NIAMS Light Imaging Facility

自成立以来，LIS一直致力于建立和提供一个非常多样化的成像方法组合，从宽视场和共聚焦显微镜到高通量活细胞延时成像，自动组织学切片采集和更多的利基技术，如双光子（2-P）和全内反射显微镜（TIRFM）。 LIS显微镜核心拥有两台共聚焦显微镜：2014年获得的Zeiss LSM 780和2018年获得的Leica TCS X SP 8。蔡司LSM 780的探测器通常比标准共焦探测器（例如光电倍增管或PMT）更灵敏，动态范围更好。该仪器配备了五条激光线，能够采集多达四种不同的颜色。蔡司共聚焦显微镜还耦合到一个定焦模块，一个加热阶段与二氧化碳注射器，使该仪器的理想捕捉动态过程中活细胞的荧光恢复后光漂白（FRAP）和福斯特共振能量转移（FRET）技术。 Leica TCS X SP 8共聚焦显微镜是目前市场上最好的共聚焦显微镜。它配备了四个混合探测器（HyD）和一个PMT，并提供了最具创新性的激光光源：徕卡白色激光器（WLL）。WLL可以激发紫外-可见光谱中的所有波长，使用户能够执行复杂的免疫荧光染色策略，并具有高达八种不同颜色的非常准确的光谱分离。此外，Leica TCS X SP 8还配备了Lightning模块，该模块使用专用算法，可以突破衍射带来的分辨率限制，生成分辨率为120 nm X-Y的共焦图像，是标准共焦分辨率的两倍。Leica TCS X SP 8共焦显微镜连接到MaiTai 2-P装置，该装置具有钛宝石激光源，能够在厚组织（例如骨骼或结缔组织）中进行激发定位，并进行活体和二次谐波生成（SHG）成像。 LIS中提供的TIRF（Leica）显微镜配备了大数值孔径镜头和高灵敏度相机，可以对离载玻片100 nm处发生的活细胞动态事件进行成像，例如膜内或胞吐作用，其清晰度在任何其他显微镜上都无法实现。此外，该系统最近配备了由16个单独控制的LED（覆盖365 nm至770 nm的范围）制成的先进光源，以基于光响应分子（如通道视紫红质）执行尖端成像方法（例如光遗传学）。 LIS显微镜核心还可以通过两台IncuCyte S3机器对活细胞进行自动化和可编程的延时成像。这些仪器基本上是能够容纳培养皿、培养瓶和培养板的盒装显微镜。它们被放置在标准组织培养箱内，并在透射光和/或荧光中收集时间序列图像，并从PC工作站完全远程控制，其中使用专用软件来确定何时何地在板上成像以及成像多长时间。该软件允许用户分析图像，并提取和分析数据。 LIS最近获得的另一个工具是全自动载玻片扫描仪（Hamamatsu Nanozoomer XR），该扫描仪对NIAMS科学界至关重要，能够捕获放大倍数高达40倍的组织学载玻片的高分辨率图像。载玻片扫描仪一次最多可扫描350张载玻片。用户可以从自己的计算机通过服务器访问他们的数据。 最后，自2020年1月以来，LIS设施为NIAMS科学界提供了蔡司晶格光片（LLS）显微镜。由诺贝尔奖获得者Eric Betzig在2010年代初开发的LLS显微镜方法使用超薄光学晶格产生亚微米“片”光，以激发连续平面中的荧光团。该机器配备三条固态激光线、一个用作活体样品培养箱的电动载物台（温度和CO2受控）和一个高灵敏度sCMOS相机，可高速采集大体积3D图像，且光毒性/光漂白非常低。Zeiss Lattice Light Sheet显微镜是NIH主校区提供的第一台此类显微镜。 在过去的一年中，NIAMS光成像部分支持了来自14个部门，实验室或分支机构的研究人员。包括在核心中进行的显微镜工作的出版物已在几个高影响力，高度竞争力的科学期刊上发表。57名研究人员接受了使用一种或多种仪器的培训，并向博士后和学士后学员介绍了显微镜和伦理成像的基础。

Since its inception, the LIS has constantly strived to build and provide a very diversified portfolio of imaging approaches ranging from widefield and confocal microscopy to high-throughput live-cell time-lapse imaging, automated histology slide acquisition and more niche techniques such as two-photon (2-P) and total internal reflection microscopy (TIRFM). The LIS microscopy core possesses two confocal microscopes: a Zeiss LSM780 obtained in 2014 and a Leica TCS X SP8 acquired in 2018. The Zeiss LSM780 presents with detectors typically more sensitive and with a better dynamic range than standard confocal detectors (e.g. photomultiplier tubes, or PMT). The instrument is equipped with five laser lines and is able to acquire up to four different colors. The Zeiss confocal microscope is also coupled to a Definite Focus module, a heated stage with a CO2 injector making this instrument ideal for capturing dynamic processes in live cells by fluorescence recovery after photobleaching (FRAP) and Forster resonance energy transfer (FRET) techniques. The Leica TCS X SP8 confocal is the best-in-class confocal microscope currently available on the market. It is equipped with four hybrid detectors (HyD) and one PMT and presents with the most innovative laser light source available: the Leica White Light Laser (WLL). The WLL can excite all the wavelengths in the UV-Vis spectrum, giving the users the feasibility to perform complex 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 that, using dedicated algorithms, can break the resolution limit