ERS-VoX spinning disk confocal microscope + widefield FURA 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. Since each beamlet is quite dim, phototoxicity is relatively low. 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 frame rates (up to 30 images per second) as opposed to the typical single frame per second achieved by point scanning devices.
Real time transport processes
A limitation comes from the fact that the pinholes required to gain confocality cannot be adjusted in size. Hence confocality is only obtained with a 100x oil immersion objective, limiting the field of view to about 66 x 66 µm. Also the penetration depth is smaller 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 (like 40x oil immersion, 60x water immersion, 20x dry). With these, confocality is somewhat reduced, but the blurring due to the extrafocal signal is still low and not comparable to a widefield (= non confocal) microscope.
Components
Nikon automated Ti inverted microscope with
- Nikon Plan Apo λ 100x NA 1.45 oil immersion objective (WD 0.13mm)
- Nikon Plan Apo λ 60x NA 1.40 oil immersion objective (WD 0.13mm)
- Nikon Plan Apo VC 60x NA 1.2 water immersion objective (WD 0.27mm)
- Nikon S Fluor 40x NA 1.3 oil immersion objective (WD 0.24mm)
- Nikon Plan Fluor 20x NA 0.75 multi immersion (water, glycerol, oil) objective (WD 0.35mm)
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 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.
The system is also equipped with an Andor Clara CCD widefield camera and separate switchable excitation- and emission-filters in automated filter wheels. It is mainly meant to be used for FURA calcium imaging with the Nikon NIS-Elements software. For this the following filters are available:
Excitation Filters (center wavelength/bandwidth):
- 340/26
- 387/11
- 510/30
