MICROSCOPY
Scientific Advisor: Dr. Martin Muggeridge
Leica TCS SP5 Spectral Confocal Microscope
This is a very flexible and fast confocal system for fixed or living samples. The system is built
with a Leica DMI 6000 CS inverted, fully automated microscope with motorized stage,
condenser, objective and filter turrets. The microscope is housed in a Ludin full enclosure
incubator with an internal Ludin Cube2 with CO2, O2 and humidity control. It is equipped with 5
lasers: blue diode (405nm), multi-line argon (458,476,488,496,514nm), green HeNe (543nm),
orange HeNe (594nm), and red HeNe (633nm). The spectral beam splitter has freely adjustable
bandwidths for the collection of signal in 5 separate detectors simultaneously or sequentially.
There is also a transmitted light detector for DIC. There are 9 available objectives, from a 2.5x
through a plan apo 100x/1.46NA oil objective. The system runs on the newest version of LAS
AF software, with FRAP, FRET, Mark & Find, 3D Visualization, Colocalization, and Live Data
Mode.
Zeiss Widefield /Apotome Microscope
The system is built with a Zeiss Axio Observer Z1 inverted fluorescent microscope, fully
automated with component recognition to minimize errors. System components include mercury
arc lamp excitation, a Zeiss AxioCamMRm CCD camera with 12-bit dynamic range and
extended sensitivity in the near infrared, fully automated xyz stage, a complement of objectives
from a 10x through 100x, 5 installed filter sets for DAPI, FITC, narrow band GFP, Rhodamine,
and far red. The Apotome attachment is designed for precise optical sectioning. The Apotome
slides easily into the optical path and projects a grid onto the image plane, which is shifted
laterally in three defined steps with an image collected at each step. A software algorithm then
removes out-of-focus signal. The acquisition software is Axio Vision v.4.6, including plug-in
options for Inside 4D, 3D Deconvolution, Colocalization, Mark& Find, Mosaic, and more.
Nikon Widefield Microscope
The system is built around a Nikon Eclipse TE300 inverted microscope with a range of
objectives for phase, DIC, and high resolution epi-fluorescent imaging. Software packages
control a Prior OptiscanTM xyz stage with a full complement of stage inserts and a Prior filter
wheel containing excitation filters from the Chroma 83000 filter set. This set includes single and
multiband excitation filters for DAPI, FITC, GFP, Texas Red, Rhodamine, or PI. Images are
acquired with a Roper Scientific CoolSNAPfxTM monochrome CCD camera with 12-bit dynamic
range, especially designed for low-light applications. High resolution color images may also be
acquired with the addition of the CRI Micro*ColorTM filter module. The stage, filters, shutters,
and camera are controlled with the user’s choice of two software packages: IPLab v3.7 for
Windows or Metavue v6.3 for Windows.
Bio-Rad Radiance2000 Confocal Microscope
The Radiance2000 is capable of multichannel fluorescence (up to 3 channels), reflectance, and
transmitted light imaging. It is equipped with the following lasers and laser lines for excitation: 4line Argon laser (457, 477, 488, 514 nm), HeNe laser (543 nm), and Red laser diode (637 nm).
The scan head is attached to a Nikon Eclipse TE300 inverted microscope, which has optics and
filters for fluorescence and DIC applications. The spectrum of objectives available on the
microscope includes 10X and 40X plan fluor dry objectives for routine observations. Objectives
for high resolution imaging include a 40x/1.30 plan apo oil, a 60x/1.40 plan apo oil, and a
100x/1.40 plan apo oil. The LaserSharp 6.0 operating software allows simultaneous and
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sequential collection of 2D, 3D, and 4D images that can be collected in a horizontal plane,
vertical plane, xyz horizontal stack, or a timed sequence. Stacks and timed sequences can be
used to create an animation series.
