Oxford Instruments Center for Advanced Microscopy – Electron Microscopy (OxCAM-EM) Core

The Andor BC43 is a benchtop spinning disk confocal that includes 4 laser lines (405/488/561/638nm) and 3 imaging modalities (transmitted (brightfield and differential phase contrast), widefield and confocal). The BC43 is a fully enclosed imaging system that does not require a dark room or individual isolation table. It has objectives ranging from 2X to 60X, with a 40X silicone objective that is optimized for reducing spherical abberations when making deep observations or imaging live cells and a 60X oil objective that is best for imaging specimens that use mounting media and are located close to the coverslip. The system has a 16-bit monochrome sCMOS camera that has a 82% QE and a 18.5mm field of view. The BC43 is run via Andor’s FUSION acquisition software that provides the necessary functionality for acquisition, rendering, visualization, and export of 4 dimensional (X,Y,Z,T) image data, as well as multi-position and montaged (stitched tile-scan) datasets. A second BC43 also accommodates live cell imaging.

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The Andor Dragonfly 502 is a modern multi-modal spinning disk confocal microscope configured for fast and gentle multi-channel confocal, widefield, and transmitted light imaging. The system is housed on a fully motorized inverted Leica DMi8 microscope that is equipped with objectives ranging from 2.5X to 63X. The Leica DMi8 has an ASI motorized linear-encoded XYZ-stage which offers 150µM of piezo-Z movement for rapid acquisition of three-dimensional confocal Z-stacks that is essential for timely calcium imaging. It also has both closed-loop and adaptive focus control for maintaining focus and avoiding drift during extended live imaging sessions and has an Okolab stage top environmental control system (temperature and CO2) that is equipped with versatile inserts supporting a wide array of common sample vessels (35mm dishes, 1x3 microscope slides, multi-well plates, etc.). The imaging platform includes four solid state lasers for excitation of fluorescent probes across the visible spectrum (405nm, 488nm, 561nm, 637nm), a separate 445nm laser for photostimulation experiments and two high-sensitivity cameras for simultaneous fluorescence detection (emCCD (iXon-888 Ultra @ 95% peak QE) and sCMOS (Zyla 4.2+ @ 82% peak QE)). The system is equipped with Andor’s Mosaic DMD (digital mirror device) that permits advanced photostimulation and photomanipulation experiments through the use of a digitally controlled patterned illumination tool. The system is run via Andor’s FUSION acquisition software that provides the necessary functionality for acquisition, rendering, visualization, and export of 4 dimensional (X,Y,Z,T) image data, as well as multi-position and montaged (stitched tile-scan) datasets.

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The Andor Dragonfly 620SR is multi-modal spinning disk confocal system with 3D SMLM and B-TIRF capabilities. It comes equipped with SRRF-stream which provides a real time super-resolution solution with resolutions down to ~100nm, while 3D SMLM methods obtain resolutions as low as 20 nm but require post-processing in Picasso. The Dragonfly 620SR is an optimal solution for both fixed samples (especially for fast volume acquisition) and for live cell imaging by providing low phototoxicity and photobleaching. The system is housed on a fully motorized inverted Leica DMi8 microscope that is equipped with 6 objectives. The Leica DMi8 has an ASI XYZ stage with 3 axis controller, a MS2000 XY stage without linear encoders, a 300µm travel Piezo top plate (50nm res/100nm repeat) and adjustable slide kit and multi-well plate holder. It also has both closed-loop and adaptive focus control for maintaining focus and avoiding drift during extended live imaging sessions. The system is housed in a full enclosure incubator for temperature stability and environmental control and has an Okolab stage top incubator for temperature and CO2 that is equipped with versatile inserts supporting a wide array of common sample vessels (35mm dishes, 1x3 microscope slides and multi-well plates). The imaging platform includes 5 solid state lasers for excitation of fluorescent samples (405/488/560/640/730nm), two high-sensitivity cameras for simultaneous fluorescence detection ((emCCD (iXon 888 Life; QEmax >95%) and sCMOS (ZL41 Cell 4.2; up to 82% peak QE)) and two 8-position emission filter wheels with 6 emission filters each (442/46nm, 521/38nm, 594/43nm, 685/47nm, 809/90nm, QUAD 405/488/561/640nm). The system is run via Andor’s FUSION acquisition software that provides the necessary functionality for acquisition, rendering, visualization, and export of 4 dimensional (X,Y,Z,T) image data, as well as multi-position and montaged (stitched tile-scan) datasets. SMLM datasets are visualized using Picasso, which supports .ims files generated in Fusion, while the 3D rendering and statistical measurements can then be delivered to Imaris.

