Michael Hooker Microscopy Facility (MHMF.ORG)

Notices:

• Training: All users of this system are required to:
  • Receive training, or an orientation, on use of the system from Neal or Michael
  • Only use your login on account
  • Multiphoton users: Complete the UNC online laser safety training and hopefully the test.
  • Follow guidelines for running humidity and temperature control in order to avoid scanhead lens condensation.
• Open house and system introduction with Robert Peterson, the Olympus confocal specialist - May 2:
  • Introduction to the system: 9:15 AM in room 3200 Thurston Bowles - coffee and snacks provided.
  • Demonstration session with your or our samples: 11:00 to 12:00 & 1:30 to 5:00 PM - please contact Neal or Michel to make an appointment.
• Location is in room 6033 Thurston Bowles.  Enter via room 6129
• Quick startup/shut down guide
• Time lapse/ Multi-area time lapse (MATL) - setting up carefully - Don't leave things inside the box that can impinge the movement of the motorized x-y stage movement.
• FV1000 booking Calendar is here:   See Neal or Michael for assistance
• Use the correct Immersion Oil - using the wrong one will give bad images:
  • 60x Standard immersion oil is in a blue bottle (RI=1.51) - carbon oil only
  • 30x Special silicon oil is in a green bottle (RI=1.43) - silicon oil only
• Please contact Michael or Neal about getting started on this system or for a demonstration
• Known problems: 
  • Loss of focus control, loss of course/fine switchover, loss of escape function - if possible please let Michael or Neal know immediately so that we can trouble shoot.  Focus control can be restored by restarting the software.
  • Dichroic turret may not position correctly if the hardware boxes (#1 to #8) are shut down before the FV-10 software is shut down.  Symptom there is bright light at the stage and the light does not correctly correspond to the selected widefield dichroic and the fluorescence shutter cannot be closed. To fix it seems that starting the hardware then starting the software will self correct the problem.  Also keep you sample off the stage so that it is not blasted by the intense light.
  • If heat is not left on long enough after the humidity is shut off then often moisture will condense on the FV1000 scan head projection lens.  Next user will see uneven illumination or no clear image of the scanned image or even no scanned image even though laser light can be seem scanning at the stage.
  • When enabling humidity heat must be turn on ~1 hour before humidifier is turned on, then humidity must be run at 40% for ~30 minutes (? Is this long enough?) then go to 60% humidity.
  • Ensure temperature probe inside the back top left of the box is fully inside the box.  If not temperature will read low (even though the temperature at the stage may be >42C!) and it will not be possible to control the humidity.

The System

• Laser Spot Scanning Confocal Microscope
• Inverted microscope stand with widefield DIC (Nomarski) and epi-fluorescence
• Visible excitation lasers switched/modulated using an AOTF
  • UV (violet) 405nm - for e.g. DAPI, Hoechst, Alexa 350, photoactivation
  • "Duke blue" 440 nm - for e.g. CFP
  • "Carolina blue" 488 nm - for FITC, Alexa 488, eGFP, CY2, Fluo-4, Calcein, FM 1-43
  • Green 514 nm - for e.g. YFP
  • Green 559 nm - for e.g. TexasRed, CY3, Rhodamine, Alexa 568, Alexa 594, FM 4-64
  • Red 635nm - for e.g. Alexa 633, Alexa 647, CY5, DRAQ5, ToPro-3
• Multiphoton excitation laser modulated with an AOM
  • Ti:Sapphire 690 to 1040 nm - many Alexa dyes, FITC, Rhodamine,
  • Pre-chirp compensation
  • Two high sensitivity non-descanned PMT detectors (NDD)
    • Dichroics for NDD
      • Green/Red
      • Blue/Green
      • Violet/Green
  • Dichroic splitters for NDD - green/red, blue/green, violet green
  • Examples of suitable dyes and 2 photon cross section absorption spectra: DRBIO  |  Arizona U |  Molecular Probes  |  More dyes  |
    • DAPI
    • Alexa 488, FITC
    • Rhodamine, CY3
    • Alexa 568
    • Alexa 594
    • Alexa 633, CY5
    • eGFP, eCFP, eYFP
    • dsRed, mCherry, mCitrine, mRFP, TagRFP e.g. see: http://pubs.acs.org/doi/pdf/10.1021/jp8087379
    • FM 1-43, FM 4-64, DiI
    • Fura-2
    • Q-Dots, Ag nano-particles
• Simultaneous transmitted light or DIC imaging while scanning confocally (single or two photon)
• Three standard Photo Multipliers Tube (PMT) light detectors with 2 spectral discriminations
• Motorized x-y stage - Can do tiling and also tiling with time lapse
• Environmental chamber for controlled temperature, gas and humidity
• FRAP/FLIP/FLIM/FRET/PhotoActivation/etc - SIM scanner
• True 12 bit acquisition - 4096 pixel grey levels

• Objectives:

  Objectives
  Mag.	NA	type	WD	corrections	cover slip	Immersion
  10x	0.40	UPlanSApo	3.1 mm		#1.5	air
  20x	0.75	UPlanSApo	550 um		#1.5	air
  30x	1.05	UPlanSApo	800 um	corr	0.13-0.19	silicon oil 1
  60x	1.42	PlanApo	150 um		#1.5	oil 2
  60x	1.00	LUMPlanFl N	2 mm		none	water	On loan from Bowles Center

  ¹ Special silicon oil is in a green bottle (RI=1.43)
  ² Standard immersion oil is in a blue bottle (RI=1.51)

Detection:

• Filtering through glass interference filters
• Spectral separation through diffraction gratings
• Two tunable detection bands (1 - 100 nm band width) with adjustable spectral windows before the three Photo Multiplier Tubes (PMT)
• Standard XY scanning with zoom and rotation
• Transmitted light and DIC (non confocal)
• FRAP, FRET, photoactivation, Emission Spectra, ratio imaging
• Two Channel non descanned detectors (NDD) - filters: blue/green, green/red, CFP/YFP, 2nd harmonic generation

Viewing Software:

• FV10-ASW 2.0c Viewer (free viewer for Windows)     FluoView_FV1000 2.0b viewer (EU web site)
• Volocity

Using the system:

• Quick startup/shut down guide

Enquiries / Reporting Problems

• Multiphoton users of this system are required to have completed the UNC online laser safety training and test
• Training and assistance: please contact facility personnel
• Olympus Confocal Service and Support 8:00 A.M. to 5:00 P.M.

		Copyright 2001-2012  Dr. M. Chua, Office of Research, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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