Welcome to the UNC Neuroscience Center Leica Confocal Microscope.

• The UNC Neuroscience Center is hosting the LeicaTCS-NT Laser Scanning Confocal Microscope.  The confocal is located in room 8104 Neuroscience Research Building and is available for use by members of the UNC neuroscience community.
• A world wide web based booking system is running to coordinate booking the confocal microscope.  Bookings may be made by users who have been approved by Dr Tushar Patel (843-8624).
• Mission Statement

 News      Last updated 2007-07-19

• June , 2003 - The confocal has been transferred to the Neuroscience Center and is located in room 8104 (843-3179) of the Neuroscience Research Building.  Questions should be directed to Dr. Tushar Patel tdpatel@med.unc.edu 843-8624 or Dr. Neal Kramarcy  kramarcy@med.unc.edu 966-0326
• Oct 1, 2001 - Dr. Neal Kramarcy is assuming the direction of the confocal facility
  Neal is a very capable and experienced microscopist, confocalist and immuno-histochemist.  Michael Chua is directing the Michael Hooker Microscopy Facility, but will continue to consult with the facility. 
• Aug 21, 2001 - The upright microscope is now the preferred system on the confocal
  The new Leica upright microscope has been attached to the confocal system and is the preferred scope.  It has  better optical efficiency (i.e. produces brighter images), an improved 10x 0.4 NA objective, 40x 0.8 NA water immersion (dipping) lens and the familiar 20x.0.7 NA and 100x.1.4 NA lenses. Users should scan with the upright scope unless their samples require the inverted format.  The scopes can be switched in a few minutes, which requires moving the scan head, galvanometer stage and reassigning the scope controlled in the software.  Please contact Dr. Neal Kramarcy if you wish to switch scopes.  
  Both the inverted and upright scopes stand on an antivibration table.  Check that the gas is turned on before scanning, and turned off if you are the last user of the day. 
• Jul 2001 - Long Term Storage Drive is Available
  The long term storage drive has been replaced.  Usage is on a first come basis.  Images older than 3 months will not be automatically removed from this drive.  However space is still finite and when the need arises a request may be made to remove data from it.  
• May 2001 - Krypton Laser - flickering out
  The Krypton laser has flickered out on rare occasions.  This is observed as interleaved scan lines that are dark in the Texas red / Rhodamine channel.  Shutting down the krypton laser, letting it cool for 10 minutes and restarting seems to remove the problem.  Please make a note in the sign in book if this problem happens while you are scanning.
• Feb 2001 - 3 Month Limit on Storing Image Files on the Confocal Scanning Computer
  Image files should be stored on the D: drive on the Leica_tcs computer.  Image files older than 3 months will be removed by an automated file deletion program.  Image files may also be left on the Mac Share drive (\\CONFOCAL\MacExchange and M: for some users).  When available, the I: drive (\\LEICA_TCS\LongTerm) may also be used.  Image files on the C: drive are subject to immediate removal, although in an emergency users may leave files in C:\USERS.  See http://confocal.med.unc.edu/wwwLeica/DataHandling/DataStoragePolicy.html  for details.
• Feb 01, 2001 - Training during February to July will be carried out by Dr. Neal Kramarcy
  Neal Kramarcy will be coordinating confocal training session during this period, while Michael Chua is spending 2 days a week working at Nation Institute of Neurological Disorders and Stroke, NIH, Bethesda.
• Jan 10, 2001 - Long Term Storage Drive is not available
  While we are awaiting the replacement of a failed hard disk drive the long term storage drive I: will not be available. See Jan 5, 2001.  If you require long term storage contact M. Chua to make arrangements.
• Jan 5, 2001 - Disk drive failure
  On the 28 of December, 2000 the second disk drive in the confocal computer \\Leica_tcs quietly failed.  This drive provided storage for "LongTerm" use on drive letter L:.  The drive has been removed and will be sent for warrantee replacement.  Any data stored on this drive has been lost.  No one has reported missing any data as yet.  Users are reminded that they alone are responsible for ensure that their data is adequately backed up, as we do not have the time nor resources to carry out backup. 
• Oct 4, 2000 - pif2.med.unc.edu is down, which means software can not be downloaded from this secure site.
  Sorry about this.  This problem results from independent software glitches in a number of computers. When time permits these problems will be rectified.  In the mean time U.N.C. users that require software items can obtain them from Dr. Michael Chua (mchuaEXPUNGETHIS@med.unc.edu)
• Jul 27, 2000 - Yet another update to tiffsplit4.exe (version 4.10)!
  Tiffsplit4.exe has been updated to version 4.10 and extends the capabilities of tiffsplit4.exe version 4.06.  This version can generate maximum intensity projection images from z-series stacks as well as extended focus images.  Also, by default, green/blue colors are removed from the split images as well as the projections.  The program is available at: confocal software page (access this page using your confocal user name and password).  Help pages are also freely available at http://confocal.med.unc.edu/wwwLeica/TIFF/TiffSplit4.html.  
• Aug 22, 2000  - New File Storage Policy
  A new for storage policy is in place.  Image files should be stored on the D: drive on the Leica_tcs computer or I: drive or J: drive or M: drive on \\CONFOCAL\MacExchange.  Image files older than 6 months will be removed by an automated file deletion program.  Image files on the C: drive are subject to immediate removal.  See http://confocal.med.unc.edu/wwwLeica/DataHandling/DataStoragePolicy.html  for details.
• April 2000 - New tiffsplit4.exe
  The LeicaTCS tiff image utility program has been updated, again, to version 4.06.  Tiffsplit4.exe may be obtained from the confocal software page  Usage instructions are also available from there.  Use your confocal user name and password to gain access.  Place tiffsplit4.exe in directory in your computer's path. e.g. c:\windows\command.  Tips on how to use it are at http://confocal.med.unc.edu/wwwleica/ImageHandling/ih_splitting_tiff_files.html.  Major new features are:

