Slide 1Tomaso Zambelli
www.lbb.ethz.ch
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development of a microinjector with force feedback to be operated in liquid environment
objective
→ only optical control → hit-and-miss
challenge
* E. Neher and B. Sakmann, 1991 Nobel Laureates in Medicine
Neher et al, Nature 260 (1976) 799 Hamill et al, Pflügers Arch 391 (1981) 85
screening cascade (GABA receptors)
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FluidFM probes Dr. Edin Sarajlic SmartTip BV (NL) - Cytosurge AG (CH)
Deladi et al, Appl Phys Lett 85 (2004) 5361 Berenschot et al, Small 8 (2012) 3823
down to 2 µm
tipless apex syringe (FIB)
SEM/FIB: PhD D. Ossola
Trends in Biotech 32 (2014) 381 4
patch-clamp vs. SICM
Neher et al, Nature 260 (1976) 799 Hamill et al, Pflügers Arch 391 (1981) 85
Hansma et al, Science 243 (1989) 641 Korchev et al, Biophys J 73 (1997) 653
scanning ion-conductance microscope
force and glass pipettes shear-force combination
Böcker et al, Nanotechnology (2007) 18:145505
AFM cantilever as sample
suspension system
Drake et al, Rev Sci Instrum (2014) 85:083706 on a tuning fork: Ito et al, Jpn J Appl Phys (2011) 50:08LB15
bent micropipette
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- custom shielding and connections to measure pA currents
- LabView for data acquisition
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Dr. Amarouch, Prof. Abriel (University Bern)
30 µm hERG
Fo rc
e (n
2µm
[1]
Force-controlled approach Whole-cell NaV1.5 current traces
• Challenging system: isolated, adult mouse cardiomyocytes
collaboration with Univ. Bern/DKF Prof. Mohamed-Yassine Amarouch
Prof. Hugues Abriel
force-controlled whole-cell mode
-100 mV
K+
disabled force-control to measure transversal contraction force
Nano Lett 15 (2015) 1743
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cardiomyocytes with force control
multiple injection, non-lethality
1s t ap
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o Enhanced mechanical stability • Non-destructive procedure
o Allows injection into cardiomyocytes o Serial patch-clamp

7GΩ / µm in 100 mM KCl

• Performance loss of voltage follower electronics
5 µm
[1]
[1] Suchyna et al, Biophys J 2009 97:738 [2] Sherman et al, Biophys J 1999 77:2590
[2]
force-controlled patch-clamp - summary
• feedback controller maintains ionic current constant o scan → substrate topography
substrate
I
z
[1]
Hansma et al, Science 1989, 243:641 Chen et al, Annu Rev Anal Chem 2012 5:507
aperture from 2 µm down to 10-40 nmSICM
scanning ion conductance microscope
Experimental Simulation (Comsol)
- solution: serum-free DMEM
«standard» SICM with FluidFM probes
no fitting parameter!
PhDs D. Ossola, L. Dorwling-Carter
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Fast scan
- 500 nm PDMS steps - scan speed: 0.33 line/s - 150 mM KCl
500 nm
• Parallel SICM and AFM controller, merged by weighted sum function
Simplified Simulink model
Phys Rev Lett 115 (2015) 238103 17
- 500 nm PDMS steps - scan speed: 0.5 line/s - 150 mM KCl
500 nm
double controller
• SICM with the FluidFM demonstrated
• smaller impedance for small apertures (Ø=50 nm 30 M vs. ~G)
• force detection facilitates set-point optimization
• double controller limits the interaction force
• higher image acquisition speed because no “hopping mode” is needed
• system simulation oversimplified (time domain, static)
• limited z-piezo range to image entire cells
• deeper understanding of double controller needed to improve performance

!
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patch-Clamp
outlook: - automatization - improving gigaseal - mechanosensitive ion channels
local chemical stimulation of neurons on MEA for recording outlook: - map of chemical synapses and their weights
novel, merged SICM & AFM imaging technique for non-contact scanning of soft samples
outlook: - in-depth modelling - smart patch-clamp (scan&patch)
conclusions and outlook
• LBB
• FluidFM
Stephen Wheeler, Martin Lanz, Dominique Textor, Paul Lüthi Prof. János Vörös → LBB head
Michael Gabi, Pascal Behr → 1st generation → CYTOSURGE Pablo Dörig, Raphael Grüter, Dario Ossola, Harald Dermutz, Dr. José Sáenz → 2nd generation
Vincent Martinez, Dr. Valeria Lotito → 3rd generation Luca Hirt, Mathias Aebersold, Livie Dorwling-Carter → 4th generation
in collaboration with Prof. Vorholt (D-BIOL, Philipp Stiefel, Eva Potthoff, Dr. Guillaume-Gentil, Max Mittelviefhaus)
Prof. Abriel (UniBern, Dr. Amarouch), Dr. Mercer (D-BIOL, F.I. Schmidt), Dr. Polesel Maris, Dr. Viel, Dr. Berthelot, Dr. Cornut (CEA Saclay, F)
CSEM SA Neuchâtel (CH, Dr. Meister), SmartTip (NL, Dr. Sarajlic), NANOSURF (CH, Dr. Frederix)
financial support → KTI-CTI.ch, SystemsX.ch, Nano-Tera.ch, SNF.ch, ETH grant
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30 µm