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Spinning disc confocal microscope
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| DescriptionLocation: Room 1.12, MRC Building |
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A light tight environmental chamber was fitted on 6th March 2013.
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Advantages: Speed, sensitivity, flexible photo-bleaching and photo-activation Disadvantages: Lower resolution; poorer optical sectioning of thick fluorescent specimens The Vox is a spinning disc confocal microscope designed for rapid acquisition of 3D |
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microscope consists of a Nikon TiE inverted |
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stand attached to a Yokogawa CSU-X1 spinning disc scan head. The detector is a Hamamatsu C9100-13 EMCCD camera |
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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 Ultraview Focus Drive (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 |
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can maintain samples in precise focus indefinitely |
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. This is most suitable for samples mounted at a glass/aqueous interface |
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but there are some exceptions. Please check specimen and objective lens compatibility before using the PFS. The Vox |
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also has a Photokinesis Unit (PKU) for spot or ROI illumination in techniques such as FRAP and |
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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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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. - Using the Nikon TiE microscope
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