Marsico Hall Microscopes Facility (MHMF.ORG)

0.  Notices

• This machine is owned by the Marsico Lung Institute/Cystic Fibrosis Research Center:
  • Use is limited to approved and trained users
  • Please arrange orientation, training and sign on access with Michael
  • All use must be logged recording 1. User; 2. Lab(s); 3. Project(s) including chart string field
  • Machine must be started correctly with particular attention to objective selection, z-position, stage clearance, upper limit setting
  • While searching and viewing samples by eye very careful attention must be made to not focus up into the sample holder, especially when near edges of samples
  • Don't focus into slide.  The stage can be buckled and the objective damaged.
  • Booking calendar  or  from http://microscopy.unc.edu/booking
  • Machine must be shut down correctly with attention to:
    • Laser shut down in LAS X software
    • Careful objective cleaning if glycerol or oil used, lowering objective turret, putting 5x or 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 power and master power (Only after lasers have been shut down in the LAS X software)
  • Environmental chamber supports temperature and humidity control, CO2 (0 - 10%) - Anoxia is possible by arrangement (Nitrogen tank will need to be ordered).
  • If environmental chamber is going to be used contact Michael about changing stage top box, objective clearance & condenser height setting in order to prevent stage crashing into condenser

1.  Operating the system

• Note: Before viewing your sample the system should be powered up and the LAS X software initialized
• Quick Guide:
  • Powering up:
    • Check that microscope table is correctly floating - ~5 mm from top of aluminum to underside of rubber disk (adjust red knobs if necessary)
    • Ensure power strip is turned on (it should be on already, and never turned off)
    • At master box
      • 1. Turn on master switch - wait >=5 seconds
      • 2. Turn on laser switch
      • 3. Turn on Emission key
    • Log into computer - use your local account
    • Objective must be lowered - push and hold Z down button (see picture on right), or set focus to course and turn the course focus knob in the down direction.
    • Place 5x, 10x or no objective in light path in order to avoid the stage crashing an objective (turning by hand is OK)
    • Ensure nothing will get in the way of stage movement - e.g. chambers, dishes, wires, etc.
      • If a stage top box is present ensure condenser will not hit the stage top box when initializing the x-y motorized stage -
      • i.e. swing the condenser back
    • Once the microscope stand display has finished initializing start the 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 - power off the LED 3 light source off for ~3 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:
      • - 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)
      • - Dye manager (more challenging, but )
    • 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 if doing simultaneous scanning. (i.e. When not acquiring all channels sequentially)
    • 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
      • Adjust PMT gain mostly or laser power. When viewing multiple channels ensure multiple channels are displayed  (Click on image panel to switch between single panel & multiple panel display)
    • Save experiments as .LIF
  • If Exports do not work correctly try using LAS X core free viewer for Windows instead
  • Shutting Down:
    • Lower stage with course focus button on right side of scope body
    • Clean oil or glycerol off objectives if used.  Lens tissue - gently blot on glass lens with different areas of dry lens tissue.  Can gently rub metal body of objective.
    • 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 hardware:
      • Emission key  #7
      • Laser power switch  #6
      • Master unit switch  #5
      • New procedure: Power strip off switches in reverse order  #4,  #3,  #2  but leave on #1 for 1 minute to allow laser unit to cool - then turn it off
    • 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/

• Inverted microscope stand DMI-8
• Laser Spot Scanning Confocal Microscope
• 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 emission 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 with pinhole=1.0 Airy unit 
WD = working distance  DIC = differential interference contrast or Nomarski

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 (Hybrid GaAsP) 410 nm to 850 nm
• 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)

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

3.  First time use (new user)

4.  Known Bugs

• For multichannel scans need to have multiple panels open for smart gain & smart laser adjustments correctly.  (With single panel mode a hidden channel will be adjusted).  On single mode double click on single image to restore multiple panels.

5.  Viewing Files

• Leica LAS X Office - This program runs under Windows 10 is available on the \\minsky.med.unc.edu  server
• Leica LAS X core - This program runs under Windows 10 & 11 is available on the \\minsky.med.unc.edu  server
• FIJI
• Imaris

8.  Leica confocal Image analysis and processing

		Copyright 2001-2024 Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
Go Back	Booking Resources	Questions/comments/problems: Michael Chua