Imaging Technology
Seeing further into the microscopic structure of biological samples in native, or near-native, conditions will provide the basis for discoveries that may change the face of the life sciences.
The Facility, which will open in June 2012, will provide services to the entire Howard Hughes Medical Institute community. The Facility will support high-throughput, high-quality data collection, and provide services for cryoEM sample preparation, and certain data processing and 3D reconstruction.
Equipment:
FEI Titan Krios: This is one of the most highly configured cryoEMs in the world. Equipped with an Image Corrector for lens aberration correction and a Falcon camera for direct electron counting, this Krios offers new possibilities for high resolution and better contrast under low-dose conditions. Other key components include a Gatan Image Filter for energy filtered transmission electron microscopy (EFTEM) imaging and an X-FEG gun for high brightness. Depending on the experiment, this Krios can be operated at four different high tensions: 80, 120, 200 and 300 kV. This microscope is optimized to perform single particle data collection, electron tomography and electron crystallography under liquid nitrogen cooling. If needed, it can also be operated at room temperature on conventional samples, such as plastic sections.
FEI Tecnai G2 Spirit: This transmission electron microscope (TEM) operates at 120 kV with a LaB6 filament. It is equipped with a Gatan 626 cryo-holder for the examination of cryoEM grids under liquid nitrogen cooling. It can also be operated at room temperature on conventional samples, such as plastic sections.
FEI Vitrobot: This is the main tool used to prepare cryoEM grids for biological samples thinner than 800 nm, such as small bacterial cells, and virus and protein complexes. It can be operated with controlled humidity, temperature, blotting force, and time to increase the reproducibility of suitable cryoEM grids.
Data processing workstations and software: There will be a Linux workstation with most cryoEM data processing software (IMOD, SPIDER, Freealign, EMAN, Bsoft, etc.) installed both for preliminary data processing and final 3D reconstruction. There will also be a Windows workstation provided by FEI with Inspect3D installed for tomogram reconstruction.
Services:
- Preparation of cryoEM grids for protein/protein complex, virus and thin cell using a Vitrobot
- Cryo-EM imaging under low-dose conditions at liquid nitrogen temperature
- Electron tomography and 3D reconstruction
- Single-particle analysis data collection
- Energy-filtered TEM
- Electron energy loss spectroscopy
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The Electron Microscopy Facility provides researchers at Janelia Farm Research Campus with state-of-the-art instrumentation, services, and training in high-resolution scanning electron microscopy, transmission electron microscopy, and image analysis. Facility staff accommodate researchers' electron microscopy needs, and are also available to empower users with training in the science and practice of electron microscopy.
The facility has 5,000 square feet of space. Sample preparation is available by conventional wet chemistry, microwave techniques, and high-pressure freezing/freeze-substitution. There are two chemical fume hoods for specimen processing. Specimen preparation equipment is available for self-use after appropriate training; however, researchers may request staff assistance for preparing and examining specimens.
Equipment:
- FEI Tecnai G2 20 TWIN electron microscope with LaB6 gun and Gatan Ultrascan 4k x 4k quad port digital camera.
- FEI Spirit BioTWIN electron microscope with LaB6 gun and Gatan Ultrascan 4k x 4k quad port digital camera.
- Zeiss Nvision 40 FIB SEM for 3D nanoscale high-resolution imaging.
- Wohlwend HPF Compact 01 high-pressure freezer for freezing suitably-sized unfixed, uncryoprotected samples without significant ice crystal damage.
- Leica EM AFS2 automatic freeze-substitution system for processing samples prepared by high-pressure freezing or progressive lowering of temperature (PLT) methods. A built-in UV lamp provides for UV-catalyzed resin polymerization at low temperature.
Leica UC6 ultramicrotomes for room temperature semi-thin and ultra-thin sectioning. Each microtome is positioned in a separate room with adequate bench space and ancillary specimen preparation equipment so as to be self-contained sectioning areas. A Leica KMR2 knifemaker is available for making glass knives.
- EMS 100 Glow Discharge unit for making support films on hydrophilic grids.
- Denton Explorer 14 Auto high-vacuum deposition system or carbon and metal coating. The unit is equipped with a quartz thin film monitor and rotary stage.
