Michael Hooker Microscopy Facility (MHMF.ORG)

Zeiss 510 Meta Laser Scanning Confocal Microscope (\\HERTZ computer)

0.  Notices

• Feb 06, 2011: The PC computer hard disk drive was replaced. 
  • User profiles will need to be recreated.  Run "New User Generic Profile" once (if not already run).
  • Old data can be restored on request.
• The confocal computer's network name is \\HERTZ  or  \\hertz.med.unc.edu  or  \\152.19.58.189
• Requirements for users with wet or live samples
• Galvanometer stage (HRZ).  Even when using the microscope fine focus for a z-series, the leveling button should be pressed in order to move the galvo to its more reliable mid range.
• Zeiss 510 confocal transmitted light condenser has been replaced with a manual unit.
• The transmitted light polarizer on top of the condenser should be set to 0 degrees for both viewing by eye and scanning.  (Note that this is different to the polarizer setup when the scan head was attacked to the side port.)
• Note that the orientation of the scanned image relative to the view observed using the eye pieces is currently not rotated 90 degrees and flipped since the scan head was moved to under the base port.
• April 27, 2004: A Chroma HQ490/30 nm bandpass filter has replaced the 505-550 nm filter in the emission filter section of PMT Channel 2.  This filter is for use with the 458 nm laser line.
• Feb. 10, 2004: Running the Argon laser
• April 21, 2003: Need to switch to Vis mode explicitly after scanning in order to see through the oculars and choose wide filed transmitted/dic/fluorescence
  
• Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Resonance Energy Transfer FRET are available on the Zeiss confocal and are also available on the Leica SP2 confocal.
• Heated Stage Adapters
  • A heated stage adaptor from 20/20 Technology Inc. is available on the Zeiss confocal. It is designed specifically for the Costar transwell inserts. Also a 2-gas curtain stage adapter is available, but still needs some minor modifications to attach it to the microscope.
  • A "Tempcontrol 37-2" heated stage and 63 x objective heater are available for long time lapse imaging.  This stage is for warming 35 mm dishes or microscope slide format chambers.  Do not set temperature greater than 40C.
• Regions of Interest (ROIs) can be stored (as files) on the Zeiss 510 full software on the \\Hertz computer.  They are used to aid FRAP and other analysis and can (and for some analysis should) be saved.  The saved regions are stored as files on the C:\AIM\ROIS directory of the \\Hertz computer.  On the network this directory is shared as \\hertz\rois.  The offline analysis software on the \\Snell image processing workstation computer can also use these ROIs if they are imported onto that computer's C:\aim\ROIS directory.  These files are automatically updated from \\Hertz to \\Snell daily at about midnight, and only newer ROIs are copied.  Updating can also be manually done if you need the ROIs updated sooner by clicking on the Update ROI icon on the desktop of Snell.  Note that ROI files are common to all users and can be overwritten by any user.  Please consider saving them to a private location.

1.  Scanning

• Basic scanning - a step by step guide to basic operation of our system
• Lambda scanning - multi dye spectral unmixing (notes still UNDER CONSTRUCTION)
• Very Basic MultiTime (draft)
• Setting up the Axiovert200 microscope stand

     Fret acquisition and analysis - Sample Fret scans
     Zeiss' manual for the software (v3.2) is available for download from the Zeiss FTP site (pdf format, 3 files). 

2.  The Zeiss 510 Meta Confocal System

• Laser Spot Scanning Confocal Microscope
• Inverted microscope
• Laser excitation bands
  • Argon: 458, 477, 488, 514 nm Important to fire this laser in "standby " mode first until "ready" indicated it is ready to be switched to "on" Also, after switching laser directly to "off", wait 5 minutes with the cooling fan running before switching fan power off (square gray remote switch )
  • HeNe1: 543 nm
  • HeNe2: 633 nm
• Detection wavelengths selected with dichroic and barrier filters
• MultiTrack (sequential scanning)
• Meta - 32 channel spectral detection in fixed wavelength 10.5 nm bands
• Standard XY scanning
• Rapid XZ scanning is possible using a galvanometer stage
• Transmitted light, DIC, FRAP, FRET

**** Note that oil lenses are color coded on the nosepiece with a red protector and water lenses correspondingly in blue.

 WD = Is working distance from front of objective to first surface of a #1.5 cover slip

Note: if objectives are changed on the turret then the setup in the LSM 510 Meta must be updated in order to reflect the changed objective parameters.  This is important for correct confocal scanning.  Contact a facility director for assistance.

Links to Zeiss objectives (Note that not all, in fact most, of these objectives are not optimized for confocal microscopy)

• Objectives for research
  • Plan-Neofluar  |  Plan-Neofluar - Multi-Immersion  |  Epiplan Neofluar  |  Plan-Apochromat  |  Epiplan Apochromat  |  C-Apochromat  |  Fluar

Filter sets (widefield epifluorescence - eye viewing only):

Viewing Your Images/Image Database/free viewing software/exporting views:

• Zeiss 510 confocal image databases (LSM image browser)

Some notes on the dichroic IDs:

• In the main dichroic pull down is a selection called NT 80/20. This is a wavelength independent light splitter used for reflectance microscopy. 20% of the laser (excitation) light is reflected to sample, 80% of the emission from the sample transmits through it to the PMT.
• HFT designation reflects 99% listed excitation wavelength to sample, but blocks transmission of that specific wavelength through it.
• NFT reflects everything below designated wavelength, transmits everything above.
• KP is a reverse convention dichroic, it reflects everything above its listed value and transmits everything below.
• Plate is ~100% transmitting glass element.
• "Change Pos" is the selection for accessing the user changeable filter behind the scan head access door (in front of PMT2 only)
• In some situations double/triple dichroics transmit and collect light more efficiently than the single dichroics.  This kind of behavior has also been noted on the LeicaTCS-NT & LeicaTCS-SP.

3.  Known Bugs

• In the UNC e-mail system *.mdb files (which are part of the Zeiss data base) can not be sent.  Attachments of .mdb files are filter out, and no warning message is given.  E-mail scanner searches for .mdb profile inside the file so simply renaming the file will not protect it from deletion.

4.  Displaying Captured Images on your PC computer.  The Zeiss Image Browser (free software)

• Install the Zeiss image browser which is available at http://mhmicroscopy.med.unc.edu/software.html   Accessible from UNC computers only.
• The Zeiss image databases may be viewed and images exported with this software.
• Note the *.mdb, *.lsm files in the directory form an integrated data base.  Do not rename files in Windows or alter the *.mdb file in Microsoft Access.  Scans may be renamed from the Zeiss Image browser

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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