NIAMS Light Imaging Facility

NIAMS 光成像设备

基本信息

批准号：
10926593
负责人：
Davide Randazzo
金额：
$ 71.73万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10926593
关键词：
Algorithms Area Automobile Driving Bone Tissue Carbon Dioxide Cell membrane Cells Color Communities Complex Computer software Confocal Microscopy Connective Tissue Core Facility Coupled Darkness Data Detection Development Dyes Electrons Elements Ensure Equipment Ethics Event Exocytosis Fluorescence Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Glass Heating Histology Humidity Hybrids Image Imaging Techniques Immunofluorescence Immunologic Immunology Incubators Investigation Journals Knowledge Label Laboratories Lasers Lateral Light Lightning Manufacturer Manuscripts Marketing Mathematics Medicine Methodology Microscope Microscopy Mission Motor National Heart, Lung, and Blood Institute National Institute of Arthritis, and Musculoskeletal, and Skin Diseases Nature New England Noise Optics Ownership Penetration Persons Philosophy Photobleaching Photons Phototoxicity Physiologic pulse Process Publishing Research Personnel Resolution Running Sampling Scanning Scientist Semiconductors Series Shapes Slide Solid Source Specimen Speed Stains Structure System Techniques Technology Temperature Thick Time Tissues Training Tube United States National Institutes of Health Visualization Work charge coupled device camera clinical investigation detector experimental study flasks flexibility fluorophore gallium arsenide gallium phosphide high resolution imaging image processing imaging approach imaging capabilities imaging facilities improved innovation instrument lens light transmission live cell imaging metal oxide microscopic imaging optical lattices photomultiplier redshift sapphire laser second harmonic generation imaging solid state submicron superresolution imaging tissue culture tool two-photon ultra high resolution virology virtual

