PerkinElmer Ultraview Vox Confocal
Location: Room 1.12, MRC Building
Advantage: Speed, sensitivity, flexible photo-bleaching and photo-activation
Disadvantages: Lower resolution; pinhole cross-talk
The Vox is a spinning disc confocal microscope designed for rapid acquisition of 3D and 4D data. The microscope consists of a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning disc scan head with a microlens design to optimise light transmission. The detector is a Hamamatsu C9100-13 EMCCD camera that multiplies the signal on the chip before reading out. This makes it more sensitive than a regular CCD but a large pixel size means that resolution is relatively low. The Prior NanoscanZ piezo focus is generally used for acquiring stacks because it is fast and does not limit the rate of acquisition. It has a maximum range of 250 µm so any specimens thicker than this will have to be imaged using the microscope internal focus motor. The TiE stand includes the Nikon Perfect Focus System (PFS), which can maintain samples in precise focus indefinitely. This is most suitable for samples mounted at a glass/aqueous interface but there are some exceptions. Please check specimen and objective lens compatibility before using the PFS. The Vox also has a PerkinElmer Photokinesis Unit (PKU) for spot or ROI illumination in techniques such as FRAP and photo-activation/photo-switching.
Suitable specimens: All culture vessels must have glass bottoms with the same thickness as a standard #1.5 cover-slip (170 µm). There are inserts for slides, Ibidi µSlides, Lab-Tek chambered cover slips (note - use Lab-Tek I not Lab-Tek II), 35 mm dishes, multi-well plates with square not chamfered corners. If you have any other requirements then you must speak to a member of the light microscopy facility before you start preparing your specimen. Note that CO2 supply is not available for all configurations so check what vessel you need before you start setting up your specimen.
In general spinning disc confocal systems perform optimally with adherent cells in culture and other relatively thin specimens. Thicker specimens with a large amount of out-of-focus fluorescence can be difficult to image because of a phenomenon called pinhole crosstalk.
- Objective lenses
- Microscope filter sets
- Lasers
- Emission filters
Using the Vox
- Start up and shut down procedure
- Using the Nikon TiE microscope
- Coming Soon - Familiarising yourself with the Vox user interface
- Further Information
Objective Lenses
Magnification | Immersion Medium | Designation | Numerical Aperture | Coverslip Correction | Working Distance(mm) |
---|---|---|---|---|---|
10X | Air | Plan Apo | 0.45 | 0.17 | 4 |
20X | Air | CFI Plan Apochromat VC | 0.75 | 0.17 | 1 |
40X | Air | CFI Plan Fluor | 0.75 | 0.17 | 0.72 |
60X | Oil | CFI Plan Apochromat | 1.4 | 0.17 | 0.13 |
100X | Oil | CFI Plan Apochromat VC | 1.4 | 0.17 | 0.13 |
60X WI | Water | CFI Plan Apochromat | 1.2 | 0.17 | 0.27 |
There is also a 100x 1.3 NA Plan Fluor oil objective that can be used if the PFS range turns out to be too narrow when using the 100x Plan Apo.
There is a choice of three immersion oils:
Oil | Refractive Index | For use with... | Kept in... |
---|---|---|---|
Cargille Type HF | 1.518 (at 23ºC) | Oil lenses at room temperature | Tray outside environmental chamber |
Cargille Type 37 | 1.518 (at 37°C) | Oil lenses at 37ºC | Tray inside environmental chamber |
Zeiss Immersol W | 1.334 (at 23ºC) | Water immersion lens only | Tray inside environmental chamber |
Note that Cargille Type 37 is optimal for use at 37ºC but introduces spherical aberrations when imaging beyond about 10 μm in aqueous media because it is not refractive indexed matched to water. Zeiss Immersol W in combination with the 60x WI lens might be a better choice in this case but its RI is corrected for use at room temperature and won't be correct at 37 degrees.
Lasers and emission filters
The Vox has diode or DPSS lasers that emit the following wavelengths: 405 nm, 488 nm, 561 nm and 640 nm.
Emission Wheel Filters
Turret Position | Emission Filter (nm) | Example Fluorophores |
---|---|---|
1 | Empty | N/A |
2 | 527(55) | GFP |
3 | 455(80) 615(70) | DAPI, Texas Red |
4 | 485(60) 705(90) | CFP, Cy5 |
5 | 587(125) | YFP |
6 | DIC | Brightfield DIC |
7 | 525(50) 640(120) | GFP, RFP |
8 | 477(45) 575(100) 705(90) | Hoechst, YFP, Cy5 |
9 | Empty | N/A |
10 | Empty | N/A |
Filter cubes in the microscope stand
The filter cubes below are for use when viewing your specimen down the microscope eyepieces. The filter turret must be in position 1 (EMPTY) when imaging with the spinning disc.
Position Number | Filter Set | Excitation Filter | Dichroic Mirror | Emission Filter | Example Fluorophores |
---|---|---|---|---|---|
1 | EMPTY | N/A | N/A | N/A | None. For use with bright field or when imaging with the spinning disc |
2 | DAPI | D350/50x | 400DCLP | D460/50m | DAPI/Hoechst/AMCA |
3 | FITC | HQ470/40x | Q495LP | HQ525/50m | Endow GFP |
4 | TRITC | HQ575/50x | Q610LP | HQ640/50m | HcRed1, mCherry |
5 | ANALY | N/A | N/A | N/A | DIC analyser (for viewing DIC in eyepieces) |
6 | EMPTY | N/A | N/A | N/A | None. Do not use. |