| Cytomics
brings together biological imaging, cell analysis tools such as
flow and image cytometry, computational resources for 3D imaging
and bioengineering resources for cellular manipulations. It links
molecular and cellular biology with the technologies of cytometry.
Cytomics embodies the essence of informatics; extracting the information
from dots, traces, curves and scans into a knowledge-base of diagnostics
and discoveries. Cytomics utilizes the specialty of cell sorting
to isolate single cells, colonies, particles or beads for further
analysis or biochemical characterization. Cytomics utilizes microscopy
in its many forms; transmission, fluorescence imaging, digital imaging,
confocal and multiphoton imaging (MP), multispectral imaging, small animal imaging and laser dissection microscopy
(LDM).
In addition to studying the impact of single cell systems, the Cytomics
core has a developing program in the field of High Content Analysis
(HCA) and the closely related area of High Throughput Analysis (HTA).
These technologies are the current driving forces in pharmaceutical
drug development.
Cell analysis & cell separation:
The Core is capable of supporting a variety of cell analysis research
needs. Cell analysis using flow cytometry allows the isolation and
subsequent analysis of individual cells using multivariate analysis.
Advanced multi-laser analysis instruments notably the Epics Altra
high speed cell sorter with 4 lasers, including a Coherent Enterprise
II UV laser and a Coherent Krypton multiline laser in addition to
the air cooled Argon and HeNe laser are present in the core. The
instrument can sort cells into sterile environments. In addition
to this, the core has an Epics Elite cell sorter which is used for
development projects, particularly related to instrument modifications,
enhancements or any alterations necessary to perform experiments
that cannot be performed in more routine instruments. For analysis,
the core has two FC500 6 color dual laser analyzers both with automated
32 tube carousels. Each instrument has multivariate analysis software.
An Epics XL 4 color single laser cytometer is also available for
basic routine flow cytometry analysis. For specialized microbial
operations, the core has a Bio-Rad Bryte and A Quanta (B-C), both of which have lamp based
excitation sources and are particularly sensitive to very small particles.
A significant analytical software suite is available in the core
for multivariate analysis.
Laser Capture Cell Microdissection:
Cell types undergoing molecular changes, such as those thought to
be most definitive of the disease progression, may constitute less
than 5% of the volume of the tissue biopsy sample. Therefore, microdissection
is essential to apply molecular analysis methods to study evolving
disease lesions in actual tissue where morphologic characterization
is required. The core has an AutoPix Automated Laser Capture Microdissection
(LCM) System from Arcturus, combining automated upright microscope
architecture, three-dimensional optical control of the dissecting
laser beam and the dissected area, non-contact tissue sampling and
post-dissection handling.
Biological Imaging: There
are multiple imaging technologies available in the cytomics &
Imaging core. Basic imaging using standards high quality microscopes
allows high resolution imaging in brightfield or fluorescence excitation.
To achieve this the core has a Nikon 1000R upright fully automated
fluorescence microscope with a SPOT high sensitivity, high resolution
digital camera is also present. A Nikon Labophot upright fluorescence
microscope and a Nikon inverted 300 fluorescence microscope with
a DVC 250 line-scanner confocal microscope is also available. More
advanced capabilities exist in other confocal microscopes which
can create 3D images using optical slicing. Systems are available
to do UV through near IR imaging. A Bio-Rad MP 1024 UV/vis confocal
microscope is mounted on Nikon inverted microscope. This unit is
ideal for UV live cell imaging. Also available is a Bio-Rad Cellmap
2 color confocal microscope mounted on a Nikon E800 upright microscope.
The unit has a 405 Violet laser and a 488 blue diode laser for excitation.
The most advanced capability in fluorescence imaging is a Bio-Rad
MP 2100 Multiphoton microscope that has the capability of advanced
live cell imaging, including live embryos, deep tissue imaging and
even functional brain imaging. This instrument also have a Becker
& Hinkl life-time module linked to two high speed PMTs that
allow for simultaneous Multiphoton imaging and lifetime imaging.
Analytical Software: The
core has a large range of software packages for advanced and basic
image processing. These include advanced packages such as Metamorph
and Image ProPlus. In addition, an extensive capability exists with
the package VoxelView.
High resolution Imaging
Atomic Force Microscopy specifically for biological samples provides
the highest possible resolution for live cell systems. Instruments
specifically designed to study biological systems operate under
very different conditions to similar technologies operating the
area of materials sciences. AFM instruments are available in the
core to provide high resolution analysis specifically for biological
samples.
Multispectral Imaging:
The latest technology under development in the center involves multispectral
imaging. We currently have several instruments available for use
that are testbeds for new technology development. For example we
have an AOTF (Acousto Optical Tunable Filter) on an upright brightfield/fluorescence
microscope. This unit can collect up to 400 images at 1.5 nm increments
from 370 to nearly 800 nm. This allows the analysis of multispectral
data sets for the potential isolation of autofluorescence or specific
target oriented molecules of interest.
Advanced Image Processing:
The core provides multidimensional image processing – 1D,
2D, 3D and 4D image analysis using sophisticated software programs.
These packages are directly linked to the detections systems and
can extract complex structures and reproduce them in multidimensional
space. Such analytical tools are both qualitative and quantitative.
We have several packages that are high level image analysis packages
such as Image ProPlus and Metamorph.
High Content-High Throughput Systems
The core is developing capabilities in high content and high throughput
analysis typical of systems where thousands of samples must be analyzed
daily. These systems require advanced computational support to provide
analysis. Current technologies include microtiter based fluorescence
imaging instruments which use sensitive cameras and advanced image
based computational analysis.
Protein Profiling:
The Core has a unique resource in a state-of-the-art automated,
two-dimensional fractionation system expressly designed for high-resolution
analysis of complex protein mixtures. The ProteomeLab PF 2D provides
an integrated solution that effectively resolves the thousands of
proteins present in cell lysates. The ProteomeLab PF 2D system generates
data from two dimensions, detailed protein maps can be constructed
for easy comparison using the ProteomeLab Software Suite supplied
with the system. Liquid fractions can be collected and stored for
future analysis or the eluent can be connected directly to ESI-mass
spectrometry. The PF 2D's unique combination of chromatofocusing
followed by non-porous reverse phase chromatography provides ultra-high
resolution of proteins, while delivering information that can be
related to 2D gel data. The ProteomeLab PF 2D's second dimension
is capable of resolving hundreds of proteins in great detail from
each first-dimension fraction. Fraction collection from the first
dimension is quality-controlled with an in-line pH monitor, then
automatically injected into the second dimension. As a result, PF
2D requires much less time and attention than traditional labor-intensive
techniques. The entire 2 dimensional process can be performed in
less than 20 hours. Liquid fractions can be stored or transferred
to a MALDI plate spotter, or directly connected to an electrospray
source.
Consulting and Data Processing:
The Core provides excellent expertise for the design, preparation
and analysis of experimental profiles. Consultants are available
to advise researchers in the correct techniques; training opportunities
are available on a regular basis.
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