Washington University Center for Cellular Imaging

WUCCI

The overarching goal of the Washington University Center for Cellular Imaging (WUCCI) is to provide reliable and affordable access to state-of-the-art cellular imaging technologies, provide professional guidance in experimental design, sample preparation and data analysis, develop and apply new imaging technologies and work collaboratively with CDI researchers to facilitate and advance research aimed at enhancing our understanding of pediatric health and disease.

Recent advances in cellular imaging enable unprecedented dynamic and spatial resolution in studies of cells, tissues, and animals, creating exciting opportunities for discovery in a broad range of research aimed an understanding childhood diseases studied in all CDI centers. The WUCCI provides education & training in the use of these advanced cellular imaging and microscopy techniques, including, but not limited to, fluorescence microscopy, confocal microscopy (both fixed and live cell), TIRF microscopy, super-resolution microscopy (both localization and structured illumination approaches), two-photon microscopy, electron microscopy (both transmission, scanning and cryo-EM) and focused ion beam microscopy and X-ray microscopy for three-dimensional ultrastructural investigations at the nanoscale. In addition, the WUCCI houses a state-of-the-art data analysis suite comprising of various commercial and custom-designed quantitative analysis algorithms along with a half-petabyte data storage server. The WUCCI also provides versatile lab space for microscopic sample preparation, sectioning and mammalian tissue culture facilities. The WUCCI is located in the basement of the recently constructed 4515 McKinley Research Building.

The CDI award will support:

  • Micro-grants for current CDI Investigators* (up to $6,000 per year) and CDI center members** (a single grant of $2,000 per year) as a mechanism to offset WUCCI hourly usage fees. 
  • Educational activities focused on emergent imaging technologies of interest.
  • Support for WUCCI technical experts to provide training and assistance in preparing samples and using WUCCI imaging and data analysis resources.

*CDI-funded postdoctoral fellows and PI’s of Educational Initiatives are not eligible to apply as a CDI Investigator for a micro-grant.

** A faculty appointment is required to apply for a micro-grant as a CDI center member.

The capabilities of the WUCCI are summarized below:

A. LIGHT MICROSCOPY

1. Basic fluorescence microscopy / stereoscopy (widefield)

Zeiss Axio Imager Z2 Fluorescence Microscope

The Zeiss Axio Imager Z2 is a motorized upright brightfield / fluorescence microscope equipped with an ApoTome 2 optical sectioning grid imager. The system is equipped with an LED illumination source and filtersets for DAPI, GFP, RFP and far-red imaging as well with the relevant prisms and sliders for Differential Interference Contrast Microscopy (DIC). The system is equipped with two cameras; a Hammatsu Orca Flash sCMOS camera for fluorescence and a color CCD camera for histological imaging.

Zeiss AxioScan Z1 Brightfield / Fluorescence Slide Scanner

The Zeiss AxioScan Z1 is a motorized 100-slide capacity brightfield / fluorescence soide scanning microscope. The system is equipped with an LED illumination source and filtersets for DAPI, GFP, RFP and far-red imaging. The system is equipped with two cameras; a Hammatsu Orca Flash sCMOS camera for fluorescence and a color CCD camera for histological imaging of whole slides.

Zeiss Stereo Lumar V.12 Fluorescence Stereoscope

The Zeiss Stereo Lumar V.12 is a motorized zoom fluorescence stereoscope equipped with LED illumination and filtersets for DAPI, GFP, RFP and far-red imaging as well with a white light source for transmitted light imaging. The system is equipped with two cameras, a Hammatsu Orca Flash sCMOS camera for fluorescence and a color CCD camera for histological imaging.

