Overview of the Physiology Imaging and Neuroscience Confocal Facility |
1.1 A LeicaTCS-NT confocal laser scanning confocal microscope with:
Three separate excitation lasers centered at 488 nm, 568 nm & 633 nm, which allow viewing of Fluorescence, Texas red/Rhodamine and/or CY5 like fluorophores and/or transmitted light, or reflectance.
A Leica DMRI inverted microscope with 20X 0.7NA, 40X 1.25NA & 100X 1.4NA oil immersion objectives for confocal scanning. Polarizes and prisms for the 40X and 100X objectives allow Differential Interference Contrast (DIC) viewing by eye or via laser scanning. A 10X 0.3NA dry objective is also available for sample location and orientation.
Simultaneously or sequentially confocal scanning on any of the three fluorescent channels may be performed along with transmitted light or DIC imaging. All such images are in geometric register, which permits the localization of fluorescent markers relative to a wide field image (transmitted light).
A mercury lamp and dichroic filters for fluorescence (FITC) and Rhodamine/Texas red (TRITC) viewing of these fluorescent bands by eye for initial sample evaluation and subject location.
A Pentium Pro PC computer running WindowsNT 4.0 for image storage, processing, analysis and for network services. Zip and Jaz drives are provided for image storage.
Software available on the system in addition to the LeicaTCS-NT control and image processing software:
PhotoShop 5.0 – general image manipulation and figure preparation.
Fortner Slicer 1.1 - 3D rendering
ThumbsPlus 4 – image cataloging
WangImage – viewing of multichannel/z-series/time lapse image sets
Tiffsplit4 – splitting multipage tiff images into single image files for handling by standard (& consumer) image processing software (e.g. PhotoShop)
A temperature controlled tissue culture stage, which holds 25 mm circular cover slips and applies CO2 (Kindly loaned by Dr. Aldo Rustioni).
1.2 A general purpose PC (\\ORAC) is available for image processing, analysis, figure preparation, movie production, CD-R & CD-RW recording. This equipment is provided by the Department of Cell & Molecular Physiology’s high speed photo diode imaging array. Confocal users from all departments have been invited to utilize this resource. This computer is also used for program development and the preparation of confocal usage reports and bills. Software on this system includes: Adobe Photoshop 5.0, ThumbsPlus 4, Tiffsplit4, VidEdit, Slicer, Voxblast, ImagePro+, EasyCD Creator 4, WinZip, LViewPro 2.1, Premiere 4.2, Cas 4.02 and various programs for handling Bitmap Image series.
1.3 A PC for Windows 2000 Server domain control services (PHYSIOLOGY domain), which is used to store confocal user account information and logon/logoff times for auditing and billing purposes. This computer also provides web page services for http://confocal.med.unc.edu, and also provides a booking calendar. The booking calendar is the means by which users can book time on the Leica Confocal system from anywhere with web access (on and off campus).
All computers are on the campus network. Users' images may be accessed via the campus network using the following protocols: Appletalk, Novel Netware, MS-Windows (Netbeui), FTP.
The facility provides a Confocal Laser Scanning Microscope system and know-how for high resolution confocal microscopic imaging, data processing and image analysis. All users receive training on how to use the Leica Confocal tailored to their prior knowledge before being allowed to use to the system. Training is directed towards producing independent users. Sample preparation is solely the users responsibility. Advice is available but the facility personnel do not perform experiments, gather data for users or are responsible for scientific outcome. Use of the confocal scanner is charged on a fee per unit time of use basis. Currently users may come from anywhere in the UNC community. Access to users from other institutions has been allowed when such users are collaborators with UNC personnel.
The facility is under the management of Dr. Michael Chua, with Dr. Robert Sealock as the senior faculty supervisor. Dr. Neal Kramarcy prepares monthly usage reports and assists with user enquiries. Hardware resources are nominally available from 7 am to 9 pm Monday to Friday and 7 am to 6pm Saturday and Sunday. Users from outside of Cell & Molecular Physiology may borrow an access card to the Medical Sciences Research Building for short term after hours access.
Training in the independent use and proper care of the confocal microscope and the associated computer equipment and software, some advice in experimental design and assistance in optimal use of the equipment and image processing and handling are provided. Once trained and checked out, users are allowed independent access to the equipment. Training, which involves major contact time, is done on Mondays. Simple user questions are answered as needed when time permits.
A World Wide Web booking calendar system is provided for the facility, and is available from any location to all authorized users (see http://confocal.med.unc.edu/scripts/display_bookings28.exe). World Wide Web pages concerning usage and information about the LeicaTCS-NT are available to all at http://confocal.med.unc.edu.
Dr. Chua & Dr. Kramarcy assure that the facility, including optical, mechanical, software, network and computer components, is an efficient, smoothly running facility. Occasionally small programs are written by Dr. Chua, to assist in the handling of image data, especially multi-channel, z-series and time series data, and made available to authorized users of the facility.
The WindowsNT computers in the facility maintain extensive logs of usage of the confocal equipment and ancillary computers used for image processing. The information from these logs is passed on to the Cell & Molecular Physiology Department business office, where personnel prepare bills for the confocal scanner time accrued by users. PIs are billed via the usual UNC billing channels.
The Cell and Molecular Physiology Department houses the facility, provides a telephone and partial salary support to Dr. Michael Chua & Dr. Neal Kramarcy in return for carrying out services for the facility. The Department also paid for the purchase of WindowsNT software and Visual Developer Software and a hard disk drive. Accounting support and management of the trust fund is provided by the Cell & Molecular Physiology business office.
Usage charges:
$6.25 per 15 minutes of scanning. Minimum charge time is 15 minutes for each and every logon.
$100 initial training session per user. Auditors are free but are charged $100 fee if they become approved users.
Subsequent training is charged at the same rate as scanning ($6.25 per 15 minutes)
Use of the general purpose PC and Confocal PC for image analysis processing and preparation (no confocal laser scanning) are provided without charge to authorized users.
Access key (optional) to 006 MSRB deposit $20.
All users/trainees are charged at the same rate regardless of Laboratory/Department/Institution.
See http://confocal.med.unc.edu/wwwleica/Operation/Booking_Policy_Charges.html
4. Location
The confocal microscope facility is housed by the Cell & Molecular Physiology Department in room 006 of the Medical Sciences Research Building.
For a map see http://confocal.med.unc.edu/wwwleica/Location/Location.html
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Dr. Michael Chua |
843-3268 |
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Dr. Robert Sealock |
966 1272 |
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Initial capital funding for the Leica Confocal system was provided by a Research Resources for Medical Schools grant obtained in 1996 by Dean Michael Simmons, PI; Stanley C. Froehner, William Marzluff, and Oliver Smithies, co-applicants.
Further information please contact Dr. Michael Chua or Dr. Robert Sealock
Created 2000-02-23 updated 2007-07-19
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© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC. All rights reserved. | |
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