Combining expansion and the lattice light sheet Optical and electron microscopy have made tremendous inroads into understanding the complexity of the brain. Gao et al. introduce an approach for high-resolution tracing of neurons, their subassemblies, and their molecular constituents over large volumes. They applied their method, which combines expansion microscopy and lattice light-sheet microscopy, to the mouse cortical column and the entire Drosophila brain. The approach can be performed at speeds that should enable high-throughput comparative studies of neural development, circuit stereotypy, and structural correlations to neural activity or behavior. Science, this issue p. eaau8302 Combined expansion and lattice light-sheet microscopy enables high-speed nanoscale molecular imaging of neural circuits. INTRODUCTION Neural circuits across the brain are composed of structures spanning seven orders of magnitude in size that are assembled from thousands of distinct protein types. Electron microscopy has imaged densely labeled brain tissue at nanometer-level resolution over near-millimeter-level dimensions but lacks the contrast to distinguish specific proteins and the speed to readily image multiple specimens. Conversely, confocal fluorescence microscopy offers molecular contrast but has insufficient resolution for dense neural tracing or the precise localization of specific molecular players within submicrometer-sized structures. Last, superresolution fluorescence microscopy bleaches fluorophores too quickly for large-volume imaging and also lacks the speed for effective brain-wide or cortex-wide imaging of multiple specimens. RATIONALE We combined two imaging technologies to address these issues. Expansion microscopy (ExM) creates an expanded, optically clear phantom of a fluorescent specimen that retains its original relative distribution of fluorescent tags. Lattice light-sheet microscopy (LLSM) then images this phantom in three dimensions with minimal photobleaching at speeds sufficient to image the entire Drosophila brain or across the width of the mouse cortex in ∼2 to 3 days, with multiple markers at an effective resolution of ∼60 by 60 by 90 nm for 4× expansion. RESULTS We applied expansion/LLSM (ExLLSM) to study a variety of subcellular structures in the brain. In the mouse cortex, we quantified the volume of organelles, measured morphological parameters of ~1500 dendritic spines, determined the variation of distances between pre- and postsynaptic proteins, observed large differences