[This review of RRC organization and services is provided for investigators who need information about the RRC for inclusion in their grant applications. There are two versions, Long Version and Short Version. Please feel free to include all or any portion of these documents into your grant as your needs require.]
The Research Resources Center (RRC) of the University of Illinois at Chicago (UIC) is a division of the Office of the Vice Chancellor for Research. Oversight of the management of the RRC is the responsibility of the Associate Vice Chancellor for Research Resources.
The RRC has been providing core support for research at UIC for decades. It provides equipment, training, and a variety of research support services for campus investigators. At present its research support services consist of confocal microscopy, flow cytometry, electron and Raman microscopy, NMR spectroscopy and micro-imaging, mass spectrometry, a DNA laboratory, a genomics facility, a protein laboratory, a macromolecular structure facility, a small molecule X-ray diffraction facility, a transgenic production service, electronic and machine shops, a computing support group and a storeroom operation. Whenever possible, service laboratories are located close to the major concentration of investigators. The electron microscopy service has laboratories on both east and west campuses. In the past few years the RRC has expanded markedly, due in part to the increase in the interest in and need for, biotechnology services and in part due to increased demand for other services, notably EM. The RRC currently has thirty-nine full-time employees.
The RRC's services are determined by faculty research needs, which mirror current scientific trends. In light of the need for faculty input, an Executive Advisory Panel consisting of six faculty members appointed by the Associate Vice Chancellor for Research Resources along with the Associate Dean of Research from the five research intensive colleges advise the RRC with respect to the long-term needs of the faculty. In addition, smaller, three-member committees consisting of funded users of each research service advise the facility directors and the RRC administration on the direction they foresee as being potentially most fruitful for the facility/service they advise.
The UIC administration supports the RRC by providing state dollars. These cover academic salaries and contribute to new equipment purchases. This level of state support permits user fees to be utilized not only for routine operating costs but for equipment upgrade and replacement. Another important source of funding is grants generated by campus investigators and when necessary supplemented with matching money from the campus, colleges, departments and/or the RRC itself for the purchase of equipment that is placed in, inventoried to, and managed by the RRC.
Nearly 1,000 investigators made use of RRC services in the last fiscal year. Income collected for that usage has increased substantially over the last five years with approximately $1.7 million being generated in FY04.
The RRC's organizational structure consists of three general components: Administrative Oversight, Research Support and Technical Support.
Since the UIC campus has two main components (east and west) which are located approximately one mile apart, research services must be made available to investigators in geographically distributed locations. The Associate Vice Chancellor for Research Resources administers the RRC with the help of two directors. The Director of RRC East has oversight for electron microscopy and spectrometry/spectroscopy while the Director of RRC West has oversight for biotechnology services. An Associate Director (MBA, MS) for administrative services oversees accounting, personnel, and the technical support units. The RRC also has several Academic Directors who are tenured faculty with active research programs who provide academic oversight of the service units.
The Confocal Microscopy Facility (CMF) is equipped with Zeiss LSM510 and LSM510 META confocal laser scanning microscopes. They are designed for analysis of living or fixed biological specimens in a multi-user environment. Using the “Z-Stack” software module, both microscopes can scan through a sample and obtain one or a series of high-resolution optical sections. Users then have the option of utilizing a Z-Stack (a series of images) to render a 3D reconstruction of the sample. The time-lapse imaging function can be used for kinetic studies of live cells with GFP, calcium indicator, FRAP, photoactivation applications, etc. Since both microscopes use the same software packages, there is no retraining necessary for use of either microscope. An advanced function of the Zeiss LSM 510 and META microscopes is the multi-tracking scanning capability that minimizes signal crosstalk while working with multiple fluorescence signals. Both microscopes also have a transmitted DIC channel.
Capabilities of the LSM 510 and the LSM 510 META:
An AIS2 automatic microinjection system is available that allows the investigator to operate the system entirely via the computer workstation and inject cells by pointing and clicking, which results in high injection rates of about 1,500 cells per hour.
The injected cells can be marked for relocating and avoiding double injection. A unique aspect of AIS2 system in our facility is its combination with the LSM 510 META confocal microscope, which allows the user to trace interesting targets with the time-lapse program. Users may rapidly switch between these two systems by clicking one button. The combination of confocal microscopy and microinjection techniques bridges the gap between in-vivo physiology and in-vitro biochemistry and molecular biology. Investigators can study complex cellular processes, structure and function in vivo using single cells.
To facilitate the production of figures for publication from digital images, an image station is available with Adobe Photoshop, NIH image PC, and Zeiss LSM5 software for processing data collected from either confocal microscope.
