NIAMS Light Imaging Facility

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

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.