• 1. Split files are output as bitmap images by default. 
• 2. The -t (lower case) command line switch causes split files to be created as tiff format images. 
• 3. Extended focus images are now named using the original file named followed by '_xf" and the channel letter.  e.g. tiffsplit4 sim.tif -e would create sim_xf_a.bmp and sim_xf_b.bmp.  (instead of sim_a_xf.bmp and sim_b_xf.bmp)  This change helps when selecting file names in bulk for many of the bitmap utility programs and other 3D rendering programs such as T3D, Slicer, VoxBlast, bmovrlay, CAS etc. 
• 4. Bug fixes, particularly when doing contrast expansion during the construction of extended focus images.
• 5. Extended focus overlays can have color channels reassigned using the -r option.  For details see the tiffsplit4 instruction pages at: http://confocal.med.unc.edu/wwwLeica/TIFF/TiffSplit4.html. 

• Long Term File Storage
  Long term storage, if space permits, is available on drive I: (\\LEICA_TCS\LongTerm) on a first come first served basis for now.  See http://confocal.med.unc.edu/wwwLeica/DataHandling/DataStoragePolicy.html  for details.
• Jan 27, 2000 - Jaz drive Unavailable
  Once again the Jaz disk drive has failed.  We are considering not replacing this drive due to its poor reliability.  If this will affect you please let mchuaEXPUNGETHIS@med.unc.edu know. 
• For Summer  2000 - New Confocal Scanning Software
  New LeicaTCS-NT software is rumored for released in Summer 2000.  This is a totally redesigned package for running the confocal microscope and will replace the current LeicaTCS-NT system.  Independent users claim that it is better and it features a new image file format.  When the upgrade is about to be installed training courses for current users will be announced via e-mail and on this web site.  Currently we do not have an expected installation date from Leica.
• Jan 07, 2000 - New file storage policy will be applied starting March 2000.  Images older than 6 months stored on D: \\Leica_tcs and M: \\Confocal will be automatically eased.
• Dec 23, 1999 - Faster and larger D: disk drive installed on \\Leica_tcs
• Dec 12 - Laser realignment by Leica service
• Dec 02 - Main disk drive crash.
  C: drive data, the operating system and Leica software are lost due to a hard disk drive crash.  Files in E:\USERS2 have been moved to D:\USERS2 and files in D:\USERS1 remain at D:\USERS1.  However files that were in C:\USERS were lost.  All users should store their images in D:\USERS1   or  D:\USERS2  or  D:\USERS3.
• New and Improved TIFFSPLIT3.EXE
  New and improve version of tiffsplit.exe is available and called tiffsplit3.exe in order to differentiate it from the old version.  For information see image handling, processing and analysis.
• May 14 - Computer system crashes (everything frozen) on the Leica TCS computer over the past week were found to be due to a failed cpu cooling fan.
• May 14 - Krypton Laser (green 568 nm) mirrors were realigned.  Maximum power at the source increased to 17 mW (power control turned fully clockwise) from 4 mW.  With the power control knob turned to minimum, fully clockwise, power is now 1.7 mW.  Power at the stage with the AOTF at 100% is 4 mW at 568 nm.
• May 7 - A new improved tiffsplit.exe is available.  Improvements include being able to send split tiff files to another drive or directory and the ability converting directly to bitmap image format or to good old standard single page tiff format.  This new version, called tiffsplit2.exe, will be available at http://pif2.med.unc.edu/software/ for the usual charge.  It runs under Windows 9x or WindowsNT from the command prompt.
• May 6 - RAM on the Leica_tcs computer has been increased to 256 Mbytes.  Operations on tiff files of large numbers of z-series and time series stacks, especially of 1024 by 1024 pixel images, will be faster.
