons. Our method provides a new research tool for studying the different biological components in the same organ and potentially facilitates the understanding of the processing mechanism of neural circuits and other biological activities.

Precision vibratome for high-speed ultrathin biotissue cutting and organ-wide imaging.

Cutting tissues into ultrathin slices is highly desired in sectioning-based organ-wide imaging. However, it is difficult to perform tissue cutting at a high speed with excellent quality. Here, we design a precision vibratome based on a paired double parallelogram flexure, which enables a vibrating blade to move strictly along a straight line. Meanwhile, we develop a high-speed cutting method that does not compromise cutting quality, which the vibratome operated at a high frequency mode. The characterized parasitic motion errors of a 180-Hz vibratome were less than 300 nm. It achieved a cutting speed six times that of an 85-Hz vibratome with acceptable quality. The capacity of the vibratome was investigated by organ-wide imaging, and the results revealed that it can be adapted in different tissues, such as the mouse brain and liver. This new vibratome shows great potential in speeding up organ-wide imaging applications especially for large volume biotissues.

A Whole-Brain Connectivity Map of VTA and SNc Glutamatergic and GABAergic Neurons in Mice.

The glutamatergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) mediated diverse brain functions. However, their whole-brain neural connectivity has not been comprehensively mapped. Here we used the virus tracers to characterize the whole-brain inputs and outputs of glutamatergic and GABAergic neurons in VTA and SNc. We found that these neurons received similar inputs from upstream brain regions, but some quantitative differences were also observed. Neocortex and dorsal striatum provided a greater share of input to VTA glutamatergic neurons. Periaqueductal gray and lateral hypothalamic area preferentially innervated VTA GABAergic neurons. Specifically, superior colliculus provided the largest input to SNc glutamatergic neurons. Compared to input patterns, the output patterns of glutamatergic and GABAergic neurons in the VTA and SNc showed significant preference to different brain regions. Our results laid the anatomical foundation for understanding the functions of cell-type-specific neurons in VTA and SNc.

Ventral Hippocampal-Prefrontal Interaction Affects Social Behavior via Parvalbumin Positive Neurons in the Medial Prefrontal Cortex.

Ventral hippocampus (vHIP) and medial prefrontal cortex (mPFC) are both critical regions for social behaviors. However, how their interactions affect social behavior is not well understood. By viral tracing, optogenetics, chemogenetics, and fiber photometry, we demonstrated that inhibition of vHIP or direct projections from vHIP to mPFC impaired social memory expression. Via rabies retrograde tracing, we found that all three major GABAergic neurons in mPFC received direct inputs from vHIP. Activation of parvalbumin positive (PV+) neurons in mPFC but not somatostatin positive (SST+) neurons can rescue the social memory impairment caused by vHIP inhibition. Furthermore, fiber photometry results demonstrated that social behaviors preferentially recruited PV+ neurons and inhibition of hippocampal neurons disrupted the activity of PV+ neurons during social interactions. These results revealed a new mechanism of how vHIP and mPFC regulate social behavior in complementarity with the existing neural circuitry mechanism.

Whole-Brain Three-Dimensional Profiling Reveals Brain Region Specific Axon Vulnerability in 5xFAD Mouse Model.

Axonopathy is a pathological feature observed in both Alzheimer's disease (AD) patients and animal models. However, identifying the temporal and regional progression of axonopathy during AD development remains elusive. Using the fluorescence micro-optical sectioning tomography system, we acquired whole-brain datasets in the early stage of 5xFAD/Thy1-GFP-M mice. We reported that among GFP labeled axons, GFP-positive axonopathy first formed in the lateral septal nucleus, subiculum, and medial mammillary nucleus. The axonopathy further increased in most brain regions during aging. However, most of the axonopathic varicosities disappeared significantly in the medial mammillary nucleus after 8 weeks old. Continuous three-dimensional datasets showed that axonopathy in the medial mammillary nucleus was mainly located on axons from hippocampal GFP-positive neurons. Using the rabies viral tracer in combination with immunohistochemistry, we found that axons in the medial mammillary nucleus from the subiculum were susceptible to lesions that prior to the occurrence of behavioral disorders. In conclusion, we created an early-stage spatiotemporal map of axonopathy in 5xFAD/Thy1-GFP-M mice and identified specific neural circuits which are vulnerable to axon lesions in an AD mouse model. These findings underline the importance of early interventions for AD, and may contribute to the understanding of its progression and its early symptom treatment.