The DNA Services (DNAS) facility provides DNA sequencing services using ABI automated sequencing instruments. Our primary instruments are an ABI 3100 and a 3730 Genetic Analyzer. With properly purified template, we provide greater than 99% accuracy over more than 600 base pairs (with total reads up to 800 bp). The services include complete sequencing from bacterial cultures, sequencing plasmids, PCR products or other templates provided by users, and performing sequence analysis on reactions performed by users with the dye terminator chemistry. As a free service, sequence chromatograms are analyzed and edited by our staff. In addition, the facility provides oligonucleotides at extremely competitive prices. The facility supports an Applied Biosystems freezer program that stocks enzymes, DNA purification kits, and reagents for investigators.
The facility also offers real time PCR and sequence detection service with an ABI 7900HT Sequence Detection System. This state of the art system allows detection and quantitation of genomic DNA or RNA thus allowing the user the ability to perform very sensitive, accurate, and reproducible measurements of gene expression levels. This is done with either the SYBR Green or TaqMan primer-probe protocols. In addition, this instrument can be used in other applications. It is able to detect single nucleotide polymorphorisms, SNPs, through the use of a fluorogenic 5' nuclease assay, viral load measurements for clinical purposes or PCR optimization conditions. After an initial training session, users are able to run and analyze their own experiments. An additional workstation with appropriate software is also available to assist with primer and probe design.
The Electron Microscopy Service (EMS) is a central facility offering instrumentation, training and service using scanning (SEM & Microprobe), transmission (TEM) and scanning transmission (STEM) electron microscopy, surface analysis (XPS), oxide film growth (MBE) and vibrational spectroscopy (Raman). EM instruments and services are located in two laboratories, one on each side of campus.
Instrumentation in the EMS includes four TEMs for materials science applications, one TEM for life science applications; two SEMs used by both life science and materials science users, a microprobe, XPS, MBE and a Raman Spectrometer. Equipment for life science specimen preparation includes freeze etching, freeze substitution, critical point drying, shadowing/coating and ultramicrotomy. Equipment for materials science specimen preparation includes coating, slicing, disc cutting, polishing, ultramicrotomy, electrochemical and chemical thinning and ion beam milling. Darkroom facilities are available in the west-side laboratory, but not on the east side where all images from the TEMs are output digitally.
The materials science TEMs are a JEOL JEM-2010F field emission TEM/STEM, a JEOL JEM-3010 TEM, a VG Microscopes HB601UX dedicated STEM and a JEOL JEM-100CX. The JEM-2010F is a 200kV instrument with a lattice resolution of 0.1nm in TEM and a minimum STEM spot size of 0.13nm. It also has a Gatan Imaging Filter which allows Electron Energy Loss Spectroscopy (EELS) and Energy Filtered Imaging, and a Thermo Noran Vantage EDX system. The JEM-3010 is a 300kV high resolution TEM with a lattice resolution of 0.14nm and is fitted with a Gatan Slow Scan CCD camera, intensified TV rate camera and a Thermo Noran Vantage EDX system. Heating (1000°C) and Cooling (-170°C) double tilt holders are available for these two microscopes. The HB601UX instrument will be used as the basis of an aberration corrected microscope with the aim of getting a sub 0.1nm spot size. The 100CX has a lattice resolution of 0.2nm and is used for teaching and training.
The Life Science TEM is a JEOL JEM-1220. It is a 120kV instrument fitted with a Gatan Slow Scan CCD camera and has a lattice resolution of 0.2nm.
The two SEMs are a JEOL JSM-6320F Field Emission SEM and a Hitachi S-3000N Variable Pressure SEM and the Electron Microprobe is a JEOL JXA-733. All are fitted with EDX systems, and the JXA-733 also has WDX. The JSM-6320F has an imaging resolution of 1.2nm at 15kV and 2.5nm at 1kV and has a Thermo Noran Voyager EDX system. The S-3000N has an imaging resolution of 3.5nm at 25kV in high vacuum mode and 5nm at 25kV in variable pressure mode and has an Oxford Inca system. The JXA-733 has an imaging resolution of 10nm and has an EDAX system interfaced to a Tracor Northern detector, which is integrated with the Advanced Microbeam WDX system, controlling five WDX spectrometers. The JEM-1220 and JSM-6320F were both installed in 1997, and the JEM-2010F and JEM-3010 in 1998, as part of a general effort to upgrade microscopy facilities on campus. The S-3000N was purchased in 2000.The surface analyzer is a Kratos AXIS-165. It is fitted with dual Mg/Al X-ray source and a monochromatic Al X-ray source. It has an ion gun for depth profiling and is capable of spectroscopy and imaging with an X, Y resolution of 30µm and depth resolution of 8-10nm.
