ERS-VoX spinning disk confocal microscope

Profile: Confocal microscope of the spinning disk type. Much higher sensitivity and faster image acquisition compared to point scanning confocals but confocality is limited to high magnification. 4 laser lines available: 405, 488, 561 and 640nm. Suitable for live imaging.

The PerkinElmer ERS-VoX is a confocal microscope of the "spinning disk" (aka "nipkow disk") type. Here about 1,000 laser beamlets continuously sweep over the field of view, excite the fluorochromes in the sample, whose emission is recorded by a ultra sensitive EMCCD camera. Because each beamlet is quite dim, phototoxicity is relatively low and at the same time the quantum efficiency of a EMCCD camera is considerably higher than that of a photomultiplier used by a "normal" point scanning confocal microscope meaning that more photons can be detected.This allows for higher framerates (up to 30 images per second) as opposed to the typical one image per second achieved by point scanning devices. A limitation comes from the fact that the pinholes required to gain confocality cannot be adjusted in size so that confocality is only obtained with a 100x oil immersion objective, limiting the field of view to about 60 x 60 µm. Also the penetration depth is lower as compared to a point scanning confocal (around 20 µm versus typically 100 µm). This means that the ERS-VoX is especially well suited for imaging of single cells, fixed or live, even for a longer period of time. Lower magnifying objectives are also available (40x oil immersion, 60x water immersion, 20x dry). With these confocality is a little bit reduced but blur resulting from extrafocal signal is still low and not comparable to a widefield (= non confocal) microscope.

Components

Nikon automated Ti inverted microscope with

  • Nikon Plan Apo VC 100x NA 1.4 oil immersion objective
  • Nikon S Fluor 40x NA 1.3 oil immersion objective
  • Nikon Plan Apo VC 60x NA 1.2 water immersion objective

Temperature-, CO2- and humidity control is provided by a TokaiHit on stage incubation system that can hold LabTek chamber slides and 35 mm dishes.

Yokagawa CSU-X1 confocal scanning unit

Hamamatsu C9100-02 EMCCD camera, 1000 x 1000 pixel, 8 µm pixel size

Lasers of 405, 488, 561 and 640nm wavelength for fluorochrome excitation.

Detection

The filter sets in the ERS-VoX allow to detect fluorochrome emission in the following ranges (center wavelength [nm]/bandwidth [nm]):

  • "Green": 527/55
  • "Blue/Red": dual pass filter 445/60 and 615/70
  • "Cyan/Far Red": dual pass filter 485/60 and 705/90
  • "Yellow"587/125

Image acquisition with PerkinElmer Volocity software (version 6.2 as of December 2012). Analysis versions of Volocity may be used free of charge in our computer room, U04.

The system is now also equipped with a CCD widefield camera and automated filter wheels. Details to come...

 

Bearbeiter: Christian Ackermann

Latest Revision: 2017-05-17

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