Marsico Hall Microscopes Facility (MHMF.ORG)

Notices:

• 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 on the movement of the motorized x-y stage.
• FV1000 booking Calendar is here:  
• Use the correct Immersion Oil - using the wrong one will give bad images and require careful cleaning:
  • 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
  • If the wrong oil is applied to an objective please see Michael or Neal about cleaning it (your images will look like bad)
• Keep 10x, 20x, 40x objectives dry and free of oil.  If oil pools on the concave lens front images will look quite cloudy or fuzzy or not crisp
• Known problems: 
  • Dichroic turret may not position correctly if FV-10 software is not shut down before the hardware boxes  are shut down.  The symptom 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.  It takes 90 minutes for the system to equilibrate!
  • When enabling humidity heat must be turn on >1 hour (preferably 90 mins) 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.
  • When reloading acquisition from the FV10 Explorer the spectral settings does not reload.  

The System

• Laser Spot Scanning Confocal Microscope
• Inverted microscope stand (IX81) with widefield DIC (Nomarski) and epi-fluorescence
  • Widefield (eye ball viewing) fluorescence filters:
    • U-MWBV2 DAPI lp (& CFP) = ex 400 - 440, dichroic 455nm, em 475 long pass
    • U-MWIG3 - TxRed/Rhod = ex 530-550 nm, dichrio 570nm, em 575 long pass
    • U-MNIBA3 - FITC bp  = ex 470-495, di 505, em 510-550
• Visible excitation lasers switched/modulated using an AOTF
  • UV (violet) 405nm - for e.g. DAPI, Hoechst, Alexa 350, photoactivation
  • "Carolina blue" 488 nm - for FITC, Alexa 488, eGFP, CY2, Fluo-4, Calcein, FM 1-43
  • Green 514 nm - for e.g. YFP
  • Green 543 nm - for e.g. TexasRed, CY3, Rhodamine, Alexa 568, FM 4-64
  • Red 638 nm - for e.g. Alexa 633, Alexa 647, CY5, DRAQ5, ToPro-3
• Simultaneous transmitted light or DIC imaging while scanning confocally
• Three standard Photo Multipliers Tube (PMT) light detectors of which 2 have spectral discrimination
• Motorized x-y stage - Can do tiling and also tiling with time lapse
• Environmental chamber for controlled temperature, gas and humidity
• True 12 bit acquisition - 4096 pixel grey levels

• Objectives:

  Objectives
  Mag.	NA	type	WD	corrections	cover slip	Immersion
  20x	0.75	UPlanSApo	550 um		#1.5	air
  30x	1.05	UPlanSApo	800 um	corr	0.13-0.19	silicon oil 1
  60x	1.00	LUMPlanFl N	2 mm		none	water	Not stored on turret
  4x	0.13	UPLFNL			#1.5	air	MLI
  10x	0.40	UPlanSApo	3.1 mm		#1.5	air	Neuroscience
  20x	0.45	LUCPLFL	6.6 - 7.8	corr	0-2 mm	air	Neuroscience
  40x	0.60	LUCPLFLN	2.7-4 mm	corr	0-2 mm	air	Neuroscience
  60x	1.42	PlanApo	150 um		#1.5	oil 2	Neuroscience
  60x	1.40	PLAPON	120 um		#1.5	oil 2	Neuroscience

  ¹ Special silicone oil is in a green bottle (RI=1.43)
  ² Standard immersion (carbon) oil is in a blue bottle (RI=1.51)
     It is important not to mix oils. (If the wrong oil is used the objective must be carefully cleaned to remove all traces of the wrong oil.  You must talk to Michael or Neal for cleaning instructions)

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)

Viewing Software:

• For Windows ($free) - FV10-ASW_Viewer4_0.zip extract to a temp directory (e.g. use winzip), run "autorun.exe" and select install  FV10-ASW Viewer (free viewer for Windows). Old versions - FV10-ASW_Viewer3_1.zip  |  FluoView_FV1000 2.0b viewer (EU web site)
• Volocity - ask Michael or Neal to have your MHmicroscopy login account enabled for Volocity license server access

Using the system:

• Quick startup/shut down guide

		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