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Grant
Information Needed
by Some of Our Clients
Center
for Microscopic Imaging
A University-wide
Service Center for high resolution light, confocal and electron
microscopy, the Center for Microscopic Imaging. CMI is located
on the first floor of the College of Veterinary Medicine,
2001 S. Lincoln, and occupies over 3,000 sq. ft. of total
space in 14 separate rooms. It is staffed with a Director,
Associate Director, full-time Supervisor and 1/2 time Secretary,
and a full-time technician.
Services
and techniques performed include: standard, microwave and
alternative media preparations for TEM, thick plastic section
for light microscopy, ultrathin sectioning, negative staining,
immunogold TEM, TEM microscope service, basic SEM preparation
service, confocal microscopy, routine darkroom service, digital
imaging for TEM and SEM negatives, LM and biochemistry gels
and digital image workstation.
Equipment
at the Center include: special microwave for processing, 4
ultramicrotomes, brightfield, phase and fluorescent light
photomicroscope, 3-lasor (Argon, Krypton and HeNe) Olympus
confocal microscope, 2 -100KV Hitachi transmission electron
microscopes , digital camera and computer workstations needed
for image capture, Vacuum Evaporator, Critical Point Dryer,
Sputter Coater, ovens, ultralow freezers, and Ultralow freezers.
Negative
Stain
The samples
were placed as a drop on parafilm and a copper grid, coated
with both formvar plastic and carbon, was placed on top of
the specimen droplet for 5-15 minutes depending upon the concentration.
The excess sample was then removed with filter paper and the
grid placed on 2% Ammonium Molybdate for 2 minutes. The grid
was then dried by removing the excess fluid with filter paper,
placed into a grid box and covered with drierite crystal for
10 minutes. The grid was then examined in the Transmission
Electron Microscope and observed at 20,000x or higher magnification.
Transmission
Electron Microscopy Using Sectioning
The tissue
or pellet was fixed in a Karnovsky's Fixative in phosphate
buffered 2% Glutaraldeyde and 2.5 % Paraformaldehyde. Microwave
fixation was used with this primary fixative and the tissue
is then washed in cacodylate buffer with no further additives.
Microwave
fixation was also used with the secondary 2% Osmium Tetroxide
fixative, followed by the addition of 3% Potassium Ferricyanide
for 30 minutes. After washing with water, saturated Uranyl
Acetate was added for enbloc staining.
The tissue
was dehydrated in an series of increasing concentrations of
ethanol. Acetonitrile was used as the transition fluid between
Ethanol and the Epoxy. Infiltration series was done with an
epoxy mixture using the epon substitute Lx112. The resulting
blocks were polymerizred at 90C overnight, trimmed and ultrathin
sectioned with diamond knives. Sections were stained with
Uranyl Acetate and Lead Citrate, and examined or photographed
with an Hitachi H600 Transmission Electron Microscope.
Confocal
Microscopy
*
Note, researcher will need to edit out unused parts of this
description
The sample
was placed under an Olympus BX50 microscope with a motorized
stage and viewed using the Olympus Fluoview confocal system
using Melles Griot Argon (488 nm), Krypton (568 nm) and HeNe
(633nm) lasers.
Objectives
included U PLan Fluorite 10X and 20X and the UP PLAN 40X and
60X Oil. A PLAN 100X oil with aperature. Nomarski prisms are available for all objective strengths.
Proprietary Olympus "Fluoview" software(FV300 v 5) is operated through
a Windows XP WorkstationWavelengths of emission are controlled by a Beam
Splitter cube, Laser emission frequencies are 488/568/633 nm, and emission filters are 510 and 550 BP, 605 and 700 bp.
Confocal Manual is at: CONFOCAL MANUAL
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