The facility provides Laser Scanning Confocal Microscopy
using either a Zeiss LSM 510 UV system, a Zeiss LSM NLO (2-photon)
Meta system or a Zeiss/Bio-Rad MRC 1024 system. Confocal software
allows simultaneous or sequential three channel (24 bit) acquisition
of 2D, 3D and 4D data. Three additional processing stations
are available for post-acquisition analysis and publication
quality prints can be produced using Codonics NP-1600 dye sublimation
printers or Epson photo quality printers.
Imaging of both fixed specimens and live cells is provided.
For live cell imaging, cells are placed in a temperature-controlled,
perfusable enclosed chamber or an open chamber allowing acquisition
over several minutes to several hours. Fluorescent Recovery
after
Photobleaching (FRAP), Fluorescence Resonance Energy Transfer
(FRET), and co-localization studies can be conducted as well
as imaging of UV excitable dyes. The IR laser of the 2-photon
system facilitates imaging of thick specimens and minimizes
tissue damage while the Meta detector provides multi-spectral
imaging.
Several software packages are available to users including
Bitplane’s
Imaris for volume/surface rendering, tracking and measurements;
Media Cybernetics’ Image Pro Plus and 3D constructor; NIH’s
ImageJ and Adobe’s Photoshop.
The core is supported by the Center for Cancer Research at
no charge to individual users. Collaborations with outside
laboratories
are also considered, time permitting. Please refer to the
publication list for examples of confocal imaging that have
been done in
this facility.
Investigators should contact Susan Garfield, Facility Manager,
to discuss their project and facility procedures. If the
project is appropriate, appointments will be made for using
the confocal.
The "Confocal Slide Submission Form" found at http://elsie.nci.nih.gov/confocal is completed when samples are ready for imaging. Consultations
on experimental design with emphasis on proper controls for co-localization,
FRET and FRAP analysis is provided along with suggestions for
optimum fluorophore selection, sample preparation and fixation,
and sample mounting and storage until image acquisition.
|
