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Spinning disc confocal microscope
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- Objective lenses
- Microscope filter sets
- Lasers
- Emission filters
- 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.
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. |
Setting up the Nikon Perfect Focus System (PFS) for multi-point acquisition in Z
The following is a procedure I've worked out for setting up the PFS for multi-point acquisition. I used fluorescent HeLa cells grown on Mat-Tek glass-bottomed 35 mm dishes and mounted in a Solent Scientific insert designed for a range of 35 mm dishes but adjusted to hold this one firmly in place. I cannot say for sure that the procedures will work for all surfaces. Nikon only recommend glass of standard #1.5 coverslip thickness (170 µm).
- Make sure that the PFS lever under the microscope stage on the right is in the In position - otherwise the PFS won't work
- Focus on the specimen. I focused on the cytoplasm of well spread cells so that my focus plane was relatively near the coverslip
- The small green LED on the front of the microscope stand should be lit, indicating that the coverslip is in range of the PFS
- If it is not lit then slowly lower the microscope focus until the LED comes on. Note that the LED may only be on in a very narrow range of focuses, so don't focus too fast or you might miss it
- Focus up and down until you are about in the middle of the range of focus where the LED is on
- Press the Focus button on the front of the microscope to turn on the PFS
- Use the Offset adjustment dial to the right of the microscope stand to refocus on the focus plane
- Bring up a live image of the plane of focus in the Vox image preview
- Use the Offset dial to readjust the focus on the screen, then turn off the Focus button
- Start marking points, making sure at each point the small green LED on the front of the microscope is on
- If the LED is not on or flickers at any of the points, adjust the microscope focus until the LED is solidly on, then mark the point
- After all points are marked, go back to the first point and turn on the PFS to make sure the focus has not drifted. Make any fine adjustments to the offset if necessary
- Turn off the PFS and set the Ultraview Focus Drive to Zero
- Set the Top and Bottom of the Z series. Note that because zero is at a plane of focus near to the coverslip, the top of the Z series may be further away from zero than the bottom (e.g. Bottom = -5.0 µm, Top = +25 µm)
- Review the points to make sure the LED is still on at all of them
- Check the Bottom and Top of the Z series settings at a few points to make sure that the focus range is adequate for all of them. Make adjustments if necessary
- Go back to the first point
- Set up the time-lapse settings, setting the autofocus method to 'Nikon Ti PFS'
- You can now either turn the PFS on before starting the time-lapse or leave it off. If you choose the former this should keep the PFS on between time-points, but this might not be necessary if your time-points are close together.
Note that Perkin Elmer don't recommend having the PFS on between time-points, but if there is a lot of focus drift between time-points it may be the only way to stop the focus drifting out of the range of the PFS. Here are some links:
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