This protocol illustrates the measurements published in Dörig et al (2013), where colloids were either picked up from the surface or grabbed from a solution with a FluidFM Micropipette, with the purpose of measure adhesion to a substrate in liquid environments.
Appropriate probe opening
The currently available FluidFM pipette opening diameters (do) are between 300 nm and 8 µm. The colloid diameter d has to be larger than the opening and at least so large, that it will not touch the upper wall of the FluidFM Micropipette. The distance between the probe opening and the upper wall is equal to the channel height h.
The FluidFM wall thickness is disregarded here, because every FluidFM pipette has a depression above the opening equaling exactly the wall thickness, due to manufacturing reasons.
When the channel height h is larger than the opening radius do/2, the equation can be ignored, and the only condition is that the colloid diameter d has to be larger than do.
For 2 N/m FluidFM probes the channel height h is 1 um, while it is 500 nm for the softer probes. Hence the following minimal colloid diameter result:
|Micropipette opening diameter do||2 N/m Micropipette - minimal colloid size||0.3 N/m Micropipette - minimal colloid size|
|2 µm||2 µm||2.5 µm|
|4 µm||5 µm||8.5 µm|
|8 µm||17 µm||32.5 µm|
This protocol was tested with polystyrene colloids (latex) from Micromod. It did not depend on the colloid coating and it used colloid sizes between 3 and 50 µm.
1. Fill a WillcoWells dish with 4 ml of buffer solution.
2. Add 1 µl of the microbead solution with a pipette and stir the Petri dish with the pipette tip.
3. Check the bead density under the microscope.
4. Add more beads if desired. The larger the beads, the more solution you need.
Picking a bead from solution
The filled probe is immersed in the Petri dish containing the beads.
1. Apply 300 mbar under pressure.
2. Wait until a bead attaches to the opening (< 1 min).
3. Check the opening smoothness: do other beads still get attracted?
- If no, continue with transfer.
- If yes, probably you have to discard the FluidFM Micropipette because of a badly shaped opening.
4. Keep under pressure conditions applied from now on, until the bead is exchanged.
Releasing the bead
1. The bead can be released with a 1 s pulse of 1 bar.
2. If the bead remains stuck to the cantilever, lift the AFM/probe out of the liquid briefly. The surface tensions should remove any remaining beads.
Transferring the bead to another dish
1. The transfer should not take more than a few seconds, such that the probe surface does not dry out.
2. If the bead is not attached after the transfer, it can have several reasons:
- The under pressure could be too weak.
- The tubing could have a leak.
- The probe opening could not be circular or have an anormal shape.
- The probe contains an air bubble at the end which prevents a tight sealing.
- The colloid dimensions could be too large or too small for the chosen FluidFM micropipette opening diameter.
Picking a bead from surface
1. Approach gently to the surface next to the bead.
2. Retract the probe such that it is a few micron above the bead level.
3. Align the probe opening and the bead.
4. Approach the bead gently.
5. Apply -800 mbar suction as soon as the probe is in contact with the bead.
6. Hold the position for 5 s.
7. Retract the probe.
8. (optional) Measure the adhesion force of the bead while retracting the probe.
References and supporting information
Dörig, P., Ossola, D., Truong, A.M., Graf, M., Stauffer, F., Vörös, J. & Zambelli, T. Exchangeable Colloidal AFM Probes for the Quantification of Irreversible and Long-Term Interactions (2013). Biophysical Journal, 105 (2), 463-472. doi:10.1016/j.bpj.2013.06.002
More information about how to use colloidal probes with FluidFM: