Michael Hooker Microscopy Facility (MHMF.ORG)

Index

0.  Notices
1.  Operating the system (a step by step guide to basic operation of the confocal)
2.  The System
3.  First time use (new user)
4.  Known Bugs
5.  Viewing Leica Files
6.  Leica confocal file analysis and processing
7.  Links
8.  Reporting Problems
9.  Password problems/German key board
10. Dealing with software crashes and microscope stand lockups

0.  Notices

• System Crashes & Recovery
  • There are multiple CPUs in the scanhead, microscope stand and main PC, which can individually crash causing the system to lockup in various ways.  The quickest route to recovery is determined which subsystem has locked up.
    • Microscope stand freezes - shut down LCS software on PC, power down stand (controller [2]), wait 15s, restart stand, then the LCS software
    • LCS software can not initialize the hardware - check that objective turret rotation is in a click position, shut down LCS software and restart it
    • Scanning freezes or can not be stopped - shut down LCS, power down Scanelectronics ("scanner" [5]), power down microscope stand (controller [2]), restart stand, restart scan electronics, run LCS software
    • If the above fails - shutdown LCS software, turn off scanelectronics ("scanner" [5]), turn off microscope stand (controller [2]), if any error involves the SCSI system, shut down Windows and power off the PC computer, but leave lasers running.  Restart the system following the standard start up procedures.
  • Please make a brief note of what happened in the log book for each crash!  This helps us keep appraised of the system's health.
  • More words on dealing with crashes and lock ups.
• Erratic movement of the motorized stage
  • On very rare occasions the motorized stage will move up unexpectedly if the objective turret is turned, even slightly.  In order to avoid injury when cleaning immersion fluids off the objective used, move it to two positions to the right (or left) away from the optical path
    1. Lower stage down using coarse focus button on right side of microscope stand
    2. Remove slide
    3. Turn objective to be cleaned from the light path to two positions to the right (or left)
    4. Gently clean objective with lens tissue.  Blot on the glass front element.  Rub slowly on the metal around the glass element.
• "New" green solid state 561 nm laser (Sept. 2004)
  • Note that power levels above ~20% may result in photobleaching and cross excitation of longer wavelength fluorophores in multiply labeled samples.  Facility personal can assist with setup.
  • More info for users from prior to Sept. 2004
• Please sign the log book in the room and note any problems.
• Switching to dual monitor mode if single screen mode comes up
• Leica Confocal Microsystems 1-866-830-0735 application questions about the LCS software if facility personnel are not available.
• Here is the SP2 TCS software manual (10 MBytes)
  • (An MHmicroscopy account is required.  Remember to put mhmicroscopy\ before your user name)
• Upper Wallaston prism (objective Wallaston) will exacerbate reflections during scanning with dry (non immersion) objectives.  Switch objective prism wheel to bright field position BF when not doing DIC/Nomarski. 
• Transmitted light scanning through the condenser can not be performed with the UV excitation laser alone.  Turn on a visible laser even if the wavelength is not required for fluorescence.

1.  Operating the system

Power up, setting up microscope, starting software, scanning, saving, shutting down, special scanning modes

2.  The System

• Laser Spot Scanning Confocal Microscope
• Upright microscope with DIC (Nomarski and epi-fluorescence) DM-RXA2
• Visible (blue, green & red) and UV excitation lasers switched/modulated using an AOTF
• Simultaneous transmitted light or DIC imaging while scanning confocally
• AOBS for splitting of excitation lines from emission bands
• Photo Multipliers Tube (PMT) light detectors with spectral discrimination
• High precision galvanometer z-axis positioning stage

• "UV" 351/364nm for e.g. DAPI, Hoechts, Alexa 350
• "Blue" Argon 458/476/488/514nm  e.g. 488 nm FITC, Bodipy,  Alexa 488. GFP, CY2, Fluo-4, Calcein; 458 nm CFP, 514 nm YFP
• "Green" Solid State Diode Pump 561nm  e.g. Texas Red, CY3, Alexa 568, Alexa 594, will excite Rhodamine, TRITC, DsRed & Alexa 543 (replaced Krypton 568 nm laser on Sept 2, 2004)
• "Greenish Yellow" Krypton 568nm was removed from service on Sept 2, 2004
• "Red" HeNe 633nm  e.g. CY5, Alexa 633, Alexa 647, TO-PRO-3, Draq-5

2.2  Objectives:

* Note: exact objective installed should be confirmed by looking at the microscope
** dipping lenses use no cover slip.  All other lenses prefer a number 1.5 (170 um) glass cover slip.
*** When not using DIC the objective Wollaston should be in the blank (BF) position and the Analyser should be out
Use only Leica immersion oil

*Counter-clockwise= increasing position number ↑
clockwise= decreasing position number ↓

2.3  Detection:

• Filtering through an Acoustical Optical Beam Splitter (AOBS)
• Spectral separation through a prism
• Three tunable detection bands with adjustable spectral windows before the three Photo Multiplier Tubes (PMT)
• Standard XY scanning with zoom and rotation (N.B. rotation is not available with UV excitation)
• XZ rapid scanning using galvanometer stage (range +/-85 um)
• XZ scanning using microscope stand's fine focus (range mm's)
• Transmitted light and DIC (non confocal)
• FRAP, Emission Spectra, ratio imaging

2.4  Analysis:

• Intensity profiles, FRAP, FRET, fluorograms, 3D renders, maximum projection, average projection

3.  First time use (new user)

Copy LCS parameter files from d:\users\_typical\lcs to d:\users\username\lcs
Do not use factory default settings since most parameters are not appropriate for our system

4.  Known Bugs

• Parameter window zoom factor and several other factors does not update when loading an old image.  To see these parameters, just right click on the image name in the experiment window to get a table of scan information.  This information can also be gleaned from the .txt file in the image database directory.

5.  Viewing Files

LeicaLCS Light - This program run under WindowsNT4/WindowsXP is available from Users' Web pages.  Note that LeicaLCS lite does not run under Windows2000

8.  Leica confocal Image analysis and processing

• scale bars

7.  Links

      www.leica-microsystems.com
       LeicaLCS-lite software FTP site - ftp://ftp.llt.de

8.  Reporting Problems

     Please contact facility personnel

     Leica Confocal Service and Support
     866-830-0735    8:00 A.M. to 5:00 P.M.
     confocal@leica-microsystems.com
     serial number 195353
     Note: if you contact Leica directly please inform facility personnel as well so that we can follow up.
      Installation date: 23 April 2002 

9.  German Keyboard mappings can prevent passwords from being recognized

German keyboard mappings translates some keyboard keys to other characters.  Basically some keys have a different meaning to the label embossed into the key cap.  Characters to avoid include: !  @  #  '   there are others.  These characters may be found in other positions on the keyboard. 

10.  Dealing with software crashes and Microscope Stand Lock Ups

• See here for information

11. Applications:

• Indo-1
  • Chroma suggested filters
  • Omega suggested filters

		Copyright 2001-08  Dr. M. Chua, Office of Research, University of North Carolina, Chapel Hill, NC 27599
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