The MBE system is an Applied EPI Mod Gen II.
The Raman Spectrometer is a Renishaw Ramascope 2000 interfaced to a Leica DM LM optical microscope with a CCD camera. Spectroscopy and imaging with a resolution of 1 micrometer is possible.
The Flow Cytometry Service (FCS) maintains six flow cytometers, two of which are also cell sorters. A Beckman-Coulter Elite ESP is used for routine sorting, while our newest instrument (acquired in 2003) is a state of the art high-speed sorter, the DakoCytomation MoFlo. All of the other instruments are bench-top flow cytometry analyzers. The Elite and MoFlo are each capable of analyzing and sorting thousands of cells/particles per second. Five to eleven parameters (relative size, granularity, and up to three/nine colors of emitted fluorescence) can be measured simultaneously and correlated particle by particle. The signals can be instantly collected into histograms for immediate results and/or stored as raw data for experimental analysis later. The data may also be transmitted to a stand-alone FCS PC workstation for analysis, either individually or in batch, and the results printed as user-designed hard copies of publishable quality.
Five of our flow cytometers can be used to analyze and quantitate autofluorescent proteins, antigen or ligand density, apoptosis, enzyme activity, DNA and RNA content, membrane potential, cytokine receptors and their synthesis, drug uptake and efflux, phagocytosis and viability of cells, isolated nuclei, organelles or microorganisms. Changes in cell cycle, intracellular pH, intracellular calcium, intracellular glutathione and oxidative burst can be also detected with these instruments. Time may be used as a parameter for kinetic studies.
Our newest flow cytometer,the Bio-Rad Bio-Plex, is a protein array system designed by Luminex to analyze multiplex microsphere arrays, which is an alternative to using ELISA assays to determine the concentration of proteins in solution (cell lysates). Standard curves of known concentration of all analytes are automatically computed, and all data from the standards and unknowns can be displayed in graphic or table form. The results can be saved and exported in raw, graphic, or table form whichever is preferred. Examples of proteins that can be analyzed with commercially prepared antibody coupled Luminex microspheres are human/murine/rat cytokines, human/murine phosphoproteins, human growth factors, human kinases, and transcription factors.
The FCS also maintains an automatic magnetic bead sorter (autoMACS from Miltenyi Biotec) that is often used as a pre-sorter to enrich rare populations of cells before sorting for greater purity on one of our sorting flow cytometers.
The Core Genomics Facility (CGF) provides state-of-the art equipment, technical resources and bioinformatics support for Affymetrix array and glass slide DNA and protein array processing, glass slide DNA array preparation, and data analysis. CGF provides the following services: Affymetrix GeneChip array hybridizaton and data acquisition, hybridization and scanning of glass slide DNA and protein arrays, custom preparation of glass slide DNA arrays, access to discount pricing on Affymetrix GeneChip arrays, access to IMAGE Consortium human cDNA clone collection, specialized software for data acquisition and analysis, and bioinformatics support. Equipment includes the Affymetrix GeneChip System (including Scanner 3000 with Workstation and Fluidics Station 450), OmniGrid Accent Array System (Genomic Solutions), a dual-laser confocal scanner (ScanArray Lite from Packard Biochip). Software solutions for data acquisition, quantification and data analysis include ScanArray and QuantArray (Packard Biochip), GeneChip® Operating Software (Affymetrix),S+Array Analyser (Insightful), PathwaysAssist (Ariadne Genomics).
The Macromolecular Structure Facility (MSF) uses x-ray diffraction for the determination of the structures of proteins, DNA or RNA, etc., at atomic resolution. A single-crystal sample of approximately 0.1 millimeters on edge is mounted in a glass capillary tube or frozen on a nylon loop. The crystal is then rotated along a single axis while being irradiated with a nearly monochromatic x-ray beam. The majority of x-rays pass through the sample, but the remaining x-ray photons are diffracted off the atoms and are then recorded on an imaging plate detector analogous to a piece of photographic film. The resulting intensities are transformed to represent the electron density of the molecule, and then the individual atoms are placed within the density to arrive at the final atomic structure of the protein, DNA, RNA, etc. The samples are typically cooled to 100K in order to reduce sample degradation by the x-ray beam.
The facility houses two area detector instruments (an older Rigaku R-AXIS-IIc and a new Rigaku R-AXIS-IV++) coupled with an x-ray generator, focusing mirror optics and low-temperature cooling systems. Both systems are computer-controlled (Windows 2000 or XP) for data collection. The processing of intensity data is carried out on a PC using CrystalClear™ or elsewhere using HKL2000, XDS, Mosflm, or d*trek. At present, the MSF is available only on a subscription basis. The RRC provides technical support services to the facility.
