Systems Neuroscience Imaging Resource

系统神经科学成像资源

• 批准号: 10703973
• 负责人: Theodore Usdin
• 金额: $ 168.55万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703973
• 关键词: 3-Dimensional; Anatomy; Antibodies; Area; Atlases; Behavior; Biology; Brain; Brain Mapping; Brain region; COVID-19; COVID-19 pandemic; Callithrix; Cells; Code; Collaborations; Computer software; Custom; Data; Data Set; Development; Dimensions; Discrimination; Dopamine; Drosophila genus; Equipment; Gene Activation; Gene Expression; Genetic; Histologic; Image; Image Analysis; Immediate-Early Genes; In Situ Hybridization; Interneurons; Intramural Research Program; Investigation; Label; Laboratories; Laser Scanning Confocal Microscopy; Lasers; Learning Skill; Light; Lighting; Macaca; Maintenance; Mediating; Messenger RNA; Methods; Microscope; Microscopy; Microtome - medical device; Midbrain structure; Mission; Molecular; Morphology; Movement; Mus; Muscle; National Institute of Mental Health; Neurons; Neurosciences; Nucleic Acid Probes; Pattern; Phase; Physiologic pulse; Pilot Projects; Population; Preparation; Procedures; Proteins; Publications; Pupa; Pythons; Qi; Ramp; Reporting; Research Personnel; Research Project Grants; Resolution; Resources; Role; Sampling; Scanning; Science; Slide; Somatosensory Cortex; Specific qualifier value; Structure of paraventricular nucleus of thalamus; System; Techniques; Technology; Thalamic Nuclei; Thalamic structure; Thick; Time; Tissue imaging; Tissues; Training; Training Support; United States National Institutes of Health; Vendor; Visualization; Work; Yang; analysis pipeline; base; cell type; cholinergic neuron; confocal imaging; cryostat; denoising; design; detector; image processing; insight; instrument; interest; light microscopy; microscopic imaging; molecular imaging; neuroregulation; operation; programs; protein distribution; reconstruction; recruit; remote interaction; response; routine imaging; software development; student training; tissue preparation; tool

Summary The mission of the NIMH Systems Neuroscience Imaging Resource (SNIR) is to make advanced light microscopy related techniques available to Intramural Program investigators. This is its sixth year of operation. SNIR functions can be divided into three interacting domains: acquisition and maintenance of equipment and software, development and implementation of procedures, and training. The COVID-19 pandemic had a major effect on its operation, but the resource is fully functional. Image processing resources, in particular, are available through remote access. Procedures have been put in place for safe training and use of equipment. Essentially all requests for use of equipment were accommodated. Major supported equipment includes: 1) Zeiss AxioscanZ1 slide scanning microscope (2016 acquisition). This is a high quality widefield microscope with transmitted brightfield and fluorescent epi-illumination capacity. Its most significant feature is the ability to program multichannel tiled acquisition of large areas from up to 100 microscope slides. It is being actively used by investigators from more than 10 intramural laboratories for projects that include whole brain mapping of gene expression profiles and the projections of genetically tagged and fluorescently labeled neuron populations. The system was used to capacity until campus occupancy was reduced because of COVID-19. Use ramped up quickly with the phased return to campus. Procedures for remote interactions with a technician who handles physical interactions with the instrument were established. 2) Zeiss LSM780 microscope (2011 acquisition, now a secondary instrument). This is a high quality inverted confocal microscope with 405, 488, 514, 561, 594 and 633 nm lasers, a 32-channel GaAsP based spectral detector and 2 conventional PMTs. 3) LaVision Ultrascope (2017 acquisition). This is a light sheet microscope optimized for low magnification (1.2 to 12X 0.5 NA objective with a minimum light sheet thickness minimum of 5 microns) imaging of large samples (up to approximately 10 x 10 x 6 mm). It has 405, 488, 552, 638, and 740 nm lasers. Whole mouse brains immunolabeled with the iDISCO technique are being imaged routinely and projects using brains cleared with CUBIC, SHIELD and other procedures are under development. 