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Example Protocol: Colloidal FluidFM

This protocol allows to reproduce the measurements published in Dörig et al., Biophysical Journal 105, 2013, where colloids are either picked up from the surface or grabbed from solution with a FluidFM micro pipette to measure adhesion to a substrate in liquid. 
 
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 micro pipette. 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 fabrication 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 regular FluidFM probes the channel height h is 1 um, while it is 500 nm for the soft probes. Hence the following minimal colloid diameter result: 
Opening Diameter do 2 N/m Micropipette - dmin 0.3 N/m Micropipette -  dmin
2 um 2 um 2.5 um
4 um 5 um 8.5 um
8 um 17 um 32.5 um
Colloid preparation

This protocol was tested with polystyrene (latex) colloids from MICROMOD.com. It worked independent of the colloid coating and the used sizes were between 3 and 50 μm. 
  • Fill a WillcoWells dish with 4 ml of buffer solution
  • Add 1 μl of the microbead solution with a pipette and stir the petridish with the pipette tip
  • Check the bead density under microscope
  • Add more beads if desired. The larger the beads, the more solution you need.

Grabbing a bead from solution

The filled probe is immersed in the Petri dish containing the beads

  • apply 300 mbar underpressure
  • Wait until a bead attaches to the opening (< 1 minute)
  • Check opening smoothness: Do other beads still get attracted?

o   If no, continue with transfer.

o   If yes, probably you have to discard the FluidFM micro pipette because of a badly shaped opening.

  • Keep underpressure applied from now on until the bead is exchanged

Release of the bead

  • The bead can be released with a 1 s pulse of 1 bar
  • 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.

Transfer bead to another Petri dish

  • The transfer should not take more than a few seconds, such that the probe surface does not dry out.
  • If the bead is not attached after the transfer, it can have several reasons

o   The underpressure could be too weak

o   The tubing could have a leak

o   The probe opening could be “ugly”/ not circular

o   The probe contains an air bubble at the end which prevents a tight sealing

o   The colloid dimensions could be too large or too small for the chosen FluidFM micro pipette opening diameter

Pick bead from surface

  •  Approach gently to the surface next to the bead
  • Retract the probe such that it is a few micron above the bead level
  • Align the probe opening and the bead
  • Approach the bead gently
  • Apply -800 mbar suction as soon as the probe is in contact with the bead
  • Hold the position for 5 seconds
  • Retract the probe
  • Optional: Measure the adhesion force of the bead while retracting the probe

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