• April 7 - LeicaTCS-NT software version 1.6.587 has been installed.  This minor upgrade is intended to fix various minor bugs with no changes in function of the software.  Hopefully it represents an overall improvement.   Please let me know if new problems arise (so that the Leica representative can be harassed, sorry called, about information concerning bugs).
• March 15 - Making extended focus images.   An extended focus image is created from a stack of serial z-sections when the images are flatted (added) into one 2-D layer.  This view differs from a conventional thick section in that all the z-levels are in focus and a crisp image is formed of all the brighter features throughout the volume  Thanks go to Jeff Dupree for discovering how to generate an extended focus image on the LeicaTCS-NT system!
• February 14 - The confocal scanning system has been serviced.  Please note that the laser optics have been realigned and power delivered to the stage has been more than doubled for all lasers. 
• Lens Cleaning 
  We are requesting everyone using an oil immersion lens to blot it immediately when changing to another lens.   Please continue to clean off oil thoroughly when finished on the microscope and replace the paper bib around the objectives used.   Also watch out for the drop of oil left on your slide.  It tends to drip onto the 10X lens.   (Reason: Immersion oil and gravity are a bad combination for the optics and electronics on the inverted Leica microscope.  Oil remaining on a lens that has been just switch out from under the optical path is no longer held up by surface tension and runs down with gravity.) 

• An efficient blotting and objective changing procedure:
  • press learn with the currently used lens still under the slide
  • clean the lens when it is in the oiling position
  • then manually rotate the next required objective into the oiling position
  • check that objective iris is fully open
  • press learn to move the newly selected objective under your slide

• Shut down the Leica_tcsNT program when finished before logging off
  When finished using the confocal system please shut down the Leica_tcsNT program by carefully doing alt-File -> eXit (or hitting the X-box on the top right hand or left hand corner of the blue title bar) before logging out of WindowsNT.  Not shutting down Leica_tcsNT does not harm the system but does cause more usage time to be billed.  Failing to exit Leica_tcsNT with alt-File -> eXit means that the time of Leica_tcsNT closing is not recorded and the timer is left running!

• Leaving the lasers and mercury lamp running: Last booked person is responsible for shutdown
  Laser life is maximized by minimization power up/down cycles.  People are asked to leave the laser on if the next person is arriving within the next 3 hours and is going to use that particular laser (see shutdown and clean up guidelines).  See available times on the confocal booking page to determine if someone is arriving soon.  Please note that the last booked person (on the day) is held responsible for making sure the system is powered down by contacting the previously booked person.  Also if you were the last person booked but cancelled within the last 24 hours you are still responsible for contacting the previous person booked to ensure they know to shut the system down or shutting it down yourself.  In other words please make sure they know you have cancelled!   To help the last booked person make contact, telephone, location and e-mail information is included in the available times on the confocal calendar and the phone number of the confocal room is 966 6117.   If in doubt please turn the system off especially at the end of the day.