imposed by diffraction and produce confocal images with a resolution of 120nm X-Y, double the standard confocal resolution. The Leica TCS X SP8 confocal is connected to a MaiTai 2-P unit with a Ti:Sapphire laser source able to perform localization of excitation in thick tissues (e.g. bones or connective tissues), and to perform intravital and Second Harmonic Generation (SHG) imaging. The TIRF (Leica) microscope available in the LIS is equipped with large numerical aperture lenses and highly sensitive cameras to allow the imaging of dynamic events in live cells occurring 100nm apart from the glass slide, such as membrane endo- or exocytosis with a clarity that cannot be achieved on any other microscope. In addition, the system has been recently equipped with an advanced light source made of 16 individually controlled LEDs (covering the range from 365nm to 770nm) to perform cutting-edge imaging approaches (e.g. optogenetics) based on light-responsive molecules like channelrhodopsins. 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 basically boxed microscopes able to accommodate culture dishes, flasks and plates. They are placed inside a standard tissue culture incubator and collect time series images in transmitted light and/or fluorescence and are fully controlled remotely from a PC workstation, where dedicated software is used to establish when and where on the plate to image, and for how long. The software allows the user to analyze the images, and extract and analyze the data on the fly. An additional tool recently acquired by the LIS that has been essential for the NIAMS scientific community is a fully automated slide scanner (Hamamatsu Nanozoomer XR) able to capture high-resolution images of histology slides with a magnification up to 40X. The slide scanner can take up to 350 slides in one run. Users can access their data through a server from their own computer. Finally, since January 2020 the LIS facility has offered the NIAMS scientific community access to a Zeiss Lattice Light Sheet (LLS) microscope. Developed by the Nobel laureate Eric Betzig in the early 2010s, the LLS microscopy approach uses ultra-thin optical lattices to generate sub-micron "sheets" of light to excite fluorophores in successive planes. Equipped with three solid-state laser lines, a motorized stage that works as an incubator for live samples (with controlled temperature and CO2), and a highly sensitive sCMOS camera, this machine allows acquisition of large 3D image volumes with high speed and very low phototoxicity/photobleaching. The Zeiss Lattice Light Sheet microscope is the first of this category available on the NIH main campus. In the past year, the NIAMS Light Imaging Section has supported researchers from 14 Sections, Laboratories, or Branches. Publications that include microscopy work carried out in the core have been published in several high impact, highly competitive scientific journals. 57 researchers have been trained to use one or more of the instruments, and one presentation on the bases of microscopy and ethical imaging was given to post-doctoral and post-baccalaureate trainees.