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The Aberrior Facility Line is a 3D-STED super-resolution imaging system that is capable of STED resolution <100x100x100 nm in 3D and of <30x30 nm in 2D (typically 80x80x90 nm and 25x25 nm, respectively). The system is housed on an inverted Evident Ix83 microscope that has 4 objectives (4x/20x dry, 60x water and 60x oil), a 200 µm piezo z motor, z-drift compensation, a universal sample holder (well plates, 35 mm dishes and slides) and a microscope table equipped with active dampening. For widefield illumination the system has a 16-color LED excitation light source, a quad band filter cube (DAPI/FITC/Cy3/Cy5) and a CCD monochrome camera (1600 x 1200 pixel). Live cell imaging is supported by an OKO BOLD LINE stage top incubator capable of maintaining temperature, CO2 and humidity. For confocal/STED imaging the system has 4 laser lines (405 nm (cw), 485/561/640 nm (pulsed)), a 775 nm STED laser and a laser scanning module with a rainbow spectral detection kit that contains 4 galvo scanners, a motorized pinhole wheel (25µm to 2mm) and a point scanner with a line frequency of up to 2.6 kHz with data collection (up to 4.2 frames per second for 512x512 pixels). The FACILITY LINE also comes with the following additional packages that improve upon image clarity. The RAYSHAPE: Adaptive Optics System improves the spherical abberations that negatively impact thick samples, making it possible to image deeper into samples with more clarity. The MATRIX Spectral Detection Unit helps reduce background levels while the RAINBOW Spectral Detection Unit enables low dark counts to render the finest details. FLEXPOSURE, DyMIN and MINFIELD are 3 strategies that aim to reduce the light dose with confocal and STED. The TIMEBOW software package implements fluorescence lifetime imaging and adds a new dimension to imaging. The TIMEBOW phasor plot allows for straightforward separation of data into different channels based on lifetime, e.g. for the separation of dyes with overlapping emission spectra or the separation of spectrally similar autofluorescence from different structures. The FACILITY Line is driven by the Lightbox Software that is capable of STED and confocal imaging and has full control of hardware and imaging settings. Lightbox can adjust ROIs and create stacks for multi-dimensional imaging such as timelapse, multi-position and tiling and can process/annotate/quantify images and present them as 3D using the viewer. For additional image processing the system also comes with the SVI Huygens Essentials package which includes the deconvolution wizard and workflow-processor.

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The Nikon N-SIM is a first-generation super-resolution microscope that delivers twice the resolution of traditional diffraction limited microscopes (~115 nm). The system is housed on a fully motorized Nikon Ti, located within an environmental control chamber. It is equipped with 4 lasers (405/488/540/633 nm), 3 objectives (20X, 60X (water), 100X SR oil) and an Andor iXon EMCCD camera. The system also has a second laser control system (a Bruker mini-scanner) that allows for simultaneous photo activation while imaging with a 405 laser. The stage has an optional Tokai hit stage top environmental chamber that can control temp, CO2, and humidity. The N-SIM is run via Nikon’s Advanced Research package of Nikon Elements and is capable of multi-dimensional acquisition including time, multi-position and z.

The Jeol 1400Flash Transmission Electron Microscope is a 120 kV transmission electron microscope equipped with AMT 14 megapixel side entry camera for a top resolution of 0.14 nm and wide area observation with image montaging system. The TEM is capable of operation between 40kV and 120kV based on necessity and sample type. The system is also equipped with a single tilt specimen holder for normal 2D imaging, in addition to a penta-specimen holder for high throughput imaging, and a high tilt sample holder capable of sample tilts from -70° to +70° at 0.5° increments for three-dimensional imaging. Because the system has optical microscope linkage function, the system is also capable of correlative light- electron microscopy (CLEM).

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The Leica EMPact 2 High Pressure Freezing Apparatus is an automated system to freeze samples of bigger volume under high pressure, in an extremely short amount of time for better preservation of morphological features and biological processes. The freezing apparatus is capable of freezing cells grown on coverslips, tissue blocks up to 500 microns thick, and suspensions of cells.

The EM Facility is equipped with three RMC PowerTome ultramicrotomes capable of sectioning resin embedded tissues and specimen from 2 microns up to 20 nanometers. The automated system of sectioning enables precise and consistent sectioning of specimen to enable serial sections for array tomography. The ultramicrotomes are equipped with a wide range of diamond knives and diamond trimmers of varying lengths and cutting angles.

The Prepmaster Specimen Preparation Robot is a fully automated system for preparation of biological specimens for TEM with robotic liquid handling. The fully programmable robot automates the processes of fixation, dehydration and resin embedding of biological specimen including tissues and cells. The programs used for automation are customizable to suit the needs of the user and sample requirements. The system is capable of simultaneous processing of up to 96 samples for TEM, while eliminating human errors.

The Quorum Technologies 150T E Sputter Coater/Turbo Evaporator (EMS 150T E) is a sputter coater/turbo evaporator for either platinum or gold for obtaining a fine grain coating of biological samples to enhance surface topology for high resolution imaging. The EMS 150T is also capable of deposition of vaporized carbon on TEM grids to obtain uniform sample support without sample aggregation. The equipment uses an Edwards RV3 rotary vacuum pump to obtain vacuum of 2 x 10-3 mbar to vaporize metals and carbon. The system is fully automated for controlling prehearing, outgassing, time and number of sputter/evaporation bursts for a wide range of use for either TEM or SEM.

The PELCO easiGlow Glow Discharge System is used for leaning TEM grids and hydrophilization of TEM carbon support films.