in postsynaptic expression at adjacent pyramidal neurons, and studied both the azimuthal asymmetry and layer-specific longitudinal variation of axonal myelination. In Drosophila, we traced the axonal branches of olfactory projection neurons across one hemisphere and studied the stereotypy of their boutons at the calyx and lateral horn across five animals. We also imaged all dopaminergic neurons (DANs) across the brain of another specimen, visualized DAN morphologies in all major brain regions, and traced a cluster of eight DANs to their termini to determine their respective cell types. In the same specimen, we also determined the number of presynaptic active zones (AZs) across the brain and the local density of all AZs and DAN-associated AZs in each brain region. CONCLUSION With its high speed, nanometric resolution, and ability to leverage genetically targeted, cell type–specific, and protein-specific fluorescence labeling, ExLLSM fills a valuable niche between the high throughput of conventional optical pipelines of neural anatomy and the ultrahigh resolution of corresponding EM pipelines. Assuming the development of fully validated, brain-wide isotropic expansion at 10× or beyond and sufficiently dense labeling, ExLLSM may enable brainwide comparisons of even densely innervated neural circuits across multiple specimens with protein-specific contrast at 25-nm resolution or better. Nanoscale brain-wide optical imaging. ExLLSM images neural structures with molecular contrast over millimeter-scale volumes, including (clockwise from top right) mouse pyramidal neurons and their processes; organelle morphologies in somata; dendritic spines and synaptic proteins across the cortex; stereotypy of projection neuron boutons in Drosophila; projection neurons traced to the central complex; and (center) dopaminergic neurons across the brain, including the ellipsoid body (circular inset). Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

中文翻译：

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具有分子对比度和纳米级分辨率的皮质柱和全脑成像

结合膨胀和晶格光片光学和电子显微镜在理解大脑的复杂性方面取得了巨大进展。高等人。介绍一种对神经元、其子组件及其大体积分子成分进行高分辨率追踪的方法。他们将扩展显微镜和点阵光片显微镜相结合的方法应用于小鼠皮质柱和整个果蝇大脑。该方法的执行速度应能够对神经发育、电路刻板性以及神经活动或行为的结构相关性进行高通量比较研究。科学，本期第 14 页。eaau8302 组合扩展和晶格光片显微镜可实现神经回路的高速纳米级分子成像。简介 大脑的神经回路由大小跨越七个数量级的结构组成，这些结构由数千种不同的蛋白质类型组装而成。电子显微镜可以在近毫米级尺寸上以纳米级分辨率对密集标记的脑组织进行成像，但缺乏区分特定蛋白质的对比度和轻松成像多个样本的速度。相反，共焦荧光显微镜提供分子对比度，但分辨率不足以进行密集神经追踪或亚微米结构内特定分子参与者的精确定位。最后，超分辨率荧光显微镜对于大体积成像而言漂白荧光团的速度太快，并且也缺乏对多个样本进行有效的全脑或皮质范围成像的速度。基本原理我们结合了两种成像技术来解决这些问题。扩展显微镜 (ExM) 创建一个扩展的、光学透明的荧光样本模型，保留其荧光标签的原始相对分布。然后，点阵光片显微镜 (LLSM) 在三个维度上对模型进行成像，光漂白程度最低，速度足以在 2 至 3 天内对整个果蝇大脑或整个小鼠皮层的宽度进行成像，多个标记的有效分辨率为∼60 x 60 x 90 nm，用于 4 倍扩展。结果我们应用扩展/LLSM (ExLLSM) 来研究大脑中的各种亚细胞结构。在小鼠皮层中，我们量化了细胞器的体积，测量了约 1500 个树突棘的形态参数，确定了突触前和突触后蛋白质之间距离的变化，观察到相邻锥体神经元突触后表达的巨大差异，并研究了方位不对称性和轴突髓鞘形成的层特异性纵向变化。在果蝇中，我们追踪了整个半球的嗅觉投射神经元的轴突分支，并研究了五只动物的花萼和侧角处的纽扣的定型性。我们还对另一个样本大脑中的所有多巴胺能神经元 (DAN) 进行了成像，可视化所有主要大脑区域的 DAN 形态，并追踪八个 DAN 的簇至其末端以确定它们各自的细胞类型。在同一样本中，我们还确定了大脑中突触前活动区 (AZ) 的数量以及每个大脑区域中所有 AZ 和 DAN 相关 AZ 的局部密度。结论 凭借其高速、纳米分辨率以及利用遗传靶向、细胞类型特异性和蛋白质特异性荧光标记的能力，ExLLSM 填补了神经解剖学传统光学管道的高通量与相应的超高分辨率光学管道之间的宝贵空白。电磁管道。假设开发出经过充分验证的 10 倍或以上的全脑各向同性扩展以及足够密集的标记，ExLLSM 可以在 25 nm 或更高分辨率下以蛋白质特异性对比度对多个样本的密集神经回路进行全脑比较。纳米级全脑光学成像。ExLLSM 对毫米级体积上具有分子对比度的神经结构进行成像，包括（从右上角顺时针方向）小鼠锥体神经元及其过程；体细胞的细胞器形态；整个皮质的树突棘和突触蛋白；果蝇投射神经元纽扣的刻板印象；投射神经元追踪至中央复合体；（中）大脑中的多巴胺能神经元，包括椭球体（圆形插图）。光学和电子显微镜在理解大脑的复杂性方面取得了巨大进展。然而，光学显微镜提供的分辨率不足以揭示亚细胞细节，而电子显微镜缺乏通量和分子对比度来可视化毫米级或更大尺寸的特定分子成分。我们将膨胀显微镜和晶格光片显微镜结合起来，对小鼠皮层或整个果蝇大脑厚度上的蛋白质之间的纳米级空间关系进行成像。这些包括树突棘的突触蛋白、轴突的髓鞘形成以及每个果蝇大脑区域多巴胺能神经元的突触前密度。该技术应该能够对神经发育、性别二态性、刻板程度以及与行为或神经活动的结构相关性进行统计丰富的大规模研究，所有这些都具有分子对比。ExLLSM 填补了神经解剖学传统光学管道的高吞吐量和相应 EM 管道的超高分辨率之间的宝贵空白。假设开发出经过充分验证的 10 倍或以上的全脑各向同性扩展以及足够密集的标记，ExLLSM 可以在 25 nm 或更高分辨率下以蛋白质特异性对比度对多个样本的密集神经回路进行全脑比较。纳米级全脑光学成像。ExLLSM 对毫米级体积上具有分子对比度的神经结构进行成像，包括（从右上角顺时针方向）小鼠锥体神经元及其过程；体细胞的细胞器形态；整个皮质的树突棘和突触蛋白；果蝇投射神经元纽扣的刻板印象；投射神经元追踪至中央复合体；（中）大脑中的多巴胺能神经元，包括椭球体（圆形插图）。光学和电子显微镜在理解大脑的复杂性方面取得了巨大进展。然而，光学显微镜提供的分辨率不足以揭示亚细胞细节，而电子显微镜缺乏通量和分子对比度来可视化毫米级或更大尺寸的特定分子成分。我们将膨胀显微镜和晶格光片显微镜结合起来，对小鼠皮层或整个果蝇大脑厚度上的蛋白质之间的纳米级空间关系进行成像。这些包括树突棘的突触蛋白、轴突的髓鞘形成以及每个果蝇大脑区域多巴胺能神经元的突触前密度。该技术应该能够对神经发育、性别二态性、刻板程度以及与行为或神经活动的结构相关性进行统计丰富的大规模研究，所有这些都具有分子对比。ExLLSM 填补了神经解剖学传统光学管道的高吞吐量和相应 EM 管道的超高分辨率之间的宝贵空白。假设开发出经过充分验证的 10 倍或以上的全脑各向同性扩展以及足够密集的标记，ExLLSM 可以在 25 nm 或更高分辨率下以蛋白质特异性对比度对多个样本的密集神经回路进行全脑比较。纳米级全脑光学成像。ExLLSM 对毫米级体积上具有分子对比度的神经结构进行成像，包括（从右上角顺时针方向）小鼠锥体神经元及其过程；体细胞的细胞器形态；整个皮质的树突棘和突触蛋白；果蝇投射神经元纽扣的刻板印象；投射神经元追踪至中央复合体；（中）大脑中的多巴胺能神经元，包括椭球体（圆形插图）。光学和电子显微镜在理解大脑的复杂性方面取得了巨大进展。然而，光学显微镜提供的分辨率不足以揭示亚细胞细节，而电子显微镜缺乏通量和分子对比度来可视化毫米级或更大尺寸的特定分子成分。我们将膨胀显微镜和晶格光片显微镜结合起来，对小鼠皮层或整个果蝇大脑厚度上的蛋白质之间的纳米级空间关系进行成像。这些包括树突棘的突触蛋白、轴突的髓鞘形成以及每个果蝇大脑区域多巴胺能神经元的突触前密度。该技术应该能够对神经发育、性别二态性、刻板程度以及与行为或神经活动的结构相关性进行统计丰富的大规模研究，所有这些都具有分子对比。电子显微镜缺乏在毫米级或更大尺寸上可视化特定分子成分的通量和分子对比度。我们将膨胀显微镜和晶格光片显微镜结合起来，对小鼠皮层或整个果蝇大脑厚度上的蛋白质之间的纳米级空间关系进行成像。这些包括树突棘的突触蛋白、轴突的髓鞘形成以及每个果蝇大脑区域多巴胺能神经元的突触前密度。该技术应该能够对神经发育、性别二态性、刻板程度以及与行为或神经活动的结构相关性进行统计丰富的大规模研究，所有这些都具有分子对比。电子显微镜缺乏在毫米级或更大尺寸上可视化特定分子成分的通量和分子对比度。我们将膨胀显微镜和晶格光片显微镜结合起来，对小鼠皮层或整个果蝇大脑厚度上的蛋白质之间的纳米级空间关系进行成像。这些包括树突棘的突触蛋白、轴突的髓鞘形成以及每个果蝇大脑区域多巴胺能神经元的突触前密度。该技术应该能够对神经发育、性别二态性、刻板程度以及与行为或神经活动的结构相关性进行统计丰富的大规模研究，所有这些都具有分子对比。