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0.  Notices

• This machine is owned by the Marsico Lung Institute/Cystic Fibrosis Research Center: 
  • Use is limited to approved and trained users
  • All use must be logged stating 1. User; 2. Lab(s); 3. Project(s)
  • Machine must be started correctly with particular attention to objective care, stage clearance
  • Only people who have had an orientation/training from Michael and have assigned accounts on this machine may use the system
  • Booking calendar  or  via http://microscopy.unc.edu/booking
  • Machine must to shut down correctly with attention to software laser shut down, careful objective cleaning, lowering objective turret, putting 10x objective in light path, exiting LAS X software and letting it shut down completely before logging off, keying off laser emission, turning off laser and master power.
  • Environmental chamber currently supports temperature and humidity control.  When tanks mountings are installed CO5 and anoxia will be available.

1.  Operating the system

• Quick Guide:
  • Powering up:
    • Check that microscope table is correctly floating - ~5 mm from top of aluminum to underside of rubber disk
    • Turn on power strip (it should be on already)
    • At master box
      • Turn on master switch
      • Turn on laser switch
      • Turn on Emission key
    • Log into computer - use MHmicroscopy domain account
    • Ensure objective focus is lowered - push Z down button (see picture on right)
    • Ensure nothing will fowl up stage movement - e.g. chambers, dishes, wires, etc.
    • Start LAS X software
    • Choose
      • Machine.xlhw
      • DMI8
      • OFF at "Load settings at startup:"
    • Initialize stage if going to use programmed x-y position control or tiling
  • Find sample by eye
    • Once LAS X software is running sample can be viewed by eye
    • On first startup cycle the power of the LED light source off for ~5 seconds then back on. Switch is on the back of the unit (not the one on the front).  Only need to do this once on first view. (Its some sort of mysterious safety procedure)
    • Find and focus on sample.  And set upper limit.
  • Simple Scanning:
    • Select settings from choice of:
      • Dye manager
      • Load a previously saved .seq file
      • Load previously scanned image and "Apply Settings"  
        • Note manually set Format, and zoom desired (Right click and choose "Properties" on previously saved scans if you want see these settings for consistency)
    • On first scan only setup "Fast Live" options: 
      • Click on circle at right of "Fast Live" button
      • ON "Fast Live Scan Settings" choose "Maximum Format" of 400 and unselect "Selected Setting Only (Between Lines Mode)"
    • Be careful of bleed through
    • Sequential scanning is preferred to ensure no bleed through
    • Set Zoom and Pixel size to adequately sample data
    • Set averaging to reduce noise
    • "Live" to view all channels
    • "Fast Live" to show only selected channels
    • Save experiments as .LIF
  • If Exports do not work correctly try using LAS X core free viewer instead
  • Shutting Down:
    • Lower stage with course focus button on right side of scope body
    • Power off lasers in software
    • Exit LAS X software
    • Put 5x, 10x or empty objective into light path
    • Transfer data to a server - e.g. SOM shared server or mhmicroscopy share
    • Wait for LAS X software to close completely  <-- THIS IS IMPORTANT
    • Turn off:
      • Emission key
      • Laser power switch
      • Master unit switch
      • Leave power strip on
    • Log off from Windows  <-- This is important too.  (Please do not just "Lock the screen" when done)
      • Left click: Window (bottom left)
        • Left click the little person
          • Left click "Sign Out"
    • Sign log book

2.  The System - see here for details  http://microscopy.unc.edu/confocal/

• Laser Spot Scanning Confocal Microscope
• Inverted microscope stand DMI-8
• Lasers: 405 nm continuous wave diode & Super continuum pulsed white light laser 485 nm (blue)  to 685 nm (far red)
• Five Hybrid Photo Multipliers Tube (PMT) light detectors with spectral discrimination
• High precision galvanometer z-axis positioning and motorized x-y positioning stage
• Environmental chamber for temperature, humidity and CO2 control with hypoxia

• "UV" Diode laser 405 nm  e.g. DAPI, photo activation
• "White Light Laser" Pulsed White Light Laser from 485 nm to 685 nm, up to 8 wavelengths can be selected  e.g. Texas Red, Alexa 568, Alexa 594, mCherry, will also excite Rhodamine, TRITC, DsRed & Alexa 543

2.2  Objectives:

** dipping lens, not kept on turret  ⁺ Approx. resolution pinhole=1 Airy unit

2.3  Detection:

• Emission spectral separation through a prism
• Five tunable detection bands with adjustable spectral windows before the five Photo Multiplier Tubes (PMT) type HyD S
• Standard XY scanning with zoom and rotation
• XZ rapid scanning using galvanometer stage (range +/-250 um)
• XZ scanning using microscope stand's fine focus (range mm's)
• Transmitted light and DIC (non confocal)
• Polarization Anisotropy

2.4  Direct Eye Viewing Fluorescent filters (Widefield, i.e. not confocal)

3.  First time use (new user)

4.  Known Bugs

5.  Viewing Files

• Leica LAS X core - This program runs under Windows 10 is available on the \\minsky.med.unc.edu server

8.  Leica confocal Image analysis and processing

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