Prepare Bead Slide
A bead slide is an essential part of everyday imaging routines as they are used for Channel Mapping Calibration during multiplex 2D imaging and 3D Mapping Calibration for 3D super-resolution imaging. ONI uses 100 nm beads routinely for these calibrations, and normally each installation comes with 2 bead slides.
A flow chamber-based bead slide, as detailed below, can be used for several months in Channel Mapping Calibration, if stored properly in freeze after every use. A fresh bead slide is highly recommended in 3D Mapping Calibration.
Materials
- Gloves.
- Standard glass slides.
- High precision 22 x 22 mm #1.5 coverslips (preferably #1.5H).
- Double-sided sticky Scotch tape.
- Razor blade.
- Clean paper.
- 1X PBS.
- 100 nm Tetraspeck beads.
- Epoxy glue to seal the open sides of the flow chamber.
- Pipette and tips.
Instructions
- Take a glass slide and put it on clean paper or clean tissue paper on a flat surface.
- Stick two strips of double-stick tape running parallel to each other with a 5~10 mm gap between them like train tracks, where the two tapes act like two pillars for the flow chamber:
- Stick a 22 x 22 mm coverslip on top of this strip. To make it stick to the tapes properly, use the rough side of your blade and gently press on the coverslip. Make sure you only press the areas of the coverslip which are in contact with the tapes. If this step is not done properly then you might end up with a leaky slide.
- Using the razor blade to cut out the unused part of the tapes and you now have a clean flow chamber:
- Make a dilution of 1:300 dilution of beads using 1X PBS.
- Flow 30-50 μL of the diluted solution from one end of the flow chamber. Remove any extra liquid which is outside the flow chamber using a very small piece of tissue paper.
- Mix both parts of epoxy with a 10 uL tip properly and put a small amount of mixed epoxy to both open ends of the flow chamber using the same tip.
- Dry the epoxy in dark for about 10 minutes.
Discussions
- If you notice the beads are clumped, try to sonicate beads for 30~60 seconds before dilution.
- If Channel Mapping Calibration or 3D Mapping Calibration fails all the time, try to prepare a new bead slide.
- If you cannot see beads in part of the FOV, check for oil or bubbles in the oil. If that does not fix the issue, contact the CX team.
If at any point you are having issues with the preparation process, please do not hesitate to contact the CX team.
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