NIAMS Light Imaging Facility

自成立以来，LIS一直致力于建立和提供非常多样化的成像方法组合，从宽视场和共焦显微镜到高通量活细胞延时成像、自动组织学切片采集以及更多利基技术，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焦显微镜：2014年获得的蔡司LSM780和2018年获得的徕卡TCS X SP8。蔡司LSM780配备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，动态范围也更好。该仪器配备了五条激光线路，能够获取多达四种不同的颜色。蔡司共焦显微镜还连接到一个特定的聚焦模块，一个带有二氧化碳注射器的加热平台，这使得该仪器非常适合通过光漂白后荧光恢复(FRAP)和福斯特共振能量转移(FRET)技术捕捉活细胞的动态过程。 徕卡TCS X SP8共焦显微镜是目前市场上最好的共焦显微镜。它配备了四个混合探测器(HYD)和一个光电倍增管，并提供了现有最具创新性的激光光源：徕卡白光激光器(WLL)。WLL可以激发UV-Vis光谱中的所有波长，使用户能够通过高达8种不同颜色的非常精确的光谱分离来执行复杂的免疫荧光染色策略。此外，徕卡TCS X SP8配备了Lightning模块，使用专门的算法可以打破衍射造成的分辨率限制，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。徕卡TCS X SP8共焦通过钛宝石激光光源连接到MAITAI 2-P单元，能够在厚组织(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(SHG)成像。 LIS中提供的TIRF(徕卡)显微镜配备了大数值孔径透镜和高灵敏度相机，可以在距离玻片100 nm处对活细胞中发生的动态事件进行成像，例如膜内吞或胞吐，其清晰度是任何其他显微镜都无法实现的。此外，该系统最近还配备了由16个单独控制的LED(覆盖范围从365 nm到770 nm)制成的先进光源，以执行基于通道视紫红质等光响应分子的尖端成像方法(例如光遗传学)。 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化和可编程的延时成像的可能性。这些仪器基本上是盒装显微镜，能够容纳培养皿、烧瓶和培养皿。它们被放置在标准的组织培养孵化器中，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，在那里使用专用软件来确定在板上何时何地成像以及成像多长时间。该软件允许用户分析图像，并动态提取和分析数据。 LIS最近获得的另一个工具对NIAMS科学界至关重要，它是一台全自动载玻片扫描仪(Hamamatsu Nanosomer XR)，能够以高达40倍的放大倍数捕捉组织学载玻片的高分辨率图像。幻灯片扫描仪一次最多可以拍摄350张幻灯片。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科学界提供了使用蔡司晶格光片(LLS)显微镜的机会。由诺贝尔奖获得者埃里克·贝齐格在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的光片，在连续的平面上激发荧光团。这台机器配备了三条固态激光生产线、一个用作活体样品孵化器的电动工作台(可控制温度和二氧化碳)，以及一个高灵敏度的sCMOS相机，可以高速采集大容量的3D图像，光毒性/光漂白非常低。蔡司晶格光片显微镜是美国国立卫生研究院主校区提供的第一台此类显微镜。 在过去的一年里，NIAMS光成像部支持了来自14个部门、实验室或分支机构的研究人员。包括在核心开展的显微镜工作在内的出版物已经在几个影响很大、竞争激烈的科学期刊上发表。57名研究人员接受了使用一种或多种仪器的培训，并向博士后和学士学位后的实习生进行了一次关于显微镜和伦理成像基础的演讲。

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.