The Small Molecule X-ray Diffraction Facility (SMXRD) uses x-ray diffraction for the determination of the structures of small organic and inorganic compounds at atomic resolution. A single-crystal sample, ranging in size from 10 to 200 microns, is cooled to –50C at the end of a glass fiber or nylon loop. The sample is then rotated while being irradiated with a nearly monochromatic x-ray beam. The resulting pattern of images is then processed and transformed to a map of electron density depicting the atomic structure of the sample compound. The raw data are archived, and a final report is prepared.
At present, data collection and analysis are only carried out by the facility manager. Individual samples are screened, and a set fee is charged for the service. The RRC provides technical support services to the facility.
The Mass Spectrometry Laboratory (MSL) serves as a research core facility and resource for the analysis of a wide variety of molecules ranging from low molecular weight volatile compounds to high mass polymers and biopolymers such as proteins. Types of measurements include qualitative and quantitative analysis, purity assessment, molecular weight determination, and high-resolution exact mass measurements for the determination of elemental composition. The hyphenated techniques of gas chromatography mass spectrometry (GC-MS) and liquid chromatography spectrometry (LC-MS) permit the composition of complex mixtures to be analyzed, and tandem mass spectrometry (MS-MS) provides information regarding molecular structure.
Several instruments are in use. The JEOL GCMate II is a double focusing magnetic sector mass spectrometer equipped with ion sources for electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) by direct insertion probe and is capable of exact mass measurements for elemental composition determinations. The spectrometer is equipped with high energy CID linked scan tandem MS-MS for use in structural analysis. The mass range is from a low of m/z 1 at full accelerating voltage up to m/z 3000 at reduced accelerating voltage. Resolution is user selectable from 500 up to 6000. An Agilent 6890 GC with auto sampler interfaced to the GCMate allows GC/MS by EI or CI for mixture analysis. The ThermoFinnigan LCQ Classic quadrupole ion trap mass spectrometer is equipped with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) ion sources and is interfaced to a Thermo Separation Products high performance liquid chromatography (HPLC) system. The HPLC consists of an auto sampler, gradient HPLC pumps and a dual-wavelength UV absorbance detector for LC-UV-MS-MS. The spectrometer is capable of sequential ion selective MS/MS up to 10 times. The mass range is from a low of m/z 15 up to m/z 200 in a special low mass range mode or from a low of m/z 50 up to m/z 2000 in normal mass range mode at unit mass resolution and a peak width of m/z 0.25. In zoom scan mode data is acquired at 10,000 resolution with a mass range of m/z 10.
The Micromass Quattro II triple quadrupole mass spectrometer is equipped with electrospray ionization, APCI, and a Waters Alliance HPLC for LC-MS-MS. The HPLC system consists of gradient HPLC pumps, refrigerated auto sampler, and a dual-wavelength UV absorbance detector for LC-UV-MS-MS. The mass range is m/z 4000 with unit mass resolution. The high-resolution Micromass QTOF-2 hybrid mass spectrometer is equipped with electrospray and nano-electrospray ionization for proteomics applications, APCI, and a Waters Alliance HPLC system for LC-UV-MS-MS. The QTOF-2 is capable of high-resolution exact mass measurements in both MS and MS-MS modes. The mass range is up to m/z 10,000 and the resolution is 10,000 full scan. The ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer is equipped with electrospray and APCI. The HPLC system consists of a refrigerated auto sampler, gradient LC pumps, and photo diode array UV/Vis absorbance detector for LC-UV-MS-MS. This is a highly sensitive and unusually high resolution triple quadrupole instrument. The Applied Biosystems, Inc. Voyager-DE PRO PS1 matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer provides high mass range, high sensitivity, and high throughput MALDI spectra of biopolymers such as proteins and oligonucleotides as well as organic prepolymers and other large organic molecules. It is equipped with post source decay (PSD) for use in peptide sequencing. An Agilent 1946A LC-MSD quadrupole mass spectrometer is equipped with electrospray, APCI and an Agilent 1100 HPLC system with refrigerated autoinjector and photodiode array detector for LC-UV-MS.
