Biological Analyses Facility Core
Equipment and Facilities
Bioimaging Facility
The equipment includes a Leica TCS-NT confocal microscope with argon, HeNe, and krypton lasers interfaced to three Silicon Graphics computer workstations. The facility also has several light microscopes fitted with brightfield, epifluorescent, Nomarski, and phase contrast optics.
The Confocal Laser Scanning Microscope (CLSM) uses a computer driven laser to obtain 0.5 mm thick optical sections from cells and tissues without physically sectioning them. Collection of optical sections eliminate the physical destruction of the specimen so large areas of tissue may be repeatedly examined at any depth up to 200 mm at magnifications up to 2,000x. Complete 2-D morphometric and three-dimensional reconstruction analyses are routinely performed. Volume rendering programs, such as VoxelView Ultra and VolWorks, and a quantitative image-processing package (ImageSpace) further enhance our powerful image analysis capabilities.
Flow Cytometry Facility
A new flow cytometry system was acquired by the facility through an NIH Shared Instrumentation Grant and installed into renovated lab space in June of 1998 and has been fully operational since that time. It consists of a Coulter EPICS ELITE analyzer (and sorter) with a Coherent 621 Enterprise II UV laser and a Cyonics air-cooled argon laser for blue excitation (488 nm). The equipment includes a PC and monitor for control, data acquisition and analysis on Expo for Elite and Multicycle for Windows software packages, as well as optional optics and PMTs for calcium flux studies using the fluorescent probe indo-1.
Electron Microscopy Facility
This facility has analytical capability for elemental analysis of biological materials. The Zeiss CEM902 with interfacing image analysis system and computer equipment for electron energy loss analysis is an outstanding transmission electron microscope with capabilities for elemental analysis primarily for lower atomic number elements. It has a resolution of 0.34 nm, magnification range of 150-400,000x, integrated imaging spectrometer, microprocessor controlled electronic stage, high camera (Dage), Soft imaging systems software and computer system for imaging, image analysis, and integrated elemental analyses, EELS detector, and network connections to a Codonics photo-quality digital image printer. Thus, the Zeiss unit is useful for analysis of elemental constituents of ultrafine particles in lung samples, as well as tracing the pathways of soluble elements within cells.
The facility has recently acquired a new, state-of-the-art scanning electron microscope (Leo 1450VP) with soft imaging systems software for digital imaging and analysis and an Oxford Energy Dispersive X-Ray Analysis system through an NIH Shared Instrumentation grant. This will allow automated and high throughput analysis of the elemental constituents of particles and fibers and is especially useful for higher atomic number elements.
Preparation equipment includes an advanced freeze slam and freeze drying unit which can be used for preparation of transmission EM samples without using liquid dehydrants. For SEM, equipment for critical point drying and carbon or heavy metal shadowing are part of the facilities. Ultramicrotomes for TEM preparation includes an RMC MT6000 and Reichert Ultracut E ultracryomicrotome. Computing facilities includes two workstations (IBM and Macintosh) and fully equipped darkrooms are included in this facility.
Proteomics Facility
This facility provides 2D Gel Electrophoresis. The majority of the projects of Center investigators are aimed at determining expression profiles of proteins. We use the "Investigator Proteomics Solution Package" (Genomic Solutions Inc.) obtained via an administrative supplement to this Center Grant in 2001. This system includes sufficient combined isoelectric focusing and SDS gel electrophoresis capacity to allow the facility to process up to 10 large format 2D gels per day.
Gel Imaging
Institutional funds provided by the Dean's office allowed acquisition of a high quality laser fluorescence imaging system (BioRad FX Pro MultiImager). The system enables detection of proteins stained with the highly sensitive fluorescent dye SYPRO Ruby as well as differential gel electrophoresis (DIGE), a newly established technique for proteome analysis, in which two pools of proteins are individually labeled with the fluorescent dyes Cy3 and Cy5, respectively. After labeling, the protein samples are mixed and separated in the same 2D gel. This technology alleviates the most frequently cited limitation of 2D gel analysis, namely gel-to-gel variation (REF).
Digital gel images obtained with the BioRad laser imager are transferred to a PC workstation for further analysis with Phoretix2D software package (Nonlinear Dynamics). The software provides rapid and precise spot detection optimization, a vital part of analyzing 2D gels, and contains five different methods for background subtraction to suit all types of applications. Further capabilities include extensive analytical and reporting features such as database querying to search for specific protein expression pattern, and data output.
Mass Spectrometry
The facility includes a Thermoquest LCQ Deca ion-trap tandem mass spectrometer (via an NSF grant). The LCQ Deca XP is an ultrasensitive, high-performance, quadrupole ion-trap instrument that accommodates either an electrospray ionization (ESI) or a nanoelectrospray probe. Scan modes include, for example, full scan, selected ion monitoring (SIM), and MS/MS product ion scan. For MS/MS, collision induced dissociation (CID) is carried out in the ion trap analyzer. The LCQ Deca is coupled to a Surveyor Integrated HPLC System, for separation of peptide mixtures resulting from proteolytic digests of picked gel plugs.
The system also includes a PC workstation with the Xcalibur software suite for experiment definition, instrument control and data analysis. The BIOMASS deconvolution software communicates with Xcalibur and allows the calculation of molecular weight of multiply charged peptide ions from their m/z values. For proteomics applications we have access to the TurboSEQUEST software package, which allows high speed protein identification using indexed database searching.
Molecular Biology Facility
Based on surveys of needs for Center investigators, this facility has been established to assist members of the NIEHS Center in the quantitation of gene expression in cell or tissue samples. This facility offers substantial expertise in molecular biology including cloning, purification of nucleic acids and proteins, gene expression analysis, DNA sequencing, and high-throughput SNP genotyping.
This facility provides expertise and access to two important technologies:
- real-time quantitative PCR (BioRad iCycleriQ RT-detection system)
- phosphoimager quantitation of Northern blots, RNAse protection assays and nuclear run-on assays (Packard Instruments).
Equipment for this facility includes a Packard InstantImager System including Pentium-based PC and required peripherals; Hewlett-Packard LaserJet printer; two Apple Power PC computer workstations, with access to the following software packages: Oligo Primer Analysis Software (National Biosciences, Inc.), GeneWorks (IntelliGenetics, Inc.), Current Protocols in Molecular Biology on CD-ROM (Teton Data Systems), and Imagequant for Windows NT (Molecular Dynamics).