December 27, 2012

WaveFX #1

Purchased from Quorum Technologies this spinning disk confocal has the following configuration:

  • Olympus IX-81 motorised microscope base
  • Yokagawa CSU 10 spinning disk confocal scan-head
    • Sedat Dichroic with reflections at 405/491/561/640 for standard 4-colour imaging
    • CFP dichroic with reflections at 440/491/561/640 for 4-colour imaging using CFP.
  • Lenses: 10X/0.3 Ph1, 20X/0.75 Dry, 40X/0.9 Dry, 60X/1.42 Oil
  • Illumination:
    • X-Cite 120 from Lumen Dynamics for visualization with eyepieces
    • LMM5 from Spectral Applied Research for laser merging.
      • 44mW 405nm pumped diode laser (for blue dyes, e.g. DAPI)
      • 40mW 440nm pumped diode laser (for CFP)
      • 50mW 491nm pumped diode laser (for green dyes, e.g. GFP, YFP, Alexa 488, FITC)
      • 50mW 561nm pumped diode laser (for red dyes, e.g. mCherry, Alexa 546, Cy3, TRITC)
      • 45mW 642nm pumped diode laser (for far-red dyes, e.g. Cy5, Alexa 633, Alexa 647)
  • Filter Cubes for use with eyepieces (BRIGHTLINE filter sets from Semrock):
    • DAPI (Ex = 377/50, DM = 409, Em = 447/60)
    • GFP (Ex = 473/31, DM = 495, Em = 520/35)
    • Cy3 (Ex = 531/40, DM = 562, Em = 593/40)
    • Analyser for DIC visualisation
  • Emission Filters for use during Confocal Imaging: The system is fitted with two interchangeable filter wheels, one for imaging standard 4 colour (Blue, Green, Red, Far-Red) “fixed” samples, and one optimised for imaging fluorophores commonly found in “live” cell experiments. Note that with these emission filters, there are no excitation filters necessary, as the lasers provide single wavelength excitation. Additionally, there is a choice of three dichroic mirrors housed in the confocal scan unit. Most commonly used, the Sedat dichroic is compatible with 4 colour fixed imaging. The second choice is a dichroic that maximises the emission from GFP (EGFP), but at the expense of separating its emission from other fluorophores. Last is a dichroic that is designed for separating CFP from YFP.
    • “Fixed” filter wheel (for use with “fixed” samples)
      • Polariser (for DIC imaging)
      • DAPI (460/50)
      • GFP (515/30)
      • Cy3 (595/50)
      • Texas Red (620/60)
      • Cy5 (690/50)
    • “Live” filter wheel (for use with “live” samples)
      • 436/24
      • CFP (470/24)
      • GFP (520/40)
      • YFP (540/30)
      • RFP (595/50)
      • Cy5 (700/75)
  • Imaging using a Hamamatsu EMCCD (C9100-13).
  • Acquisition using Perkin Elmer’s Volocity
  • Chamlide TC-A Live Cell Chamber (37°C incubator + 5% CO2 atmosphere). Can accommodate either 18mm or 25mm diameter coverslips.
  • ASI MS-2000 motorised XY stage with a piezo Z insert that has 100µm travel.

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