SQUID Sensors for SQUID Sensors for Detecting Some Detecting Some

Electrophysiologic Electrophysiologic Phenomena in PlantsPhenomena in Plants

Zvonko TronteljZvonko TronteljPhysics Dept., University of LjubljanaPhysics Dept., University of Ljubljana

andandInstitute for Mathemtics, Physics and Institute for Mathemtics, Physics and

Mechanics, LjubljanaMechanics, Ljubljana

Outline of talkOutline of talk

IntroductionIntroduction On SQUID sensorOn SQUID sensor Basic Basic ssteps in data analysis and modelingteps in data analysis and modeling of of

sources of bioelectric activitysources of bioelectric activity Examples from the world of plants:Examples from the world of plants: a) a) Chara corallina Chara corallina (single cell(single cell studies)studies) bb) ) Vicia fabaVicia faba (plant injury study)(plant injury study) ConclusionsConclusions

Objectives:Objectives:

To apply SQUID(s) in order to obtain the noninvasive To apply SQUID(s) in order to obtain the noninvasive information on:information on:

a)a) ionic currents in electrically stimulationic currents in electrically stimulat.. Chara corallina,Chara corallina,

b) the influence of visible light on AP and ob) the influence of visible light on AP and onn AC in AC in C.c.C.c.

and to relate the ionic kinetics to and to relate the ionic kinetics to chemical nature of chemical nature of AP.AP.

c) the injury induced ionic currents in the plant organs -c) the injury induced ionic currents in the plant organs -

leaves in the higher developed plant leaves in the higher developed plant Vicia faba.Vicia faba. To model the transmembrane pot. difference under To model the transmembrane pot. difference under

different conditions and to support the suggest. different conditions and to support the suggest. hhypothypoth.. of light action on the cytoplasmic of light action on the cytoplasmic Ca Ca ion. conc.ion. conc.

SQUID sensorsSQUID sensors

1.1. What is SQUID?What is SQUID?

2.2. What they offer to us?What they offer to us?

3.3. Where we can use them?Where we can use them?

4.4. Why SQUID sensors in electrophysiology?Why SQUID sensors in electrophysiology?

Ad 1 and Ad 2Ad 1 and Ad 2

Superconducting QUantum Interference DeviceSuperconducting QUantum Interference Device Magnetic flux-to-voltage convertor (the most Magnetic flux-to-voltage convertor (the most

sensitive sensor for quasi dc magnet. fields m.)sensitive sensor for quasi dc magnet. fields m.) Measured m. field; via Amper’s law the sourceMeasured m. field; via Amper’s law the source Based on 3 facts described by QMBased 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

From Josephson jct. to From Josephson jct. to closed sc. circuitclosed sc. circuit

Dc SQUID configurationDc SQUID configuration

Outer magnetic field is Outer magnetic field is present at the SQUIDpresent at the SQUID

Ad 3 and ad Ad 4Ad 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. shieldedSQUID has to be in m. shielded env. env.

Basic steps in analysis and Basic steps in analysis and modelling of current modelling of current sources in living mattersources in living matter

Distribution of ionic currents in tissue. ComplicatedDistribution of ionic currents in tissue. Complicated Direct and inverse problemDirect and inverse problem The direct problem – a unique solutionThe direct problem – a unique solution TTZZTTEE

The inverse problem is ill-posed problemThe inverse problem is ill-posed problem EEZZETETTT

Simple geometry – analyt. solutions, Simple geometry – analyt. solutions, otherweise modeling otherweise modeling

Simple geometrySimple geometry

Single cylindrically shaped cell (1D case)Single cylindrically shaped cell (1D case)

Bound. cond.: Bound. cond.: mm(z) = (z) = iia,z) – a,z) – eea,z)a,z)

nn..JJii (a,z) = (a,z) = n n..JJee(a,z)(a,z)

Simple geometry Simple geometry (contin.)(contin.)