Zeiss LSM 510 NLO Confocal/Multiphoton Microscope
The Zeiss LSM 510 NLO system is configured to enhance living tissue research. The scanning
system is connected to an upright Axioskop 2 FS MOT microscope with a set of objectives
selected for physiological measurements and live animal studies. The stage remains in a fixed
position, and the objectives have motorized focus control. It is equipped with the following lasers
and laser lines for excitation: Argon (458, 477, 488, 514 nm), HeNe (543nm), HeNe (633nm),
and the Coherent Chameleon-XR Ti:Sapphire laser (tunable from 705 through 980 nm). The
ultrafast pulsed Chameleon laser emitting NIR radiation allows imaging up to 500 μm deep
within tissue. . There are three PMTs for visible wavelength detection, a transmitted light
detector, and two non-descanned detectors for multiphoton imaging. The LSM 5 operating
software includes the Physiology v3.5 and Image Visart v3.5 options that permit 2D, 3D, and
4D image collection and processing, 3D/4D animation, calibration and measurement of ion
concentrations, time series analysis, and graphical mean-of-ROI analysis.
Offline Image Analysis Stations
There are two computer stations in the Core Facility Computer Lab reserved for microscope
users, which are loaded with specialized imaging software. The newest system has two
processors, four terabyte hard drives, and an ATI Fire GL V7350 video card with 1GB of onboard memory. It is loaded with Media Cybernetics’ AutoQuant AutoDeblur deconvolution
software, including the AutoVisualize option, and full offline versions of Leica LAS AF, Zeiss
Axio Vision 4.6, and Zeiss LSM software. Another system is loaded with Bio-Rad’s LaserSharp,
IPLab, Metamorph, Photoshop, and ImageJ.
DNA ARRAY (CHIP) ANALYSIS
Scientific Advisor: Dr. Rona Scott
Affymetrix GeneChip System
The Research Core Facility is equipped with a state-of-the-art Affymetrix GeneChip Instrument
System. This system consists of the following components:
1. A GeneChip Hybridization Oven 640 for automated control of hybridization to the
GeneChip arrays.
2. A GeneChip Fluidics Station for automated washing of chips and labeling of hybridized
probes. This station can wash and stain four arrays simultaneously.
3. A GeneChip Scanner 3000 for obtaining high-resolution images of hybridization signals.
4. A GeneChip Workstation that controls the operation of the system, data collection and
processing of initial raw data.
5. A bioinformatics system including a GCOS server, GCOS 1.2 software, Data Mining Tool
3.0, Spotfire 7.0, GeneSpring 8.0 and GeneSifter.
This system is suitable for global gene expression studies using the Affymetrix GeneChip Probe
arrays. Oligonucleotide arrays, prepared on glass, are hybridized to biotinylated probes
prepared from biological samples and detected with a fluorescent label. Probes for these
experiments are derived from a single source, and differentially expressed genes are identified
by comparing the results of experiments performed with different chips. A major advantage of
this approach is the ready availability of pre-prepared arrays representing a large number of
sequences from a number of species.
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GenePix 4000B Microarray Slide Scanner
The Research Core Facility has a GenePix 4000B microarray scanner from Axon Instruments.
It is capable of acquiring and analyzing expression data from DNA microarrays, protein
microarrays, tissue arrays and cell arrays. Unlike most commercially available microarray
scanners, the GenePix 4000B scanner acquires data at two wavelengths simultaneously, 532
nm and 635 nm, greatly reducing scan time. Accepting standard microscope slides (1” x 3”), it
acquires data at resolutions between 5 microns and 100 microns. Other features include a
user-selectable focus position, user-selectable laser power, a dynamic detection range of four
orders of magnitude and line averaging mode for extra-high signal-to-noise ratios. The system
is fully integrated with GenePix Pro software. Acuity 4.0 is also available for analysis and
visualization of multi-platform array data.
FLOW CYTOMETRY
Scientific Advisor: Dr. Robert Chervenak
Analytic Instruments
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 fluorochrome
excitation: a) an argon ion laser for 488-nm excitation; and b) a Red Diode laser for 635-nm
excitation. This instrument uses CellQuest software for acquisition and analysis.
BD LSR II (#1)
The LSRII (#1) is capable detecting up to 14 parameters (Forward Scatter, Side Scatter, and
twelve fluorescence detectors). It has four lasers for excitation of fluorochromes: a) a Coherent
Sapphire laser for 488-nm excitation; b) a JDS Uniphase HeNe laser for 633-nm excitation; c) a
Coherent VioFlame for 405-nm excitation; and d) a Lightwave UV laser for 355-nm excitation.
