Zeiss 510 Meta Laser Scanning Confocal Microscope (\\HERTZ
- 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
- Requirements for users with wet or
- 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.
510 confocal transmitted light condenser has been replaced with a
- 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
- 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
- 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.
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
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
- 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
(These are the objectives shipping with the
confocal. Other objectives will be available)
||xy resolution (ph=1.0)
||z res (ph=1.0)
||dic 44-44-31 (I)
||0.17 mm (#1.5)
||44 01 45
||0.14-0.18 (#1 to #1.5)
||44 00 52
(good for deconv)
||0.15 to 0.17 (#1 to #1.5)
||44 06 68
|| 0.17 (#1.5)
||44 07 82
||40 00 90-01
**** 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):
|position in carousel
||beam splitter (dichroic) nm
Viewing Your Images/Image Database/free viewing
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
- 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
4. Displaying Captured Images on your PC computer. The
Zeiss Image Browser (free software)
- Install the Zeiss image browser which is available at
Accessible from UNC computers only.
- The Zeiss image databases may be viewed and images exported with this
- 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 2001-2013 Dr. M. Chua,
Program in Molecular Biology & Biophysics, School of Medicine, University of North Carolina, Chapel Hill, NC 27599