1. SQUID Sensors for Detecting Some Electrophysiologic Phenomena in Plants Zvonko Trontelj Physics Dept., University of Ljubljana and Institute for Mathemtics, Physics and Mechanics, Ljubljana

2. Outline of talk  Introduction  On SQUID sensor  Basic steps in data analysis and modeling of sources of bioelectric activity  Examples from the world of plants:  a) Chara corallina (single cell studies)  b) Vicia faba (plant injury study)  Conclusions

3. Objectives:  To apply SQUID(s) in order to obtain the noninvasive information on: a) ionic currents in electrically stimulat. Chara corallina, b) the influence of visible light on AP and on AC in C.c. and to relate the ionic kinetics to chemical nature of AP. and to relate the ionic kinetics to chemical nature of AP. c) the injury induced ionic currents in the plant organs - leaves in the higher developed plant Vicia faba. leaves in the higher developed plant Vicia faba.  To model the transmembrane pot. difference under different conditions and to support the suggest. hypoth. of light action on the cytoplasmic Ca ion. conc.

4. SQUID sensors 1.What is SQUID? 2.What they offer to us? 3.Where we can use them? 4.Why SQUID sensors in electrophysiology?

5. Ad 1 and Ad 2  Superconducting QUantum Interference Device  Magnetic flux-to-voltage convertor (the most sensitive sensor for quasi dc magnet. fields m.)  Measured m. field; via Amper’s law the source  Based on 3 facts described by QM - superconductivity with Cooper pairs - superconductivity with Cooper pairs - C. pair tunneling - C. pair tunneling - m. flux quantization - m. flux quantization

6. From Josephson jct. to closed sc. circuit

7. Dc SQUID configuration

8. Outer magnetic field is present at the SQUID

9. Ad 3 and ad Ad 4 M. flux has to be transp.to SQUID M. flux has to be transp.to SQUID SQUID has to be in m. shielded env. SQUID has to be in m. shielded env.

10. Basic steps in analysis and modelling of current sources in living matter  Distribution of ionic currents in tissue. Complicated  Direct and inverse problem  The direct problem – a unique solution   T  Z T E    The inverse problem is ill-posed problem   E  Z ET  T  Simple geometry – analyt. solutions, otherweise modeling

11. Simple geometry  Single cylindrically shaped cell (1D case)    Bound. cond.:  m (z) =  i  a,z) –  e  a,z)  n.J i (a,z) = n.J e (a,z)

12. Simple geometry (contin.)  From Ampere law:  B i = Integr. [G(  a,z – z’)J i (a,z’)]dz  Applying the Fourier and the inverse Fourier transformations one can come from potential to mag. field and v.a.v.

13. Some methods in modeling of current sources  Current multipole expansion  Current distribution with the minimum norm estimation  Covariance method (to extract the dc component of the measured modulated magnetic field data)

14. Examples from the world of plants:  a) Simple plant cell – Internodal cell of green algae Chara corallina green algae Chara corallina

15. Our cell culture

16. Chara corallina internodal cell

17. Multi-SQUID measuring configuration (37 channels) - schematically

18. C.Corallina intern. cell: stimulus location and measuring points; intra-,extracell. curr.; m. field

19. The time evolution of magnetic field (vert. comp.) measured in 37 points above the C. corallina

20. The isofield lines representation  3 particular time values (1.3s, 1.6s, 1.9s) after the stimulus

21. The isof. representation (at 1.4s, 2.5s, 3.6s); model. calc. of current dipol and current density along the C.c. intern. c.

22. Measured and calculated AP and B

23. Some results  Spreading of excitation along the cell: v ~ 4cm/s  Conductivity:  i = 1.2  -1 m -1,  ex = 0.025  -1 m -1   Length of the depolarized area: l ~ 50 mm  Maximal intracellular current: I i = 1  A

24. Examples from the world of plants: b) the influence of visible light on AP and on B in Chara corallina: The chemical nature of AP obtained from the non- invasive observation (by SQUID microscope) of the intracellular current under the influence of light

25. SQUID microscope prepared for the C.c. inernodal cell studies (schematically)

26. Part of SQUID microscope and C.c. internodal cell holder  We measure:  Electric AP  K + anesthesia technique  Magnetic field  SQUID Microscope  Both measurements are simultaneous

27. Protocol of the C.c. experiment with white light illumination Light OFF reference Light ON 10 min Light ON 20 min Light ON 60min AP as function of light exposure

28. The influence of ilumination on the measured B and AP of electrically stimulated C.c. internodal cell

29. Model which explains the illumination experiment in the context of 2nd messenger system  [Ca 2+ ] c is altered under the influence of light/dark transitions (Miller@Sanders 1987) Miller@Sanders  AP can be described by an electrically stimulated release of Ca 2+ from internal store: -a) the voltage depend. synthesis/breakdown of the 2nd mesenger IP 3. -b) the joint action of IP 3 and Ca 2+ on the gating of the receptor channels, which conduct Ca 2+ release from internal stores. -c) modification: cells move excess Ca 2+ from the cytoplasm back into internal stores by an endogeneous Ca 2+ pump system (described by the Hill function. - Quantitative evaluation follows the Othmer model.

30. Simulated [Ca +2 ] c transients in response to a single electrical stimulation

31. Some results  Assuming that the activation of the Cl - channels, that cause the depolarization, is the direct consequence of the change in [Ca 2+ ] c, the measurements quantitavely agree well with the model. is the direct consequence of the change in [Ca 2+ ] c, the measurements quantitavely agree well with the model.

32. Examples from the world of plants: c) the injury induced ionic currents in the plant organs - leaves in the higher developed plant Vicia faba, detected magnetically by the multichannel SQUID system.

33. Measuring setup

34. The position of injury (panel B cut)

35. Time evolution of magnetic field in all channels: panel A 15 min. before injury, panel B 1-16 min after injury, panel C time evolution of field RMS value, panels D end E isofield maps 10 min before and 1.5 min after injury.

36. Some results Some results  All measured injured Vicia f. plants responded to injuries with detectable quasi-d.c. magnetic signals. injuries with detectable quasi-d.c. magnetic signals.  Large injury leads to easily localizable current source of dipolar pattern. The characteristic time delay is of dipolar pattern. The characteristic time delay is about 10 min. about 10 min.  No long-distance spreading of electrical activity was generally observed. generally observed.

37. Conclusions  Magnetic measurements offer also in the world of single plant cells and plants valuable noninvasive information.  Both, multichannel SQUID system and SQUID microscope can be applied. The last option offers good spatial resolution.  Results from magnetic measurements can be considered as complementary to the existing electric measurements in plants.  SQUID measurements provide direct information on the behavior of ionic currents.

38. Participating Researchers University of Ljubljana: Vojko Jazbinsek, Matjaz Slibar, Ales Stampfl, Robert Zorec PTB Berlin: Sergio Erné (now at Univ. Ulm), Wolfgang Mueller, Gerd Wuebbeler TU Darmstadt: Gerhard Thiel, Michael Wacke Vanderbilt University: Franz Baudenbacher, Luis Fong, Jenny Holzer, John Wikswo