项目摘要

The LIS offers three confocal microscopes. The Zeiss LSM780 is equipped with highly sensitive gallium arsenide phosphide (GaAsP) and low dark noise photomultiplier tube (PMT) detectors. The instrument presents with five laser lines and is also equipped with a Definite Focus module to stabilize the field of acquisition, a motorized stage with a heating module and a CO2 injector, making this instrument ideal for capturing dynamic processes in live cells stained with fluorogenic dyes or for imaging techniques such as fluorescence recovery after photobleaching (FRAP) or Forster Resonance Energy Transfer (FRET). The Leica TCS X SP8 confocal is equipped with three extremely sensitive hybrid detectors (HyD) and two low dark noise PMTs. It presents with a solid state 405nm laser and one of the most innovative light sources: the Leica White Light Laser (WLL). The WLL can excite all the wavelengths in the UV-Vis spectrum in the range between 470nm and 670nm, which gives users the feasibility to employ complex multiplexed immunofluorescence staining strategies with a very accurate spectral separation of up to eight different colors. In addition, the Leica TCS X SP8 is equipped with the Lightning module in the LAS X software driving the TCS SP8 X system, which breaks the resolution limit imposed by diffraction by using adaptive deconvolution algorithms and provides a lateral resolution of 120nm (XY), i.e., double the standard confocal resolution. In January 2021, the Leica TCS X SP8 confocal was further upgraded with the Leica tauSTED module. STED (for Stimulated Emission Depletion) is a purely optical approach that circumvents the limit imposed by diffraction by switching off the fluorescence of emitting molecules in the outer regions of the excitation focus using high intensity (i.e., depletion) lasers. The specimen is illuminated by two synchronized, ultrafast, co-linear sources consisting of an excitation laser pulse followed by a red-shifted depletion laser pulse (the latter referred to as the STED beam). By spatially arranging the STED beam in a doughnut shape, the fluorescence emitted by molecules at the periphery of the excitation focus is quenched. In the center of the doughnut, where the STED laser intensity is zero, fluorescence remains unaffected. Finally, since STED images are produced optically during the confocal scan process no mathematical post-acquisition processing is required, which makes STED the best super-resolution technique for imaging high-speed live-cell events. The tauSTED system available in the LIS integrates the lifetime information of the fluorophores to discriminate photons based on their arrival time, providing an even better resolution (30nm laterally and 130nm axially) than conventional STED. The Leica TCS SP8 X is also coupled to a Mai Tai two-photon unit with a Ti:Sapphire laser source able to perform localization of excitation in thick (>50um) tissues (e.g. bones or connective tissues), as well as intravital and Second Harmonic Generation (SHG) image acquisition. In February 2023, the LIS acquired the new Zeiss LSM980, a highly advanced confocal system capable of imaging even the most challenging samples. Equipped with 8 solid-state laser lines, a beam path with up to 36 simultaneous channels and full spectral flexibility into the near-infrared (NIR) range, this microscope is ideal for multicolor experiments including living samples. The LSM 980 can image multiple labels simultaneously, covering a wide emission range from 380nm up to 900nm. Importantly, the Zeiss LSM980 is equipped with a state-of-the-art 32-channel detector, namely the AiryScan 2, and a dual-channel NIR detector. The Zeiss AiryScan 2 is an area detector with 32 circularly arranged detection elements. Each of these acts as a small pinhole, contributing to super-resolution imaging, while the complete detector area collects more light than in a standard confocal setting. This reduces the distance that can be resolved between two points even further, specifically up to 90nm laterally (XY) and 270nm axially (Z) so that imaging experiments will benefit from an improved separation of multiple labels. Expanding the spectral range into the NIR allows the use of more labels in parallel and visualization of additional structures with more dyes in multi-color experiments, efficiently supporting spectral multiplexing experiments. Considering that NIR fluorescent labels are less phototoxic for living samples due to the longer wavelength, this allows investigation of living samples for longer periods of time while limiting the influence of light. Additionally, light of longer wavelength ranges is less scattered by cells and tissues increasing the penetration depth. The dual-channel NIR detector combines two different detector technologies (the extended red gallium arsenide phosphide-GaAsP and the gallium arsenide-GaAs) for optimal sensitivity. Finally, the microscope stand of the LSM980 system is fully insulated to achieve optimal control of humidity, CO2 level and temperature during live imaging. The TIRF microscope available in the LIS (based on the Leica DMi6000 unit) is equipped with two (63X and 100X) large numerical aperture lenses (1.47NA) and a highly sensitive Electron Multiplying Charged Coupled Device (EMCCD) camera to allow imaging of low intensity dynamic events in live cells occurring 100nm apart from the glass slide (e.g., endo- or exocytosis on the plasma membrane) with a resolution that cannot be achieved on any other microscope. The LIS microscopy core also offers the possibility to perform automated and programmable time-lapse imaging of live cells through two IncuCyte S3 machines. These instruments are "boxed" microscopes placed within a tissue culture incubator and can accommodate culture dishes, flasks, and plates. The IncuCyte S3 machines collect time series images in transmitted light and/or fluorescence, and are fully controlled remotely from a PC workstation to determine when and where to image on a specific plate, and for how long. An additional tool acquired by the LIS in 2018 and that has been essential for the mission of the NIAMS scientific community is a fully automated slide scanner (the Hamamatsu Nanozoomer XR), which enables the acquisition of high-resolution images of histology slides (up to 320 slides in a single run) with a magnification up to 40X. Since January 2020, the LIS facility has included in its portfolio a Zeiss Lattice Light Sheet (LLS) 7 microscope (in co-ownership with NHLBI and NCI). The LLS microscopy approach employs ultra-thin optical lattices to generate sub-micron "sheets" of light to excite fluorophores across multiple consecutive planes (up to 300um in depth). Equipped with three solid-state laser lines, a motorized stage that works as an incubator for live samples (with controlled humidity, temperature and CO2) and two highly sensitive scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras, this machine allows acquisition of large high-resolution 4D volumes with high speed and very low phototoxicity/photobleaching. The Zeiss LLS7 microscope is currently the only one available on the NIH main campus. From January 2023 up to the present time, the NIAMS LIS has supported researchers from all NIAMS Sections and their Laboratories or Branches ensuring full access to the instruments, constant support to users, as well as full training of new microscopy users through virtual and in person sessions. Manuscripts including imaging data obtained in the core have been published or are already submitted for consideration in high impact factor journals including The New England Journal of Medicine, Nature Immunology, Developmental Cell, Journal of Clinical Investigation, Journal of Virology, and Structure.

