Michael Hooker Microscopy Facility (MHMF.ORG)

Perkin Elmer Yokogawa Nipkow disk confocal

Introduction:

This confocal system has a Yokogawa multiple lens Nipkow spinning disk scanner (CSU10), allowing it to capture confocal images rapidly with a minimum of excitation light.  Images are captured using a Hamamatsu OrcaER camera driven by the SimpelPCI software from Hamamatsu.  Excitation light is provided by a ~100 mW Krypton/Argon laser, which can excite at 488, 568 & 647 nm.  Imaging of FITC-like, Rhodamine-like, Cy5-like and similar fluorophores, including GFP, Calcium Green & Texas Red can be carried out.  A filter wheel changes emission filters.  A Prior motorized drive is attached to the fine focus knob for software control of z- position.  Transmitted light shutter permits widefield, DIC or phase contrast images to be interlaced with fluorescent image capture.  Acquisition modes can be combinations of z-series, time lapse, &/or multiple fluorophores.

The confocal head is mounted on a Nikon TE2000U inverted microscope.  Analysis software includes the Volocity 3D rendering & quantitation package & C-Imaging (including deconvolution on the \\Nomarski image processing workstation).  Images can be transferred directly over the network.

Notes:

• The acquisition software on this system runs SimplePCI v 6.6.0.7 (Hamamatsu Photonics)
• The focus drive systems allows DIC imaging to be done correctly, and also allows convenient focus control on any objective (0.2 um minimum step size).  Formerly this system utilized a piezo drive collar, which, inconveniently, had to unscrewed and reattached when the objective was changed.  Also the drive moved the Wollaston prism away from the objective back aperture, which severely interfered with the operation of DIC imaging.
• This work station should be considered as an acquisition only platform, since little software is available on it to perform 3D viewing and rendering.  Other software is available on our off-line platforms for image manipulation and volume rendering. See Volocity software and the computer called \\Snell 
• Due to the risk of damage the oil immersion and water dipping lenses should not be left on the microscope turret. 

Operating the System

• Confocal mode
• Widefield mode

Equipment:

• Laser - Argon/Krypton with lines at blue 488nm, green 568 and red 647nm.
• Xenon XBO 75W lamp.  Alternate metal halide lamp may be available
• Microscope
  • Nikon TE2000-U
• Condenser: LWD 0.52NA
• Objectives (and others are available)

Objectives  (These are the objectives shipping with the confocal.  Other objectives will be available)
Mag.	NA	type	WD	corrections	cover slip	Immersion	part no.	dic/phase	condensor Wollaston
20x	0.75	S-Fluor	1.0 mm		0.17 (#1.5)	air	93127
40x	1.3	S-Fluor	0.22 mm		0.17 (#1.5)	oil	93130
100x	0.7-1.3mm	S-Fluor	0.2 mm	NA	0.17 (#1.5)	oil

• Camera:
  • Hamamatsu Orca-ER  (Spectral Response Curve)
                            
• Scanhead
  • Yokogawa CSU-10 spinning disc confocal (Perkin Elmer, LAS)
•  Wavelength selection for imaging
  • Sutter Lamda 10-2 Filterwheel controller with two filterwheels (excitation and emission)
  • High performance Semrock filters are installed
• Focus control
  • Prior z-stepper motor

Filters

• Widefield (epifluorescence) filters.
          These are housed in a rotating carousel enclosed in a black drum beneath the stage. Selection is made by rotating the notched wheel to the appropriate index on the right hand side of the microscope. Just behind this index is a lever labeled "C___O".  This shutter can be closed to prevent photobleaching of your sample

Position #	Label	Fluorophore	Filterset	Specifics
1	----	Trans	-	-
2	UV	DAPI	Nikon UV-1A
3	B	FITC, GFP	Nikon B-2E/C	Specifics
4	G	Texas Red	96322 TR C109382
5		CY5	96324M CY5 HQ
6		no installed YFP	96345 HQ: Y GFP C74135	ex=HQ500/20 60341,em=HQ535/30 60361

Position #	Label	Fluorophore	Filterset	Specifics
1	B	FITC, GFP	Nikon B-2E/C	Specifics
2	G	Rhodamine	Nikon G-2E/C	Specifics
3
4
5
6

• Laser/Imaging Filters
   

  Position    Ex                    Em

  1              488/10            525/50

  2              568/10            600/45

  3              PARK            PARK

  4              647/10            700/75

  Triple Dichromatic mirror specifications

Related Links:

• Improvision Volocity 3D, multi-channel & time-lapse imaging
• Compix C-Imaging

		Copyright 2001-09  Dr. M. Chua, Office of Research, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
Go Back	Booking Resources	Questions/Comments: Michael Chua

Last Updated: 2009-09-08

---