Marsico Hall Microscopes Facility (MHMF.ORG)

0.  Notices

• This confocal system 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
  • Use should be logged recording 1. User; 2. Lab(s); 3. Project(s) including chart string field
  • System 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 taken 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%), hypoxia
  • 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
  • Connecting to file shares (SOM server), e.g. CF Groups/shared, MHI groups, Peds groups

1.  Operating the system

• Note: Before viewing your sample the system should be powered up and the LAS X software initialized as described below
• 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)
    • Turn on power strip switches in order #1 (wait ~5 seconds) then #2 to #4 each with 3 second wait
    • At master box
      • 1. Turn on master switch (#5) - wait >=5 seconds
      • 2. Turn on laser switch (#6)
      • 3. Turn on Emission key (#7)
    • 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 larger 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. ESPECIALLY TO THE LEFT OF HE STAGE.
      • 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 left of the unit.  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 previously used settings )
      • - Dye manager (more challenging)
    • 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 much 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 objective(s) 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
    • 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
    • Sign out from Windows  <-- This is important too.  (Please do not just "Lock the screen" when done)
      • Right mouse click: Window (bottom left)
        • Click "Shut down or Sign out"
          • Click "Sign Out"
    • Report any problems encountered - e.g. email Michael.  Take screen captures &/or phone pictures. 
    • Fill out 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 2001-2026 Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
Go Back	Booking Resources	Questions/comments/problems: Michael Chua