4) Leica SP8 confocal/multiphoton system (2017 acquisition). This is an upright microscope equipped with long working distance dipping objectives designed for work with thick cleared samples. It is equipped with 405, 488, 552 and 638 nm fixed lasers and an Insight X3 tunable IR laser, and both internal, and external non-descanned, PMT and HyD detectors. It has capacity to perform fluorescent lifetime imaging microscopy (FLIM). 5) Nikon A1HR confocal system (2020 acquisition). This is set up for both widefield epillumination and laser scanning confocal imaging and includes both galvanometer and resonant scanning ability. It is equipped with 405, 488, 561, 630 and 750 nm lasers. 6) Leica Stellaris confocal instrument (2020 acquisition). This is equipped with a tunable pulsed white light laser as well as 5 tunable high sensitivity detectors, providing precise control of excitation wavelength and emission windows from 405 to 730nm. Image acquisition can be done in galvanometer or resonant modes. 6) Nikon Biopipeline Slide System (2020 acquisition). This is a slide scanning instrument that will provide highly customizable control over acquisition, including imaging of selected regions of interest in either widefield or confocal mode (using confocal components repurposed from a Nikon C2 system). The vendor is working on software implementation. Pilot projects are underway. Major supported software includes: Microbrightfield Brainmaker and Neurolucida 360. These packages facilitate reconstruction and analysis of the distribution and morphology of labeled neurons. Arivis Vision4D is available for visualization of large - dimensional datasets and implementation of analysis pipelines. In addition custom python-based code was developed within the group for denoising, deconvolution, dehazing, stitching, and segmentation of massive data sets, taking advantage of the NIH Biowulf computational cluster. Training provided this year included: 1) Initial use of each of the microscopes and the software packages described above. 2) Ad hoc assistance during microscope and software use. 3) Use of iDISCO- and SHIELD based clearing for whole mouse brain mapping of immediate early gene distribution. 4) Use of a custom pipeline for atlas registration and whole brain analysis of cells with immediate early gene activation. Publications that used images generated on SNIR microscopes include: 1. Helseth AR, Hernandez-Martinez R, Hall VL, Oliver ML, Turner BD, Caffall ZF, Rittiner JE, Shipman MK, King CS, Gradinaru V, Gerfen C, Costa-Mattioli M, Calakos N (2021) Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice Science. 372 eabe1931. PMID: 33888613 2. Naskar, S., Qi, J., Gerfen, C.R., and Lee, S. (2021). Cell-type-specific recruitment of GABAergic interneurons in the primary somatosensory cortex by long-range inputs. Cell Reports 34(8): 108774. PMID: 33626343 3. Inagaki HK, Chen S, Ridder MC, Sah P, Li N, Yang Z, Hasanbegovic H, Gao Z, Gerfen CR, Svoboda K. A midbrain-thalamus-cortex circuit reorganizes cortical dynamics to initiate movement. Cell. 2022 Mar 17;185(6):1065-1081.e23. doi: 10.1016/j.cell.2022.02.006. Epub 2022 Mar 3. PMID: 35245431; PMCID: PMC8990337. 4. Qi J, Ye C, Naskar S, Inacio AR, Lee S. The role of a higher-order thalamic nucleus in perceptual discrimination. (Under revision, PLOS Biology, PBIOLOGY-D-21-03329R1) Elliott AD, Berndt A, Houpert M, Roy S, Scott RL, Chow CC, Shroff H, White BH. Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila. Elife. 2021 Jul 8;10:e68656. doi: 10.7554/eLife.68656. PMID: 34236312; PMCID: PMC8331185. 5. Ma J, du Hoffmann J, Kindel M, Beas BS, Chudasama Y, Penzo MA. Divergent projections of the paraventricular nucleus of the thalamus mediate the selection of passive and active defensive behaviors. Nat Neurosci. 2021 Oct;24(10):1429-1440. doi: 10.1038/s41593-021-00912-7. Epub 2021 Aug 19. PMID: 34413514; PMCID: PMC8484052.

概括 NIMH系统神经科学成像资源（SNIR）的任务是使高级光学显微镜相关的技术可用于壁内计划研究者。这是其运营的第六年。 SNIR功能可以分为三个相互作用的域：设备和软件的获取和维护，程序的开发和实施以及培训。 COVID-19大流行对其运作有重大影响，但资源完全有效。尤其是图像处理资源，可通过远程访问获得。已经制定了安全培训和设备使用的程序。从本质上讲，所有使用设备的请求均已容纳。 主要支持的设备包括：1）Zeiss Axioscanz1幻灯片扫描显微镜（2016年收购）。这是一个高质量的广场显微镜，具有传播的Brightfield和荧光表演能力。它最重要的功能是能够编程多通道瓷砖从最高100个显微镜幻灯片进行大面积采集。来自10多个壁内实验室的研究人员正在积极使用它，用于包括基因表达谱的整个大脑映射以及遗传标记和荧光标记神经元种群的投影。该系统用于容量，直到由于19号而降低了校园入住率。随着分阶段返回校园，使用迅速增加。建立了与处理与仪器进行物理互动的技术人员进行远程互动的程序。 2）Zeiss LSM780显微镜（2011年收购，现在是次要仪器）。这是具有405、488、514、561、594和633 nm激光器，一个基于32频道GAASP的光谱检测器和2个常规PMT的高质量倒置共聚焦显微镜和633 nm激光器。 3）Lavision Ulterscope（2017年收购）。这是用于优化低放大倍率的轻型显微镜（1.2至12x 0.5 Na物镜，最小的轻度厚度最小厚度为5微米）的大型样品（最高约为10 x 10 x 6 mm）。它具有405、488、552、638和740 nm激光器。用IDISCO技术进行的整个小鼠大脑正在定期成像，并使用Cubic，Shield和其他程序清除大脑的项目正在开发中。 4）Leica SP8共聚焦/多光系统（2017年收购）。这是一个直立的显微镜，配备了较长的工作距离浸入目标，该目标是为厚实的样品工作而设计的。它配备了405、488、552和638 nm固定激光器和洞察力X3可调IR激光，以及内部和外部非偏见，PMT和HYD探测器。它具有执行荧光寿命成像显微镜（FLIM）的能力。 5）尼康A1小时共聚焦系统（2020年收购）。这是针对广场癫痫发光和激光扫描共聚焦成像设置的，并包括仪表仪和谐振能力。它配备了405、488、561、630和750 nm激光器。 6）Leica Stellaris共聚焦仪器（2020年收购）。它配备了可调的脉冲白光激光器以及5个可调的高灵敏度检测器，可精确控制激发波长和发射窗口的405至730nm。图像采集可以在仪表仪或谐振模式下完成。 6）Nikon Biopipeline幻灯片系统（2020年收购）。这是一种幻灯片扫描仪器，将对采集进行高度定制的控制，包括在广场或共聚焦模式下对选定的感兴趣区域进行成像（使用从尼康C2系统重新使用的共聚焦组件）。供应商正在研究软件实施。试点项目正在进行中。 主要支持的软件包括：Microbrightfield Brainmaker和Neurolucida 360。这些软件包有助于重建和分析标记神经元的分布和形态。 Arivis Vision4D可用于可视化大维数据集和分析管道的实现。此外，在小组内开发了基于自定义的Python代码，用于利用NIH Biowulf计算集群，用于降解，反卷积，去卷积，缝制和分割。 今年提供的培训包括：1）最初使用每个显微镜以及上述软件包。 2）在显微镜和软件使用过程中进行临时帮助。 3）使用基于IDISCO和屏蔽的清除量用于立即的早期基因分布的整个小鼠大脑映射。 4）使用自定义管道进行地图集的注册和整个大脑分析细胞，并具有早期基因激活。 使用SNIR显微镜上生成的图像的出版物包括： 1. Helseth AR, Hernandez-Martinez R, Hall VL, Oliver ML, Turner BD, Caffall ZF, Rittiner JE, Shipman MK, King CS, Gradinaru V, Gerfen C, Costa-Mattioli M, Calakos N (2021) Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice Science. 372 EABE1931。 PMID：33888613 2。Naskar，S.，Qi，J.，Gerfen，C.R。和Lee，S。（2021）。通过远程输入，特定于细胞类型的GABA能中间神经元在原发性体感皮层中的募集。细胞报告34（8）：108774。PMID：33626343 3。InagakiHK，Chen S，Ridder MC，SAH P，Li N，Yang Z，Hasanbegovic H，Gao Z，Gerfen CR，Svoboda K. Midbrain-Thalamus-Cortex Circuit Reorange Reorlandical cortalical Dynamilics timallike cortical Dynamilics以开始运动。细胞。 2022年3月17日； 185（6）：1065-1081.E23。 doi：10.1016/j.cell.2022.02.006。 EPUB 2022 3月3日。PMID：35245431; PMCID：PMC8990337。 4。QiJ，Ye C，Naskar S，Inacio AR，LeeS。高阶丘脑核在感知歧视中的作用。 （根据修订版，PLOS生物学，PBIology-D-21-03329R1） Elliott AD，Berndt A，Houpert M，Roy S，Scott RL，Chow CC，Shroff H，White BH。果蝇神经调节响应于非结构化的肌肉活性。 Elife。 2021年7月8日； 10：E68656。 doi：10.7554/elife.68656。 PMID：34236312; PMCID：PMC8331185。 5。MaJ，Du Hoffmann J，Kindel M，Beas BS，Chudasama Y，Penzo MA。丘脑的旁牙核的分歧投影介导了被动和主动防御行为的选择。 Nat Neurosci。 2021年10月； 24（10）：1429-1440。 doi：10.1038/s41593-021-00912-7。 EPUB 2021 8月19日。PMID：34413514; PMCID：PMC8484052。