- Pelco BioWave Pro microwave oven with ColdSpot, SteadyTemp, and vacuum chamber options for maximum microwave sample preparation flexibility.
Services:
- Operation of microscopes and acquisition of images
- Sample fixation and processing
- Sample sectioning and staining for TEM
- Development of protocols for special research needs
- Training
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Drosophila brain reconstructed by confocal microscopy.
The Facility enables the development and use of state-of-the-art methods like super-resolution imaging and the newest-generation optical equipment, plus software and instrument solutions for specimen control and image analysis. The Facility also serves as a place for researchers to experiment with a wide variety of approaches and options, for example, selecting systems that will benefit the whole campus, or assisting in the adaptation of new microscopes for Janelia researchers. The facility also provides courses and instruction on optics and imaging principles that strengthen education and research potential.
The Light Microscopy Facility has 2,000 square feet of space with state-of-the-art equipment to support the needs of researchers in light microscopy. The manager and staff supervise three point-scanning confocal microscopes, one with optics for 2-photon imaging; three widefield microscopes for preparing montages, live cell, and color imaging; a high-throughput scanning system; and other specialized microscopes. In addition there are three workstations dedicated to image analysis that can handle large image files. Most of the time, microscopes are reserved for self-use on an on-line equipment scheduler; however, researchers may request staff assistance for acquiring and analyzing images. The facility staff also offer a course on microscope optics and provide training on how to operate specialized software for image acquisition and processing.
Equipment:
- Zeiss 710 confocal microscopes (two complete systems), a versatile point scanning/PMT-based system with built-in spectral detector and seven laser lines. One system is equipped for optional 2-photon imaging with two non-descanned detectors and a highly sensitive GaAsP detector.
- Zeiss 510 confocal microscope with Meta multi-spectral imager, versatile point scanning/PMT-based system with seven laser lines and optical/computational system for eliminating bleedthrough when using multiple fluorochromes.
- Zeiss Axioimager with ApoTome. This station is frequently used for image tiling (montages) of component images for large sections, such as mouse brain and rat brain sections. Apotome is an attachment for structured illumination microscopy for improved S/N performance when imaging moderately bright specimens.
- Olympus IX81 inverted microscope. A fully motorized inverted microscope with sensitive EM-CCD camera is available for high-sensitivity live-cell detection, deconvolution, and highest quality fluorescence imaging. There is also an Olympus DSU (disk spinning unit) with Rolera MGi EM-CCD camera for sensitive spinning disk confocal imaging. Brightfield and DIC modes are also available.
- Olympus/Hamamatsu Nanozoomer. An automated slide scanning system can hold up to 210 slides and scan images at 20x or 40x magnification, in three colors, and at 8-bit or 12-bit resolution. Nanozoomer image files can be exported in .tif and other file formats.
- Neurolucida system. An Olympus BX51 upright microscope with Neurolucida software is used for tracing neurites and constructing neuron contours in tissue sections.
- Color histology/pathology. An Olympus BX51 upright microscope with a 12-bit Olympus DP71 color camera for convenient accurate color photography.
- Fluorescence dissecting microscopes (two). Olympus MVX-10 “macroscope” with giant 0.5 NA lenses and highest fluorescent throughput to CCD camera. With 0.5-50X zoom range on DP71 color camera, these microscopes allow zooming to moderately high magnification without a decrease in brightness.
- Abbe refractometer and HP Designjet 90r inkjet printer
Software:
The facility uses the following image analysis software:
- Zen and LSM confocal software, and Axiovision (Carl Zeiss Microimaging)
- Volocity (Improvision), V3D (JFRC), and ImageJ (NIH) software for 3D display and voxel quantitation
- iVision for image segmentation and quantitation (BioVision)
- Slidebook for image capture with fluorescence ratio-imaging and deconvolution extensions (3I)
- DPI imaging for color histology (Olympus)
- Neurolucida for neuron tracing (MicroBrightfield)
Services:
- Training for self-use of confocal and widefield microscopes
- Operation of microscopes and acquisition of images
- Assistance with image processing and display
- Performance of image analysis and measurement and counting operations
- Assistance with the setup of equipment for live cell imaging
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