Michael Hooker Microscopy Facility (MHMF.ORG)

Notices:

• Training: All users of this system are required:
  • To have completed the UNC online laser safety training and test if using multiphoton excitation
  • To receive training or an orientation on use of the system from Neal or Michael
  • Only use your own login account
• Location is in room 6033 Thurston Bowles.  Please enter via room 6129
• FV1000 booking Calendar is here:   See Neal or Michael for assistance

Quick Start:

• Call a few hours ahead if incubator box heating is going to be used so that we can get it warmed up and equilibrated ahead of time.  Complete warm takes about 90 minutes.
• Check Nitrogen tank (through doorway and on right) pressure is ~60 psi & main gage not at zero
• Turn on HBO Mercury arc lamp [#1] - do not turn on if lamp has been on in the last ~20 minutes or is warm
• Turn on other control boxes in order listed just below.  Note: Only turn on lasers #4 or #5 if required for you samples
• Turn on other control box [#2] then [#3]
• Turn power switch [#4A] on if using 488 nm (blue) laser, wait 3 seconds then turn on key [4B].
• Turn power switch [#5] on if using 559 nm (green) laser.  Wait until green LEDs stops flashing before turning key on.
• Turn on box [#6]
• Turn on box [#7] - rocker power switch & key to on
• Leave [#8] off unless using the SIM scanner, e.g. for photo activation or bleaching experiments - power switch on & key to on
• Turn on [#9] x-y stage controller.  Usually left on, but check that it is actually on.
• Log on to computer (called \\Sutton) - username, password & MHmicroscopy
• Start FV10 software - icon is on the desktop
• Choose a user profile

• If and only if Ti:Sapphire laser is going to be used for multiphoton scanning turn it on in the laser control window

• Press "Escape" on scope body to lower objective
• Use FV10 software or the control box to select objective
• Use software to turn on/off transmitted light or fluorescence
• Use microscope remote box to select fluorescence cubes for eye ball viewing
• Put slide on stage (cover slip down)
• Press "escape" to raise objective
• Focus on sample
• Use joy stick to position* slide
• Setup Kohler illumination if transmitted/DIC image is going to be acquired with laser confocal scanning
• Setup DIC and adjust if required with transmitted light viewing
• In filter wizard press clear all and choose dyes - press apply
• Press continuous Focus x2 or Focus x4 or xy repeat with Kalman unchecked to locate sample
• Press ^h (control + h) for red overload and blue under load
• Set laser power using AOTF if required
• Adjust Offsets (usually 7 is optimum) and PMT gain - put mouse over box in window to be adjusted and turn mouse wheel or click on area above or below blue interface or click on buttons with small triangles - avoid blue black clipping and red overload
• Ensure pin hole is on Auto
• Check for bleed through (and also cross excitation)
• Choose pixel size (usually 512 by 512 or 1024 by 1024)
• Select or deselect sequential (line sequential is usually preferable)
• Choose Kalman averaging and set to about 4 (typically 2 to 6)
• Press XY scan to take a final snap
• View image and right click to select "save as" either as .iob (single file per scan) or .oif (multiple files - suitable for Volocity)
• Right click on live image and choose add view to display an overlay

Quick Shutdown:

• If next user is arriving in about next hour:
  • Clean all objectives with dry & clean portion of lens tissue (never with Kim wipes)
  • Exit FV10
  • Log off windows
  • Sign log book
• Or of no user expected in the next hour or so:
  • Key off Argon laser 488/515 nm [#4B] - Important: leave rocker switch [#4A] on for ~3 minutes in order to allow for cooling, then turn of switch [#4A]
  • Key off 559 nm diode laser then turn off switch
  • Use laser software control to turn off Ti:Sapphire laser if it is on.
  • Carefully clean immersion objectives used with dry & clean portion of lens tissue (never with Kim wipes)
  • Exit FV10 software
  • Optionally copy data files to \\Minsky (file server)
  • Log off windows
• Turn of control boxes in reverse order [#8] to [#1] - remember to leave [#4A] on for 3 minutes to let 488 nm Argon laser cool (Please leave #9 on)
• Sign log book

Running the  System:

• XY scans
• bleed through
• Sequential versus simultaneous
• Adequate sampling resolution with given optical resolution
• Z-series
• Time lapse
• Multi area scanning
• Multi area time lapse
• FRAP/Photo activation

		Copyright 2001-2014  Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
Go Back	Booking Resources	Questions/comments/problems: Michael Chua