Gene Therapy - Core Facilities: Morphology

Morphology
Core Facility

     The Morphology Core Facility, located on the sixth floor of the Biomedical Research Institute, and currently offers instrumentation for nine separate technologies. Each is operated by a skilled Research Associate.

Fluorescence Microscopy
     The fluorescent microscope in the core facility is a Nikon TE300 inverted microscope, which has fluorescence capacity for FITC and Rhodamine excitation-emission wavelengths, differential interference contrast (DIC) optics and a temperature regulated stage. The objectives include 10, 20 and 40X CF infinity plan fluor dry objectives. High-resolution immersion objectives include a CF1 plan fluor 40X oil (N.A. 1.30), a DF1 plan apo 60X oil (N. A. 1.40) and a Cf1 plan apo 100X oil (N.A. 1.40). Images are captured with a Photometrics Synsys digital camera (Roper Photometrics, Tuscon, AZ) and analyzed with Metamorph V4 image analysis software (Universal Imaging Corp, Trenton NJ).

Confocal Microscopy
     The confocal microscopy component is built around a Bio-Rad Radiance 2000/AGR-3 (Q) with a three-channel, three-laser, three-detector system mounted on a Nikon TE 300 inverted microscope. The instrument has three lasers with ATOF for balancing the laser lines: a) a 25-mw argon ion laser emitting at 457, 476, 488, 514nm; b) a 1-mw green HeNe laser emitting at 543nm; and c) a red diode laser emitting at 638nm. The software consist of Lasersharp I 32-bit acquisition, display and 3D volume rendering. The TE300 inverted microscope has fluorescence and differential interference contrast capacity with 10, 20 and 40X CF infinity plan fluor dry objectives, as well as CF1 plan fluor 40X oil (N.A. 1.30), DF1 plan apo 60X oil (N. A. 1.40) and CF1 plan apo 100X oil (N.A. 1.40) objectives. A separate computer with MetaMorph image software is used for additional image analysis.

Flow Cytometry
     Three instruments with supporting workstations make up the flow cytometry component of the Core Facility. 1. FACSVantage SE. The FACSVantage flow cytometer/cell sorter is capable of 8-parameter (two laser light scatter and up to six fluorescent colors) analysis and cell sorting. It is equipped with three lasers for fluorochrome excitation. The sorter is equipped with the TurboSort option, permitting high speed sorting up to 35,000 events per second. The sorter is also equipped for direct sorting into tissue culture plates of any configuration, permitting direct cell cloning and low frequency response analyses. 2. FACSCalibur. The FACSCalibur is an ultra sensitive flow cytometer, capable of 6-parameter (two laser light scatter and up to four fluorescent colors) analysis. It uses two lasers for fluorchrome excitation. 3. BD LSR. This flow cytometer is capable of 8-parameter (two laser light scatter and up to six fluorescent colors) analysis. It has three lasers for excitation of flourochromes.

Mass Spectrometry/Proteomics
     The mass spectrometry component of the Core Facility consists of a high resolution (10 ppm) Voyager DE PRO mass spectrometer. The particular type of mass spectrometry performed is Matrix-Assisted Laser Desorption Ionoziation - Time-Of-Flight (MALDI-TOF). Coupled to this instrument is a 2-GHz LeCroy computer for on-line database-searching. The third component is a robotic device (Symbiot) for spotting MALDI plates.

Automated Cellular Imaging System
     The ACIS system (Automated Cellular Imaging System) developed by ChromaVision uses proprietary, color-based imaging technology and automated microscopy to perform quantitative image analysis of immuno-histochemically stained slides. The system consists of two major subassemblies: a microscope with its associated and electromechanical hardware and a computer with a frame grabber and image processor. The ACIS system is capable of DNA ploidy analysis of cells based on DNA content and nuclear morphology and is approved by the FDA for clinical diagnostics in conjunction with immuno-histochemical staining.

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