| Overview
Fundamental research into the causes of environmentally related
diseases often benefits from state-of-the-art laboratory techniques,
especially those that use human cells and tissues grown in a biological
culture medium.
| Hepa
1 Microtubules |
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Analysis
and measurement of the structural components of tissues, as well
as direct biochemical analysis of many specific functions in living
cells and tissues in culture, can be performed with remarkable
sensitivity and specificity using techniques such as flow cytometry,
laser-cytometry–confocal microscopy, and digital image analysis.
Collectively, these experimental approaches form the basis of
analytical cytology. These tools have become indispensable for
assessing the effects of toxic chemicals on cell physiology, structure,
and function and new and exciting approaches continue to be developed
in this field. The expense and technical expertise required to
master these tools, however, often present barriers to wide use
of cytometry equipment.
| Topo
plot of glutathione
content in cells |
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The
goal of the Analytical Cytology Facility Core is to make analytical
cytology services more accessible by reducing costs and providing
technical assistance and consultation for CEEH investigators,
thereby providing significant incentives to use this advanced
instrumentation. By providing access to and training in the use
of advanced cytological tools, the Core supports CEEH research
into the mechanisms of action of environmental agents affecting
human health. |
| The
Analytical Cytology Facility Core maintains a confocal
laser microscope, has upgraded the Core digital imaging
system, and now provides access to a two flow cytometers.
Confocal Laser Microscope
The
Core maintains
an Adherent Cell Analysis
and
Sorting
(ACAS)
Ultima
Laser Cytometer,
a
confocal
laser
microscope/image
analysis system specifically designed for fluorescence
analysis
and sorting of cells that are attached to solid surfaces, such
as tissue culture plates or glass slides (adherent cells).
| Mouse
Fetus stain with
Mercury Orange |
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UV and visible light excitation); in situ hybridizations, DNA
content/analysis; in situ PCR quantitation and localization; high
resolution confocal microscopy with 3-D reconstructions/rotations
of optical slices in 1 or 2 colors; reflectance confocal images
for quantitative imaging of silver grains (autoradiography), The
instrument also has the ability to microscopically focus at different
depths of the tissue (confocal). The types of measurements and
interactive studies that can be performed with the instrument
on cells/tissues/structures include bio-imaging applications such
as quantitative fluorescence microscopy in one or two colors (with
simultaneous UV and visible light excitation); in situ hybridizations,
and DNA content/analysis; in situ PCR quantitation and localization;
high-resolution confocal microscopy with 3-D reconstructions/rotations
of optical slices in one or two colors; reflectance confocal images
for quantitative imaging of silver grains (autoradiography), colloidal
gold, peroxidase-diaminobenzidine, geimsa, etc.; and cell physiology/pathology
studies including dye exclusion (viability), kinetic measurements
of calcium and other ions, pH, mitochondrial membrane potential,
glutathione and glutathione transferase, NAD(P)H, gap junctional
intercellular communication, membrane fluidity, endoplasmic reticulum,
membrane pumps (MDR, organic ion transport), antigen tracking/capping,
cytochrome P450 activity, beta-galactosidase activity, bioluminescence,
reactive oxygen species, respiratory burst, lipid peroxidation,
and apoptosis.
|
Liver
stain for
cytochrome
P450 1A1 activity |
|
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The
instrument can also be used for interactive studies such as cell
sorting/enrichment (while cells are attached to dishes); FRAP
(fluorescence recovery after photobleaching); FRET (fluorescence
resonance energy transfer); laser surgery on cells and organelles;
photo-activation of "caged" compounds (calcium, IP3,
fluorescent dextrans); quantitative irradiation of cells/organelles
with UV or visible light; laser poration of macromolecules into
cells; and laser-induced cell membrane fusion.
Digital Imaging
The
Core also houses a digital image analysis system full color and
densitometric analysis of samples (both macroscopic and microscopic).
The system consists of
of a cooled CCD camera (Spot R/T Slider) attached to a
Nikon Optiphot microscope with fluorescence, DIC and dark field
illumination,
and associated image analysis software (Metamorph V4.6, Universal
Imaging).
Flow
Cytometer
Recently, Kavanagh and Rabinovitch were successful
in obtaining a DRR Shared instrumentation Grant to purchase a
high-speed
cell
sorter (MoFlo, DakoCytomation) that is capable of sorting up
to 70,000 cells per second and is available to CEEH investigators
for their studies. This instrument replaced a fully functional
Coulter Elite Flow Cytometer/Cell Sorter, which was moved to
the laboratory of the Analytical
Cytology Facility Core. The Core upgraded the system's laser,
the operating software, and the computer system (Pentium 3 Flowcenter
Work
Station with Expo32 Multicomp Software, Beckman/Coulter). Thus,
both instruments are available to fully serve CEEH investigator
needs in the area of flow cytometry and fluorescence-activated
cell sorting. |