• November 18 - 1998
  After hours use
  The confocal system is now available to trained users for longer hours.  Times the system may be used are from Monday to Friday 7:00 to 21:00 (9 PM) and weekends 9:00 to 17:00 (5 PM).  These hours include times when the doors to the Medical Science Research Building are locked after-hours (6 PM to 7 am).  Users from outside of Physiology needing after hours access should contact Michael Chua for further details.

• November 18
  File storage - the Grim Reaper will be coming to harvest excessively old files
  Image directories on the Leica confocal computer are filling up.  The time is rapidly approaching when the grim reaper will need to heartlessly and remorselessly remove files older than 2 months from users' directories.  If you have valuable old files on the Leica confocal computer please back them up and remove them soon or make special arrangements with M.C. (a few Jacksons will help).  You have been notified of the inevitable!  A 3-day warning will be given before files are actually removed.   The Grim Reaper prefers that users manage their own files.  Storage areas that need to be cleaned on the Leica confocal computer are at c:\users (\\Leica_tcs\users) & e:\users2 (\\Leica_tcs\users2).  Files in the MacExchange computer at M:\ (\\Confocal\MacExchange), for most Macintosh users, do not need to be cleaned out just yet.

• New user training
  Training for new confocal users has been held on a on demand basis.  This will continue to be done.  However I ask that training be scheduled for when you have an immediate need to use the confocal.  The system is somewhat complex and the best learning is done while driving the system soon after the training sessions.

• Techniques in Confocal Microscopy - Monthly Courses
  When there is a demand a "Techniques in Confocal/imaging" course can be arranged.  Topics such as introductory theory of confocal microscopy, multichannel scanning display and interpretation, 3D-rendering and processing, basic operation of a compound microscope can be presented.  Sessions would be, perhaps on a monthly basis, for new and current confocal users.  The format of the 60 minutes session would be flexible and depend on the nature of the topics covered and may include external experts, lectures, demonstrations and paper reviews.  The first session on introductory theory of confocal microscopy will be scheduled for early 1999.  If you have any areas of particular interest please let Michael Chua or Bob Sealock  know.  

• ThumbsPlus 3.31 for PC
  ThumbsPlus version 3.31 is now available on the Leica_tcs confocal computer.  This program allows the viewing of images inside a directory as small thumbnail images, which may be clicked on to open up to a full size image.  Operations such as click and drag moving or copying and deleting may also be done from the thumbnail views.  This is a very convenient program to use to quickly view and organize directories loaded with images.  This program can also view all the images in multipage tiff files.  So it is possible to view and separate all the images in multichannel fluorescence scans, or a z-series or time lapse stacks saved using "alt-File -> save As".   Two spare PC licenses for this program are available for the cost price.  See M.C. for information.

• Norton Anti Virus 4.0 for WindowsNT workstation
  The confocal facility has Norton Anti Virus 4.0 for WindowsNT 4.0 Server.  This package includes 10 Norton Anti Virus licenses for WindowsNT 4.0 Workstation only.   Some of these licenses are available to confocal users.  If there are spare licenses anyone in the Department is welcome to use a license.  See M.C. to borrow the Norton Anti Virus CD.

• September - We are noticing periods of heavy booking on the confocal, which is good.  To help users plan their experiments effectively we are requesting that all carefully book ahead, especially when reserving large blocks of time.  We understand that biological experiments, especially with live cells, are not always cooperative.  However please appreciate that reserving time on the confocal may be causing others to delay their experiments.  In addition please remember that there are cancellation penalties (see Charges).  We may be applying them starting October 98.