The Nuclear Magnetic Resonance Laboratory (NMRL) offers NMR techniques for both spectroscopy and imaging (microMRI). Several spectroscopy probes are available for determining the structure of compounds, the interactions of drugs with proteins, cellular pH, in vivo metabolite levels and the metabolic fate of nonradioactive tracers. The microMRI accessory can accommodate samples up to 1 cm in diameter. It can provide a proton image in 3 dimensions, allow the study of diffusion of molecules, and acquire proton spectra in a specific volume within the sample. Both spectroscopy and imaging are nondestructive and are available on the Bruker AVANCE-500. A Bruker AVANCE-360 MHz spectrometer is available for routine spectroscopy for H-1, F-19, P-31 and C-13 nuclei. Both NMR spectrometers are broadbanded and are linked to the Web so that data can be off-loaded via SFTP to computers running user supplied NMR/MRI processing software.
The Protein Research Laboratory (PRL) provides comprehensive services related to protein studies. Services include peptide synthesis, protein sequencing, protein purification, antibody production, amino acid analysis, western blotting, PCR, ELISA, circular dichroism spectroscopy, 1-D and 2-D gel electrophoresis, proteomics, and recombinant protein production. Major instruments include an Applied Biosystems 477 automatic n-terminal protein sequencer with Edman degradation chemistry; an ABI 433 peptide synthesizer and a Rainin Symphony multiple peptide synthesizer; ABI 392 and 394 oligo synthesizers; a Waters PICO-TAG amino acid analysis system; an ABI Voyager DE-Pro MALDI-TOF Mass Spectrometer; ; a Agilent MSD ion-trap XCT with on-line nano LC and nano electrospray ion source (access through a laboratory at NWU); a Finnigan TSQ mass spectrometer with electrospray ionization and triple quadruple detector; a Jasco 710 circular dichroism spectropolarimeter with programmed temperature control for measuring the secondary structure and transition temperature of a protein; a Micro-Tech MicroLC HPLC system with a flow rate as low as 300 nl/min for a 100 micrometer column; an HP1100 HPLC system with variable-wavelength UV-visible, photodiode array, fluorescent, light-scattering and mass spectrometric detectors; a BioRad isoelectric focusing (IEF) Rotofor; 2-D SDS-PAGE systems using immobilized pH gradient strip for the first dimension (Pharmacia, BioRad) and precast SDS-PAGE gels as the second dimension (Novex, BioRad); a BioRad Chemi-Doc system fluorescent gel imager with a cooling CCD camera; a Bio-Rad Proteomeworks System including high-throughput 2-D gels, a Gel Spot Cutter and a Bio-Rad FX Pro Plus Multi-Imager; a Hybaid PCR machine with temperature gradient; an Awareness Tech ELISA reader; a Linde liquid nitrogen cell storage system; and an Olympus fluorescent inverted microscope. All services include consultation and assistance according to the researcher's needs. Users are strongly encouraged to discuss their specific requirements with the staff before submitting their samples.
The Transgenic Production Service (TPS) generates transgenic and gene-targeted (knockout) mice. Services include pronuclear microinjection of the DNA construct (transgene) into mouse zygotes for the production of transgenic founders, injection of the targeted ES cell lines into blastocysts to produce chimeric mice, and re-derivation by embryo transfer to remove murine pathogens from existing mouse strains. Investigators submit purified transgenes or targeted ES cells for microinjection into zygotes or blastocysts to produce transgenic founders or chimeric mice, respectively. The staff is currently establishing embryo cryopreservation and storage services to provide insurance against the loss of a colony or to preserve seldom-used strains - please contact the facility director for updates. The facility maintains a Leitz Diavert inverted microscope fitted with a range of phase contrast and HMC objectives, mechanical inertia-free Leitz manipulators and an Eppendorf microinjector. Other equipment includes a micropipette-puller and microforge for fashioning microinjection and embryo-transfer needles, and a Leitz Zoom stereomicroscope for embryo collection and surgical procedures.
In addition to the Research Support Services, the RRC provides technical support in the form of a Scientific Electronics Shop (SES), Scientific Instrument Shop (SIS), Scientific Computing Support group (SCS) and the RRC Scientific Supply Center (SSC).
The SES repairs and maintains electronic equipment and designs electronic devices to the specifications of campus investigators' research needs. The SIS repairs, designs and constructs physical devices and machinery to investigators' specifications as their research needs require. The SCS group is primarily an in-house computer service facility, but computing staff are sometimes available to outside departments for expert help in solving problems with virtually any type of computer, network or service, especially those that are Unix/Linux driven. The RRC Scientific Supply Center carries a wide array of items that are sold at a discount from many major vendors . Freezer programs include: Biorad, Fermentas MBI, Invitrogen/Gibco/Life Sciences, Promega, Fisher, Mediatech, Stratagene, and Midwest Scientific. The adjacent Protein Research Laboratory also carries Sigma, Pierce, and Clonetech products.