2. Confocal microscopy (both fixed and live cell)

Nikon A1Rsi Confocal Microscope (inverted, live cell capable)

The Nikon A1Rsi is a scanning confocal microscope is suitable for a broad range of applications including spectral and rapid imaging. The system is based on a motorized inverted Nikon Ti-E microscope frame equipped with the PerfectFocus stability mechanism. The system is equipped with a unique hybrid scan head, incorporating both a high-resolution galvo scanner and an ultra-high speed resonant scanner, enabling simultaneous photo-bleaching / photo-activation and image acquisition. The scan head is also equipped with two GaAsP PMTs, and two high-sensitivity PMT detectors for four-color confocal imaging as well as a transmitted light PMT for simultaneous DIC imaging. The enhanced spectral acquisition capabilities include a 32-channel detector for spectral acquisition at full image (512 x 512 pixel) resolution in 0.6 seconds.

Nikon Spinning Disk Confocal Microscope (inverted, live cell capable)

The Nikon Spinning Disk (SD) confocal microscope is designed for high-magnification 4D imaging of living cells with minimal effects of photo-damage. The system is based on a motorized inverted Nikon Ti-E microscope frame equipped with the PerfectFocus stability mechanism. The system is fitted with a Yokagawa CSU-X1 variable speed Nipkow spinning disk scan head for the confocal illumination and imaging of an entire field of view (FOV) simultaneously on a camera. The scan head is equipped with an Andor TuCam two-camera adapter that enables simultaneous two-color confocal imaging at video-rate (30fps) on two separate Andor Zyla 4.2 Megapixel sCMOS cameras. The system also boasts an LED-based DMD system for ultrafast photo-stimulation and a separate galvo-based laser scanner for slower FRAP and photo-activation studies.

Olympus FV1200 Confocal Microscope

The Olympus FV1200 scanning confocal microscope is a versatile imaging platform for both fixed and living samples based on an upright BX61WI frame. The FV1200 scan head is equipped with two GaAsP PMTs and three high-sensitivity PMT detectors (2 spectral and 1 filter-based) for five-color confocal imaging, and a transmitted light PMT for simultaneous DIC imaging. The spectral PMTs are capable of acquiring spectral image data at 2nm resolution. The system is also equipped with a separate high-speed SIM laser scanner enabling simultaneous photo-bleaching / photo-activation and image acquisition

3. Two-photon microscopy (both fixed tissue and in vivo intravital)

Zeiss LSM 880 Airyscan Two-Photon Microscope (inverted, live cell / tissue capable)

The Zeiss LSM 880 Airyscan NLO two-photon microscope is suitable for a broad range of applications including spectral and rapid imaging as well as deep tissue imaging. The system is based on a motorized inverted Zeiss Axio Observer Z1 microscope frame. The system is equipped with a unique scan head, incorporating a high-resolution galvo scanner along with two PMTs and a 32-element spectral detector as well as a transmitted light PMT for simultaneous DIC imaging. In addition, the Airyscan unit provides sub-diffraction limited imaging down to 140nm lateral resolution. The system is also equipped with four non-descanned detectors (NDDs) for two-photon imaging in thick tissue samples.

4. Super-resolution microscopy (both localization and structured illumination)

Nikon n-SIM Structured Illumination Microscope (inverted)

The Nikon n-SIM microscope allows for sub-diffraction super-resolution imaging using high-frequency structured illumination in three-dimensions for resolutions beyond 100nm. The system is based on a motorized inverted Nikon Ti-E microscope frame equipped with the PerfectFocus stability mechanism. Fluorescence detection is provided by two Andor Zyla 4.2 Megapixel sCMOS cameras fitted to an Andor TuCam dual camera adapter, which facilitates simultaneous two-color and sequential four-color super-resolution imaging.

Nikon n-STORM TIRF Microscope (inverted, live cell capable)

The Nikon n-STORM TIRF Microscope allows imaging close to the surface of a coverslip facilitating imaging of endo- and exo-cytic processes as well membrane trafficking events. The system is based on a motorized inverted Nikon Ti-E microscope frame equipped with the PerfectFocus stability mechanism. Fluorescence detection is provided by either an Andor Zyla 4.2 Megapixel sCMOS camera when imaging in TIRF mode or an Andor iXon 897 EM-CCD when imaging in single-molecule or STORM (STochastic Optical Reconstruction Microscopy) super-resolution imaging mode.