LIS 提供三种共焦显微镜。 Zeiss LSM780 配备高灵敏度砷化镓磷化物 (GaAsP) 和低暗噪声光电倍增管 (PMT) 探测器。该仪器具有 5 条激光线，还配备了用于稳定采集区域的定焦模块、带加热模块的电动载物台和 CO2 注射器，使该仪器非常适合捕获用荧光染料染色的活细胞中的动态过程。或用于成像技术，例如光漂白后荧光恢复 (FRAP) 或福斯特共振能量转移 (FRET)。 Leica TCS X SP8 共焦配备了三个极其灵敏的混合探测器 (HyD) 和两个低暗噪声 PMT。它配备了固态 405 nm 激光器和最具创新性的光源之一：徕卡白光激光器 (WLL)。 WLL 可以激发 470 nm 至 670 nm 范围内的 UV-Vis 光谱中的所有波长，这使用户能够采用复杂的多重免疫荧光染色策略，并能够非常准确地分离多达八种不同的颜色。此外，徕卡TCS X SP8在驱动TCS SP8 X系统的LAS X软件中配备了Lightning模块，利用自适应反卷积算法打破了衍射带来的分辨率限制，提供了120nm（XY）的横向分辨率，即，标准共焦分辨率的两倍。 2021年1月，徕卡TCS X SP8共焦进一步升级，配备徕卡tauSTED模块。 STED（受激发射损耗）是一种纯光学方法，通过使用高强度（即损耗）激光关闭激发焦点外部区域中发射分子的荧光，从而规避衍射所施加的限制。样品由两个同步、超快、共线源照明，该源由激发激光脉冲和红移耗尽激光脉冲（后者称为 STED 光束）组成。通过将 STED 光束在空间上排列成环形，激发焦点周围的分子发射的荧光被猝灭。在甜甜圈的中心，STED 激光强度为零，荧光不受影响。最后，由于 STED 图像是在共焦扫描过程中以光学方式生成的，因此不需要数学后采集处理，这使得 STED 成为高速活细胞事件成像的最佳超分辨率技术。 LIS 中提供的 tauSTED 系统集成了荧光团的寿命信息，根据光子的到达时间来区分光子，从而提供比传统 STED 更好的分辨率（横向 30 nm，轴向 130 nm）。 Leica TCS SP8 X 还与带有 Ti:Sapphire 激光源的 Mai Tai 双光子单元耦合，能够在厚（>50um）组织（例如骨骼或结缔组织）以及活体和第二组织中执行激发定位谐波发生 (SHG) 图像采集。 2023 年 2 月，LIS 购买了新型蔡司 LSM980，这是一种非常先进的共焦系统，甚至能够对最具挑战性的样品进行成像。该显微镜配备 8 条固态激光线、具有多达 36 个同步通道的光束路径以及近红外 (NIR) 范围内的完整光谱灵活性，非常适合包括活体样本在内的多色实验。 LSM 980 可以同时对多个标签进行成像，覆盖从 380 nm 到 900 nm 的宽发射范围。重要的是，Zeiss LSM980 配备了最先进的 32 通道探测器（即 AiryScan 2）和双通道 NIR 探测器。 Zeiss AiryScan 2 是一款区域探测器，具有 32 个圆形排列的探测元件。每一个都充当一个小针孔，有助于超分辨率成像，而整个探测器区域比标准共焦设置收集更多的光。这进一步缩短了两点之间可分辨的距离，特别是横向 (XY) 达 90 纳米，轴向 (Z) 达 270 纳米，因此成像实验将受益于多个标签的改进分离。将光谱范围扩展到 NIR 允许在多色实验中并行使用更多标签，并使用更多染料可视化其他结构，从而有效支持光谱复用实验。考虑到近红外荧光标记由于波长较长，对活体样品的光毒性较小，因此可以在更长的时间内研究活体样品，同时限制光的影响。此外，较长波长范围的光较少被细胞和组织散射，从而增加了穿透深度。双通道 NIR 检测器结合了两种不同的检测器技术（扩展红色砷化镓磷化物 - GaAsP 和砷化镓 - GaAs），以实现最佳灵敏度。最后，LSM980 系统的显微镜支架完全绝缘，可在实时成像过程中实现湿度、二氧化碳水平和温度的最佳控制。 LIS 中的 TIRF 显微镜（基于 Leica DMi6000 装置）配备了两个（63X 和 100X）大数值孔径镜头 (1.47NA) 和一个高灵敏度电子倍增电荷耦合器件 (EMCCD) 相机，可对低活细胞中距离载玻片 100 nm 处发生的强度动态事件（例如，质膜上的内吞作用或胞吐作用），分辨率无法达到在任何其他显微镜上。 LIS 显微镜核心还提供了通过两台 IncuCyte S3 机器对活细胞进行自动和可编程延时成像的可能性。这些仪器是放置在组织培养箱内的“盒装”显微镜，可容纳培养皿、烧瓶和培养板。 IncuCyte S3 机器收集透射光和/或荧光的时间序列图像，并通过 PC 工作站进行完全远程控制，以确定何时何地在特定板上成像以及持续多长时间。 LIS 于 2018 年获得的另一个工具是全自动载玻片扫描仪（Hamamatsu Nanozoomer XR），它对于 NIAMS 科学界的使命至关重要，它能够采集组织学载玻片的高分辨率图像（高达单次运行 320 张载玻片），放大倍率高达 40 倍。自 2020 年 1 月以来，LIS 设施在其产品组合中加入了蔡司晶格光片 (LLS) 7 显微镜（与 NHLBI 和 NCI 共同拥有）。 LLS 显微镜方法采用超薄光学晶格来生成亚微米“片”光，以在多个连续平面（深度高达 300 微米）上激发荧光团。该机器配备了三条固态激光线、一个用作活体样品培养箱的电动载物台（湿度、温度和 CO2 受控）和两个高灵敏度科学互补金属氧化物半导体 (sCMOS) 相机，可以采集大型高光谱图像。 - 分辨率 4D 体积，速度快，光毒性/光漂白性极低。 Zeiss LLS7 显微镜是目前 NIH 主校区唯一可用的一台。从 2023 年 1 月至今，NIAMS LIS 为所有 NIAMS 科室及其实验室或分支机构的研究人员提供支持，确保充分使用仪器、为用户提供持续支持，以及通过虚拟和面对面方式对新显微镜用户进行全面培训会议。包含核心成像数据的手稿已在高影响因子期刊上发表或提交供考虑，包括《新英格兰医学杂志》、《自然免疫学》、《发育细胞》、《临床研究杂志》、《病毒学杂志》和《结构》。