• August 31 - A 20X 0.7 NA plan apo oil/glycerol/water immersion lens has been installed.   Note that this lens has a correction collar that must be set to the appropriate immersion medium.  This lens is suitable for low magnification and multiple fluorophore confocal scanning.

• August 18 - The latest version of LeicaTCS-NT v 1.6.551d is installed.  It will take some time to come to grips with the new features and idiosyncrasies (see M.C. for the latest information).   Some improvements worth noting are:
  • image saving now returns to the same directory as the last successful save
  • a new ZoomIn button on the tools box zooms up an image area outlined by the mouse
  • sequential scanning with preset gain/offset, pinhole, AOTF & filters is possible
  • photo bleaching protocols including spot bleaching can be saved
  • illumination can be blanked during raster rescanning
  • "F9" sets the pinhole to 1 Airy disk unit (given that the objective is correctly specified)
  • "F1" help files have been updated

• June 14 - To help with the alignment of samples viewed by eye with the confocal scanned view an eye piece graticule has been installed in the right ocular of the Leica microscope.

• May 29 - The LeicaTCS-NT confocal 3-dimensional and analysis package seems to be functional once again.  It has not been functional since last December!   By the way we do have other 3-D rendering (Slicer) & analysis (Voxblast) and movie making (e.g. Adobe Premiere) programs available on the PIF computer.

• May 22 - The Iomega Jazz drive (1 gigabyte) on the Leica_tcs is back! Hopefully it will keep functioning for a while.

• May 20 - A WindowsNT graphics imaging workstation is available in the Confocal Cave (006 MSRB).  Sorry, it is not an Apple G3 system, but it does run Adobe PhotoShop 5.0 as fast.  The computer network name is PIF (an acronym for Physiology Imaging Facility).   It is connected to the Leica_tcs (confocal) and Slave (photodiode) image acquisition computers on the PHYSIOLOGY Domain via the campus network.   Other networks, such as Apple Talk or FTP may be serviced by arrangement.  It is part of the Cell & Molecular Physiology Department fast photodiode imaging system.  Adobe Premiere 4.0, Slicer and other image processing software will be available.  A CD-Recordable and CD-ReWritable drive is on this computer.  Qualified users of the confocal system may log onto PIF using the same user-name, password and domain as used on the confocal computer.

• Passwords and security: Passwords are required for a number of reasons.  
  • Firstly user accounts are used to logon onto either of the Physiology Imaging Facility computers.   Logging on opens up WindowsNT and identifies who is using the computer.  On the Leica_tcs this is the person/PI who gets billed for time when the confocal TCS_RUN program is used. 
  • Secondly accounts open up network access.  Several networks are available.  The Departmental/campus Novell system, the Physiology peer domain, AppleTalk and TCP/IP (includes FTP, the world wide web).  Rights and access generally depend on the serving machine(s) knowing the username and password.  Access is made more convenient by having multiple servers known to the user.  For example having the same name and password on the Leica_tcs and Departmental Novell server can make connecting to the Novell server automatic.  Confocal network access is restricted to those who can provide a valid user-name password and domain.  This restriction ensures that data files are not available to others in the network world.
  • Please note that when logging on from Apple machines the maximum password length is 8 characters.  By the way on the PC side (e.g. Leica_tcs computer) 8 character passwords is the minimum size to ensure security so there is little room for variation from 8 characters.

• Commonly used fluorochromes with excitation and emission information (David Chambers, Salk Flow Cytometry).
  Version of fluorochrome table for printing.  Biorad have a great fluorochrome table too (see the 'reference' then 'fluorescence' section in that page).
  Characteristic Argon laser and Krypton laser emission lines (David Chambers, Salk Flow Cytometry).

• The updated Leica-TCS-NT software version 1.5.451 is installed.  Most operations are the same as in version 1.4 with some improvements.  Be aware that the units of pinhole size has changed and is now in Airy disk units, which normalizes for objective numerical aperture and magnification.  A pinhole of 1.0 is a good value to start at while locating structures.  A pinhole of 0.6 is a good balance of resolution, optical section thickness and light transmission.

Last updated 2007-07-19

					© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC.  All rights reserved.
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