NIAMS Light Imaging Facility

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

The LIS currently offers access to two confocal microscopes. The Leica TCS X SP8 confocal is equipped with three 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 in the confocal microscopy field: 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 with increments of 1nm, which gives users the feasibility to perform easily complex, multiplexed fluorescence 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 embedded 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 pinhole size reduction providing 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. 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 virtually 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. Finally, the Leica TCS SP8 X is also coupled to a Mai Tai 2-P unit with a Ti:Sapphire laser source to perform localization of excitation in thick (>50um) as well as intravital and Second Harmonic Generation (SHG) image acquisition. In February 2023, the LIS acquired the latest confocal system available from Zeiss, the 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 up to the near-infrared (NIR) range, this microscope has proved ideal for multicolor experiments especially in living samples. Furthermore, the whole microscope is fully insulated to achieve optimal control of humidity, CO2 and temperature levels during live imaging. 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 whole 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). Furthermore, expanding the spectral range into the NIR allows for the use of more labels in parallel in highly multiplexed imaging projects. The TIRF microscope available in the LIS (based on a Leica DMi6000 stand) is equipped with two (63X and 100X) large numerical aperture lenses (1.47) and a highly sensitive Electron Multiplying Charged Coupled Device (EMCCD) camera to allow imaging of low intensity dynamic events in live cells occurring 100nm above the glass slide (e.g., endo- or exocytosis events happening 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 inside an incubator and can accommodate 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 clinical scientific community is a fully automated slide scanner (the Hamamatsu Nanozoomer XR), which enables for the acquisition of high-resolution images of histopathology slides (up to 320 in a single run) with a magnification up to 40X. Since January 2020, the LIS facility has included in its portfolio a Zeiss Lattice LightSheet (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 working simultaneously, this machine allows for the acquisition of large, high-resolution 4D volumes with high speed and very low phototoxicity/photobleaching. Finally, at the beginning of 2025, the LIS completed the acquisition of the most powerful widefield setup currently available in the NIH main campus composed by the Leica DMi8 Thunder Imager, equipped with highly-corrected, long and short working distance lenses to allow imaging of cells and tissues in culture and slides, the latest Leica Scientific Complementary Metal Oxide Semiconductor (sCMOS) K8 camera, the most advanced Leica high-speed light source LED8 able to excite at wavelengths ranging from 395nm to 730nm, the Adaptive Focus Control to reliably maintain focus while performing live-imaging and long slide-scanning experiments, and the ability to achieve blur-free images through the new opto-digital method created by Leica called Instant Computational Clearing. Plus, a preexisting stand (the Leica DM6000B) has been custom improved in collaboration with Leica with a fully motorized stage, a new advanced LED light source, a highly-sensitive color camera and a new workstation to drive the scope equipped with the latest Leica software, making this unit the ideal system to perform the imaging required for the various platforms of spatial transcriptomics available in NIAMS, such as those available from 10X Genomics, in collaboration with the NIAMS Genomic Technology Section led by Dr. Stefania Dell’Orso. From October 2024 up to the present time, the NIAMS LIS has supported researchers from virtually all NIAMS Sections and has been able to establish collaborations outside the IC by actively contributing to imaging projects of groups in NICHD, NHLBI, NHGRI, NIAID. Many of these collaborations are currently ongoing.