项目成果

期刊论文数量（72）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Sirtuin 1 enzymatic activity is required for cartilage homeostasis in vivo in a mouse model.

DOI：
10.1002/art.37750
发表时间：
2013-01
期刊：
ARTHRITIS AND RHEUMATISM
影响因子：
0
作者：
Gabay, Odile;Sanchez, Christelle;Dvir-Ginzberg, Mona;Gagarina, Viktoria;Zaal, Kristien J.;Song, Yingjie;He, Xiao Hong;McBurney, Michael W.
通讯作者：
McBurney, Michael W.

Identification of Interleukin-1β-Producing Monocytes That Are Susceptible to Pyronecrotic Cell Death in Patients With Neonatal-Onset Multisystem Inflammatory Disease.

DOI：
10.1002/art.39307
发表时间：
2015-12
期刊：
Arthritis & rheumatology (Hoboken, N.J.)
影响因子：
0
作者：
Edwan JH;Goldbach-Mansky R;Colbert RA
通讯作者：
Colbert RA

Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein.

DOI：
10.1016/j.bone.2015.05.007
发表时间：
2015-09
期刊：
BONE
影响因子：
4.1
作者：
Foster, B. L.;Ao, M.;Willoughby, C.;Soenjaya, Y.;Holm, E.;Lukashova, L.;Tran, A. B.;Wimer, H. F.;Zerfas, P. M.;Nociti, F. H., Jr.;Kantovitz, K. R.;Quan, B. D.;Sone, E. D.;Goldberg, H. A.;Somerman, M. J.
通讯作者：
Somerman, M. J.

A Muscle-Specific Enhancer RNA Mediates Cohesin Recruitment and Regulates Transcription In trans.

DOI：
10.1016/j.molcel.2018.06.008
发表时间：
2018-07-05
期刊：
Molecular cell
影响因子：
16
作者：
Tsai PF;Dell'Orso S;Rodriguez J;Vivanco KO;Ko KD;Jiang K;Juan AH;Sarshad AA;Vian L;Tran M;Wangsa D;Wang AH;Perovanovic J;Anastasakis D;Ralston E;Ried T;Sun HW;Hafner M;Larson DR;Sartorelli V
通讯作者：
Sartorelli V

Differences in neuromuscular junctions of laryngeal and limb muscles in rats.