B. ELECTRON MICROSCOPY

1. Transmission Electron Microscopy

JEOL JEM-1400 TEM

The JEOL JEM-1400Plus is a 120kV Transmission Electron Microscope (TEM) that boasts high resolution and high contrast imaging on an easy to use platform. The system features 0.38nm point resolution and is configured for imaging at either 80kV or 120kV. It has a 5-axis high-precision Piezo motorized stage suitable for large-area montages and can accommodate tilt angles up to + /-10˚ for anaglyph. The system is equipped with an AMT XR111 high-speed 4k x 2k pixel phosphor-scintillated 12-bit CCD camera, which features vibration-free Peltier cooling and 12µm pixel sizing.

FEI Titan Krios G2COMING SOON - MARCH 2017

The FEI Titan Krios G2 is a 300kV Cryo-Transmission Electron Microscope (Cryo-EM) that is the ultimate platform for the atomic resolution imaging of cryogenically preserved samples. It is a state-of-the-art, dedicated imaging robot, optimized for both the high-resolution three-dimensional imaging of proteins and macromolecular assemblies, and the high-resolution, dual-axis, tomographic imaging of cellular samples. It combines excellent performance with ease of operation in the default TEM mode, as well as additional imaging modes, such as STEM, TEM/STEM tomography, EFTEM and Phase Contrast. The platform is equipped with a unique, automated sample loader/unloader for sample handling at liquid nitrogen temperatures.

2. Scanning Electron Microscopy (SEM)

Zeiss Crossbeam 540 FIB-SEM

The Zeiss Crossbeam 540 is a Focused Ion Beam Scanning Electron Microscope (FIB-SEM) workhorse that is available for both analytical imaging of whole samples and 3D nanotomography of embedded samples. Equipped with a GEMINI II column, this SEM has superb low kV performance, and the ability to fine-tune lens parameters for optimizing imaging quality and acquisition speed. The voltage can be adjusted from 0.02-30 kV (10pA to 40nA probe current) with 3.0nm resolution at 0.2kV, 1.8nm resolution at 1kV, and 0.9nm resolution at 15kV. In order to accommodate a large array of sample types, the system is equipped with the following detectors: 

  •        Everhart-Thornley Secondary Detector
  •        In-lens Secondary Detector
  •        4-quadrant Solid State Backscatter Detector
  •        In-lens Energy Selective Backscatter Detector
  •        Solid State STEM Detector 

The Crossbeam 540 is also equipped with a Capella Ga-Liquid Metal Ion Source (Ga-LMIS) column that is capable of 3nm milling resolution at 30kV. The column can be set to 0.5-30kV (1pA to 100nA probe current). When coupled with the unique nature of the GEMINI II column, imaging can be performed using only the SEM column, only the FIB column, or simultaneous SEM imaging/FIB imaging or milling for easy monitoring of routines. For masking and charge compensation purposes, the instrument is equipped with two Orsay gas injection systems with both Pt and C precursor gasses.In addition to the included imaging software, the Crossbeam 540 is also equipped with the ATLAS 5.0 scan engine which boasts gigapixel imaging capabilities (50k x 40k pixels) for large-area imaging projects. In addition, the software comes with two imaging modules:

  • Array Tomography – This module allows the automated imaging of biological serial sections for 3D analysis of large volumes.
  • 3D Tomography – This module offers an automated solution with unique, high-speed proprietary workflows for the automatic acquisition of FIB tomography datasets.