In its present configuration, the instrument is set up to detect 6 colors excited by the 488-nm
laser, 2 colors excited by the 633-nm laser, 2 colors excited by the 405-nm laser and 2 colors
excited by the UV laser. This instrument uses BD Biosciences FacsDiva software for
acquisition and analysis.
BD LSR II (#2)
The LSRII (#2) is capable detecting up to 14 parameters (Forward Scatter, Side Scatter, and
eleven fluorescence detectors). It has three lasers for excitation of fluorochromes: a) a
Coherent Sapphire laser for 488-nm excitation; b) a JDS Uniphase HeNe laser for 633-nm
excitation; and c) a Coherent VioFlame for 405-nm excitation. In its present configuration, the
instrument is set up to detect 6 colors excited by the 488-nm laser, 3 colors excited by the 633nm laser, 3 colors excited by the 405-nm laser. This instrument uses BD Biosciences FacsDiva
software for acquisition and analysis.
Cell Sorters
FACSAria II
This instrument was upgraded from a FACSAria to a FACSAria II in the Fall of 2009. The
FACSAria II cell sorter is capable of 15-parameter (Forward Scatter, Side Scatter, and 13
fluorescence detectors) analysis and cell sorting. It has 3 solid state lasers for excitation at 407nm, 488-nm and 633-nm wavelengths. The instrument is configured so that 7 colors can be
detected from the 488-nm laser, 3 colors can be detected from the 407-nm laser and an
additional 3 colors can be detected from the 633-nm laser. This is a digital high speed sorter,
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capable of sorting up to 70,000 events per second. It is capable of standard “bulk” sorting of up
to 4 user-defined cell populations simultaneously, or can be used for direct deposition of a
counted number of cells directly into tissue culture plates for cloning, frequency response
assays, or other single cell analyses. This instrument uses BD Biosciences FacsDiva software
for acquisition and analysis.
FACSVantage Diva
The FACSVantage Diva flow cytometer/cell sorter is capable of 11-parameter (two laser light
scatter and up to nine fluorescent colors) analysis and cell sorting. It is currently equipped with
three lasers for fluorochrome excitation: a) a solid state 488 nm laser; b) a helium/neon laser for
633-nm excitation; and c) a solid-state 354-nm UV laser. The FACSVantage Diva is an alldigital upgrade of an older analog sorter (FACSVantage SE) that enables higher speed sorts
(up to 35,000 events per second) and the sorting of 4 (rather than 2) user-defined populations in
a single experiment. A recent optical upgrade of this sorter (Multicolor Detector Option) has
significantly increased the sensitivity of this sorter, making it more useful in the detection of
weaker fluorescent signals. In addition, the sorter is also equipped for direct sorting into tissue
culture plates of any configuration, permitting direct cell cloning and low frequency response
analyses. This instrument uses BD Biosciences FacsDiva software for acquisition and analysis.
Workstations and Software
There is one G4 Macintosh computer, one MacPro dual quadcore Macintosh, and two Dell PCs
for off-line analysis of data. The off-line Macintosh computers provide Cellquest Pro, while the
off-line PCs have the latest version of FacsDiva software for data analysis. In addition, FlowJo
is available on the Macintosh and Dell workstations for specific data analysis needs. For the
analysis of cell cycle data, ModFit LT is available on both Macintosh and PC workstations.
LASER CAPTURE MICRODISSECTION
Scientific Advisor: Dr. Michael Mathis
PixCell II
Major scientific and medical advances are transforming the field of translational laboratory
research. Developments in gene sequencing and amplification techniques, among others, now
allow investigators to extract DNA or RNA from tissue biopsies and cytological smears for
pinpoint molecular analysis. The efficacy of these sophisticated genetic testing methods,
however, depends on the purity and precision of the cell populations being analyzed. Simply
homogenizing the biopsy sample results in an impure combination of healthy and diseased
tissue. Using mechanical tools to manually separate cells of interest from the histologic section
is time-consuming and extremely labor-intensive. None of these methods offers the ease,
precision and efficiency necessary for modern molecular diagnosis. A new method, Laser
Capture Microdissection (LCM), provides research and pathology laboratories with the ideal
microdissection technology. LCM was conceived and first developed as a prototype research
tool at the National Institute of Child Health and Human Development (NICHD) and the National
Cancer Institute (NCI) of the NIH. LCM is being used in the Cancer Genome Anatomy Program
(CGAP) to catalog the development of cells from a normal to a diseased state. It can be applied
to any disease process which is accessible through tissue sampling, such as premalignant
cancer lesions, multiple sclerosis, arteriosclerosis, and Alzheimer's disease. Research
applications include: genomics (differential gene profiling, loss of heterozygosity, microsatellite
instability, and gene quantification) and proteomics (two-dimensional protein gels, western
blotting, and immuno-quantification of proteins).
