Marsico Hall Microscopes Facility (MHMF.ORG)

Notices:

• Training: All users of this system are required to have received training/orientation from NSC research director or use the system with Michael
• Location is in room 8104 Neuroscience Research Building
• FV1000 booking Calendar is a google calendar.  Please ask Michelle in order to be invited to it

Quick Start:

• If going to be used, incubator box needs to be preheated and equilibrated well ahead of time.  Complete warm up takes more than 90 minutes.  Important: Keep humidity off during preheat time.  (Note: If not adequately warmed up for 90 minutes image will fade away due to condensation on the scan lens)
• Leave humidify and CO2 off until ~10 minutes and ~3 minutes before use respectively
• Check Nitrogen tank (through doorway and on right) pressure is ~50 psi & main gage not at zero
• Turn on HBO Mercury arc lamp - 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 Argon 488 nm (blue) or HeNe 543 nm (green) lasers if needed.
• Turn on other control boxes
• If 488 nm blue laser (FITC) is going to be needed turn power rocker switch on, wait 3 seconds then turn on key.
• Turn power key switch on if using 543 nm (green) laser. 
• Turn on box
• Turn on microscope stand box
• Turn on box - rocker power switch & key to on
• Turn on Prior x-y stage controller.  On floor right of table. Usually left on, but check that it is actually on.
• Log on to computer (called \\Sutton) - onyen username and password
• Start FV10 software - icon is on the desktop
• Choose a user profile

• 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 (or use virtual channel check box with Phase Control Window)
• Press continuous Focus x2 or Focus x4 or xy repeat with Kalman unchecked to locate sample
• Press ^h (control + h keys) for red overload and blue under load
• Set laser power using AOTF if required
• Adjust Offsets (usually 9 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
• Note PMTs tend to go into low gain overload if voltage is above ~900V.  Usually see faint diagonal pattern or no signal of noise when in overload mode. Reset by turning PMT voltage back down to very low (< ~300 V).
• 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 or 1600 by 1600)
• 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
  • Turn off master N2 valve
• Or of no user expected in the next hour or so:
  • Key off Argon laser 488 nm - Important: leave rocker switch on for ~3 minutes in order to allow for cooling, then turn of switch
  • Key off 543 nm diode laser - no cool down procedure
  • 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.med.unc.edu (file server) or other server e.g. \\ad.unc.edu\med\cf or USB drive
  • Log off windows
• Turn of control boxes in reverse order - remember to leave 488 nm Argon rocker switch on for 3 minutes after turning its key off (lasing stopped with key off, but fan continues to run) to let 488 nm Argon laser cool.  Leave Prior controller on floor on
  • Sign log book
  • Turn off master N2 valve

Running the  System:

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

		Copyright 2001-2021 Dr. M. Chua, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
Go Back	Booking Resources	Questions/comments/problems: Michael Chua