Spinning Disc Confocal Microscope

Application

The UltraVIEW VoX (Perkin Elmer) spinning disc confocal (SDC) microscope combines high rates of acquisition with minimal photobleaching and is ideally suited for live imaging of rapid cellular processes. This inverted microscope is equipped with an auto focus system, a motorized XY stage and a Piezo Z stage for 4D imaging. The UltraVIEW VoX also includes a PhotoKinesis Unit for Fluorescence Recovery After Photobleaching (FRAP) and photoswitching experiments. Image acquisition, visualization and quantification is powered by the Volocity software (Improvision).

Specifications

Microscope

Nikon Ti-E Eclipse inverted microscope equipped with Perfect Focus (auto focus system), motorized XY stage and Nano Focusing Piezo Stage (NanoScanZ, Prior Scientific).

Objectives

  1. 4x CFI Achro Flat Field, NA 0.1, air
  2. 20x CFI Plan Apo, NA 0.75, air
  3. 40x CFI Plan Apo, NA 1.0, oil
  4. 60x CFI Plan Apo VC, NA 1.4, oil
  5. 100x CFI Plan Apo VC, NA 1.4, oil
  6. 100x CFI Apo TIRF, NA 1.49, oil

Light sources

  1. Transmitted light source: 12 V, 100 W Halogen EVA
  2. Wide-field epifluorescence source: Intensilight 130 W mercury lamp. Illumination delivered through a liquid guide.
  3. Lasers:
  • Melles Griot solid state 405 nm/50 mW diode laser.
  • Melles Griot solid state 445 nm/40 mW diode laser.
  • Coherent solid state 488 nm/50 mW diode laser with DPSS module.
  • Cobolt solid state 514 nm/25 mW diode laser with DPSS module.
  • Cobolt solid state 561 nm/50 mW diode laser with DPSS module.
  • Melles Griot solid state 640nm/50 mW diode laser.

Confocal spinning disc head

CSU-X1 (Yokogawa)

Camera

1000x1000 pixels, 14 bit Hamamatsu (C9100-50) electron-multiplied, charge-coupled device (EMCCD).

Workstation

Dell 5400 with Raid Motherboard P4 2x2, 4GHz processors, and 4 Gb RAM. Operating system: Windows XP. Acquisition software: Volocity (Improvision).

Wide-field epifluorescence filter cubes

  1. CFP Zero shift.
  2. GFP Zero shift.
  3. Texas Red Zero shift.
  4. FITC/Texas Red.

Dichroic Mirrors in the Scan Head

  1. 405/488/568/640: transmission 80% min, reflection 90% min
  2. 405/440/514/640: transmission 80% min, reflection 90% min
  3. 405/440/488: transmission 80% min, reflection 90% min

Emission Filters in the Scan Head

  1. Fast sequential (double band pass) #1: 500-548 nm and 582-700 nm
  2. Fast sequential (triple band pass) #2: 455-500 nm, 526-623 nm, and 664-750 nm
  3. Green (single band pass): 527 nm band pass center wavelength (CWL), 55 nm half-power bandwidth (FWHM).
  4. Blue/Red (dual band pass): 445 nm CWL/60 nm FWHM, 615 nm CWL/70 nm FWHM
  5. Cyan/Far Red (dual band pass): 485 nm CWL/60 nm FWHM, 705 nm CWL/90 nm FWHM
  6. Yellow (single band pass): 587 nm CWL/125nm FWHM
  7. DIC Polarizer