Zeiss Merlin SEM COMING SOON - OCTOBER 2016

The Zeiss Merlin SEM is a workhorse scanning EM that is available for analytical imaging of whole samples. Equipped with a GEMINI II column, this SEM has superb low kV performance, and the ability to fine-tune lens parameters for optimizing imaging quality and acquisition speed. The voltage can be adjusted from 0.02-30 kV (10pA to 40nA probe current) with 3.0nm resolution at 0.2kV, 1.8nm resolution at 1kV, and 0.9nm resolution at 15kV. In order to accommodate a large array of sample types, the system is equipped with the following detectors: 

  •          Everhart-Thornley Secondary Detector
  •          In-lens Secondary Detector
  •          4-quadrant Solid State Backscatter Detector
  •          In-lens Energy Selective Backscatter Detector
  •          Solid State STEM Detector 

C. X-RAY MICROSCOPY

Zeiss Versa 520 X-ray Microscope

The Zeiss Versa 520 is a highly versatile platform allowing sub-micron imaging of large sample volumes. The Versa is unique in that it offers Resolution at a Distance (RaaD) in addition to being a completely non-destructive imaging modality. The systems offers 700nn resolution over inches of sample volume.

D. SAMPLE PREPARATION

1. Thin Sectioning

Leica UC7 Ultramicrotome

The thin sectioning room houses two UC7 ultramicrotomes for routine and ultrathin EM sectioning. These units both have touchscreen control, Multi-LED illumination, eucentric movement for the microscope, and automated routines for trimming and sectioning. In addition, each unit features a digital camera coupled to a 50” LED TV for ergonomic and teaching purposes. For Tokuyasu or cryo-sectioning, the center also has a Leica FC7 cryo stage attachment for the UC7. This unit boasts three temperate zones for precise sectioning control, anti-contamination airflow, and a CRION antistatic device with discharge mode.

Leica KMR3 Glass Knife Maker

The Leica EM KMR3 knifemaker is a complete tool-free unit that scores, clamps, and breaks glass strips into knives for ultramicrotomy. Available glass strips are 6.4mm thick.

2. Sample Preparation

Leica EM ICE High Pressure Freezer

The EM ICE is a unique workstation that combines both high-pressure freezing and light stimulation with millisecond precision. Unlike previous approaches, in this case no alcohol or cryoprotectants are required for operation, and an automated loading system allows for one-second fresh-to-frozen cycles. In addition, the system allows for freezing specimens up to 5mm in diameter, and accommodates a variety of sample holders including sapphire disks (3 mm and 6 mm), freeze fracture holders, and membrane carriers. Light stimulation is achieved via easily exchangeable LED light modules. Currently, the system has blue (450 λ), green (523 λ) and amber (597 λ) modules. Light stimulation is achieved by easy to program routines through the system software and can be customized for any duration or number of pulses. After stimulation, the sample is instantly frozen within one millisecond, precisely capturing any dynamic process. 

Leica AFS2 Freeze Substitution Unit

The AFS2 is a fully automated freeze substitution system for processing samples after they have been high pressure frozen. It also includes the EM FSP automatic robotic processor with integrated UV LEDs for user-free operation. 

Leica CPD300 Critical Point Drier

The EM CPD300 is a fully automated critical point drier for the gentle drying of samples for SEM analysis. It is compatible with samples that have been dehydrated in ethanol, methanol, acetone, and isopropyl alcohol. This unit can accommodate small to medium (~75 x 40mm) sized samples, and has dedicated holders for drying multiple coverslips. Runs can be fully customized with number of cycles and fluid exchange speeds to accommodate a large variety of sample types. 

Leica ACE600 Sputter / Carbon Coater

The ACE600 coater is a high-resolution turbo-pumped combination sputter coater, carbon thread evaporator, and glow discharge device. It features a motorized, planetary drive stage with quartz crystal thickness monitor. Foil targets for sputtering include: Iridium, Platinum, and Gold.

E. OTHER FACILITIES

  •          Wet / chemical lab space for sample prep including sample freezing
  •          Sectioning space for thin section production for TEM
  •          BSL-2 tissue culture space including BSCs and incubators
  •          Animal holding closet for short-term (< 24hr imaging studies)
  •          High-end Data Analysis Workstations for 3D/4D image analysis
  •          500TB Data Storage Server

 

For additional information please email the WUCCI Scientific Director, James Fitzpatrick, Ph.D. at fitzp@wustl.edu.


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