NIAMS Light Imaging Facility

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

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 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 2012 and a Leica TCS X SP8 acquired in 2018. The Zeiss LSM780 is equipped with highly sensitive GaAsP detectors and low darn noise PMTs detectors. The instrument is presents with five different laser lines and is also coupled to a Definite Focus module, 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 fluorescence recovery after photobleaching (FRAP) and Frster 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 three extremely sensitive detectors (HyD) and two low dark noise PMTs, and presents with a solid state 405nm laser and one of the most innovative laser light sources: the Leica White Light Laser (WLL). The WLL can excite all the wavelengths in the UV-Vis spectrum (470-670nm), giving users the feasibility to perform 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 that, using adaptive deconvolution algorithms, can break the resolution limit imposed by diffraction producing images with a lateral resolution of 120nm XY, double the standard confocal resolution. On January 2021, the Leica TCS X SP8 confocal has been further upgraded with the Leica tauSTED module. STED (for Stimulated Emission Depletion) is one of the best super-resolution microscopy techniques available. STED 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 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 (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 STED laser intensity is zero, fluorescence remains unaffected. Consequently, almost all the excited molecules in the outer regions of the excitation focus return to the ground state and the only fluorescence captured by the detectors derives from the remaining excited molecules in the center of the excitation focus. Finally, since STED images are produced optically during the confocal scan process, no mathematical processing is required making STED the best super-resolution technique for imaging high-speed live cell events. The system present in the Light Imaging Section is an advanced version of the original/conventional STED approach and is called tauSTED as it integrates the lifetime information of the fluorophores to discriminate photons based on their arrival time and get an even better resolution than conventional STED. This translates into an achievable resolution of 30 nm laterally (XY) and 130nm axially (Z). The Leica TCS SP8 X is also coupled to a Mai Tai 2-P unit with a Ti:Sapphire laser source able to perform localization of excitation imaging in thick (>50um) tissues (e.g. bones or connective tissues), as well as intravital and Second Harmonic Generation (SHG) imaging. The TIRF microscope available in the LIS (based the Leica DMI6000 unit) is equipped with two (63X and 100X) large numerical aperture lenses (1.47NA) and a highly sensitive 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. 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 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, 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 on the fly, and extract data for further analysis or time lapse videos for presentations and publications. An additional tool recently acquired by the LIS that has been essential for the mission of NIAMS scientific community is a fully automated slide scanner (the 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 a single run. Users can access their data through a server within the NIAMS network from their own computer. Since January 2020, the LIS facility has included in its portfolio a Zeiss Lattice Light Sheet (LLS) 7 microscope. Developed by the Nobel laureate Eric Betzig in the early 2010s, the LLS microscopy approach employs ultra-thin optical lattices to generate sub-micron sheets of light to excite fluorophores across multiple successive 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 temperature and CO2) and a highly sensitive sCMOS camera, this machine allows acquisition of large high resolution 3D image volumes with high speed and very low phototoxicity/photobleaching. The Zeiss Lattice Light Sheet 7 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. Even during the COVID-19 pandemic and despite the restrictions imposed by the necessary safety rules, the Light Imaging Section has been able to guarantee full access to the instruments, constant support to all users and full training of new microscopy users through hybrid sessions (virtual and hands-on). Manuscripts including imaging carried out in the core have been published in several high impact scientific journals.