The PixCell II® instrument performs Laser
Capture Microdissection from heterogeneous tissue samples simply, quickly and precisely. In
minutes the investigator can locate a single cell or large groups of cells and, using a simple aimand-shoot method, extract them for subsequent molecular analysis. LCM preserves the exact
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morphologies of both the captured cells as well as the surrounding tissue. The PixCell II
transfers cells from paraffin-embedded and frozen tissue samples, stained and immunolabelled
slides. The entire process can be monitored and documented, and the images can be stored in
an archiving workstation. Microdissection of fluorescently-stained cells is also possible with a
fluorescence package that has been purchased.
MASS SPECTROMETRY
Scientific Advisors: Dr. Beniam Berhane, Tammy Dugas
Voyager DE PRO MALDI-TOF Mass Spectrometer
The mass spectrometry component of the RCF consists of a high resolution (10 ppm) Voyager
DE PRO mass spectrometer. The particular type of mass spectrometry performed is MatrixAssisted Laser Desorption Ionoziation – Time-Of-Flight (MALDI-TOF). Coupled to this
instrument is a 2-GHz LeCroy computer for on-line database-searching. Proteins excised from
denaturing polyacrylamide gels are digested with trypsin or other proteolytic enzymes and
identified by matching peptide masses to the theoretical peptides derived from all proteins in
databases such as SwissProt and NCBI. This automated system is capable of identifying up to
70 proteins per hour. The entire system is marketed by Applied Biosystems as a package
termed Proteomics Solution 1.
Thermo Finnigan Deca XP Max nanospray LC/MS Mass Spectrometer
The Research Core Facility is equipped with a Thermo Finnigan Deca XP Max nanospray
LC/MS instrument, fitted with an electrospray ionization (ESI) source and an ion trap mass
analyzer. Electrospray ionization enables the study of a wide range of molecular types, and the
instrument’s nanospray capabilities provide enhanced sensitivity. In addition, the LC/MS is
interfaced to software that enables both small molecule identification and peptide/protein
sequencing. The instrument’s ion trap mass analyzer is particularly useful in cases where the
molecular mass of the analyte is completely unknown. While enabling MSn capabilities, the ion
trap provides structural information, allowing the analyst to “walk down” the structure of the
molecule. Finally, data dependent scanning allows the user to identify protein/peptide
modifications.
REAL-TIME PCR
Scientific Advisor: Rona Scott
ABI 7900HT Fast Real-Time PCR System
The Applied Biosystems 7900HT Fast Real-Time PCR System is the only real-time quantitative
PCR system that combines 96- and 384-well plate compatibility and the TaqMan® Low Density
Array. With optional Fast real-time PCR capability, this system reduces run time to about 35
minutes in a standard 96-well format, or about 55 minutes in a 384-well plate. Key applications
include gene expression quantitation and the detection of single nucleotide polymorphisms
(SNPs) using the fluorogenic 5' nuclease assay.
To induce fluorescence, the 7900HT system distributes light from an argon laser excitation
source to all sample wells via a dual-axis synchronous scanning head. It then directs the
resulting fluorescent emission through a spectrograph to a CCD camera. Emission wavelengths
from 500-660 nm are monitored allowing the simultaneous detection of multiple fluorophores.
The system is compatible with FAM/SYBR Green I, VIC/JOE, NED/TAMRA/ Cy3, ROX/Texas
Red and Cy5 fluorescent dyes.
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The Sequence Detection Software for the 7900HT system runs on the Windows XP operating
system and is used for instrument control, data collection, and data analysis. The software
includes a plate set-up wizard for easy experimental design.
ICycler iQ (2 Instruments)
The iCycler iQ real-time PCR detection system is a sophisticated optical system that fits directly
above the iCycler thermal cycler. It covers the widest range of excitation/emission wavelengths
available, facilitating the greatest array of fluorescent PCR strategies.
Features:
 Real-time analysis can be viewed online during the PCR.
 Range of fluorophore excitation and emission from 400-700 nM
 4 different fluorophores can be multiplexed per sample tube
 96 sample can be tracked simultaneously
 Sample data can be reanalyzed at any time
 ICycler iQ software is designed to automate analysis options, including
quantitative and melt-curve analysis.
 Closed-tube detection reduces the risk of sample contamination and eliminates
the use of electrophoresis
SMALL ANIMAL IMAGING FACILITY
Scientific Advisor: Dr. Mike Mathis
Concorde microPET R4
MicroPET provides high performance, functional imaging for studies involving animal models of
disease, genetically engineered animals, pharmaceutical development, and radiotracer
development. The facility has purchased a microPET R4 device from Concorde Microsystems,
Incorporated. This unit allows non-invasive serial and longitudinal studies to be performed in
the same animal. Isotopes are provided to investigators in cooperation with the Biomedical
Research Foundation (BRF) of Northwest Louisiana PET imaging center cyclotron.
Imtek MicroCT
MicroCT generates anatomic reference for the MicroPET data sets, as well as provides the
capability of bone morphology and density measurements, tumor identification and
classification, fat pad distribution and volume measurements, as well as many other types of
studies. The center has a MicroCT unit custom built by Imtek, Inc. This unit uses the same
gantry as the microPET machine, which allows superimposition of images for anatomic
correlation of functional PET studies. Resolution is up to 45 microns and real-time image
reconstruction is possible with this unit.
FLEX Triumph (PET, SPECT, CT)
The FLEX Triumph multi-modality pre-clinical platform combines three different imaging
modalities (PET, SPECT, CT) in a single platform. This pre-clinical imaging system is employed
by primary medical research and drug development teams in both academia and the
pharmaceutical and biotech industries to study disease processes, and to track interactions of
pharmaceuticals at a molecular level. Key application areas include cardiology, neurology,
oncology, and bone analysis. The platform is designed and manufactured to be easily
upgradeable, allowing configuration of a low-priced entry-level system that can be fieldupgraded to fit growing needs. Accessories for imaging such as vital sign monitoring,
anaesthesia delivery and exhaust system, automatic injection system and blood monitoring
system are available. A unique user interface controls all modalities. It is easy to operate, and
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has been specifically designed for pre-clinical imaging. The interface helps the user gather
important scan information. All data is saved in DICOM format. Vital signs and specific scan
information are also saved. Pre-defined imaging protocols can be designed to ensure that the
same imaging parameters are used for each subject studied. GM-I’s new VIVID™ (Volumetric
Image Visualization, Identification and Display) software package has been developed
specifically for FLEX Triumph™ with streamlined user interface to provide One-Step Fusion™
which provides automated fusion and co-display of up to four co-acquired images
(SPECT/SPECT/PET/CT). Other features of our VIVID software include automated loading of
SPECT, PET and CT image data, and automated 2D and 3D visualization modules. VIVID also
provides higher pixel definition, faster camera maneuvers for projection data and streamlined
image segmentation and quantification features, as well as an automated module for timeactivity curves of dynamic data for biodistribution studies. A working project directory to prevent
loss of analytical results/data and DICOM push capabilities are also among the obvious
advantages of VIVID.
Xenogen IVIS imaging system
The Small Animal Imaging Facility offers two optical imaging options, photon detection from
chemiluminescent systems such as luciferase, and detection of fluorescence from models
containing systems such as green fluorescent protein. Both of these modalities are imaged
from the Xenogen IVIS imaging system. This technology combines specially designed imaging
chambers and software with a charged-coupled device (CCD) camera. Investigators will be
able to monitor and record cellular and genetic activity within a living organism in real time.
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