From Ampere law:From Ampere law:

BBii = Integr. [G( = Integr. [G(a,z – z’)Ja,z – z’)Jii(a,z’)]dz(a,z’)]dz

Applying the Fourier and the inverse Applying the Fourier and the inverse Fourier transformations one can come Fourier transformations one can come from potential to mag. field and v.a.v.from potential to mag. field and v.a.v.

Some methods in modeling Some methods in modeling of current sourcesof current sources

Current multipole expansionCurrent multipole expansion Current distribution with the minimum norm Current distribution with the minimum norm

estimationestimation Covariance method (to extract the dc Covariance method (to extract the dc

component of the measured modulated component of the measured modulated magnetic field data)magnetic field data)

Examples from the world of Examples from the world of plants:plants:

a) Simple plant cell – Internodal cell of a) Simple plant cell – Internodal cell of

green algae green algae Chara corallinaChara corallina

Our cell cultureOur cell culture

Chara corallinaChara corallinainternodal cellinternodal cell

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

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

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

The isofield lines representationThe isofield lines representation

3 particular time values (1.3s, 1.6s, 1.9s) 3 particular time values (1.3s, 1.6s, 1.9s) after the stimulusafter the stimulus

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

Measured and calculated AP and B Measured and calculated AP and B

Some resultsSome results

Spreading of excitation along the cell: v ~ 4cm/sSpreading of excitation along the cell: v ~ 4cm/s

Conductivity:Conductivity:i i = 1.2 = 1.2 -1-1m m -1 -1 , , ex ex = 0.025 = 0.025 -1-1m m -1-1Length of the depolarized area: l ~ 50 mmLength of the depolarized area: l ~ 50 mm Maximal intracellular current: IMaximal intracellular current: I ii = 1 = 1AA

Examples from the world of Examples from the world of plants:plants:

b) the influence of visible light on AP and b) the influence of visible light on AP and oonn B B in in CChara hara ccorallina: orallina: The chemical The chemical nature of AP obtained from the non-nature of AP obtained from the non-invasive observation (by SQUID invasive observation (by SQUID microscope) of the intracellular current microscope) of the intracellular current under the influence of light under the influence of light

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

Part of SQUIDPart of SQUID microscope and microscope and C.c. C.c. internodal cell holderinternodal cell holder

We measure:We measure: Electric AP Electric AP

KK++ anesthesia anesthesia techniquetechnique

MagneticMagnetic field field SQUID MicroscopeSQUID Microscope

Both measurements Both measurements are simultaneousare simultaneous

Protocol of the Protocol of the C.c.C.c. experimentexperiment with white light illuminationwith white light illumination

• Light OFF reference• Light ON 10 min• Light ON 20 min

• Light ON 60min

AP as function of light exposure

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

Model which explains the Model which explains the illumination experiment in the illumination experiment in the context of 2nd messenger systemcontext of 2nd messenger system

[Ca[Ca2+2+]]c c is altered under the influence of light/dark transitions (is altered under the influence of light/dark transitions (Miller@SandersMiller@Sanders 1987) 1987)

AP can be described by an electrically stimulated release of CaAP can be described by an electrically stimulated release of Ca2+2+ from from internal store:internal store:-a) the voltage depend. synthesis/breakdown of the 2nd mesenger IPa) the voltage depend. synthesis/breakdown of the 2nd mesenger IP3 3 . . -b) the joint action of IPb) the joint action of IP3 3 and and CaCa2+2+ on the gating of the receptor channels, on the gating of the receptor channels, which conduct Cawhich conduct Ca2+ 2+ release from internal stores.release from internal stores.-c) modification: cells move excess Cac) modification: cells move excess Ca2+2+ from the cytoplasm back into from the cytoplasm back into internal stores by an endogeneous Cainternal stores by an endogeneous Ca2+2+ pump system (described by the pump system (described by the Hill function.Hill function.

- Quantitative evaluation follows the Othmer model.Quantitative evaluation follows the Othmer model.

Simulated [CaSimulated [Ca+2+2]]c c transients in response to a transients in response to a single electrical stimulationsingle electrical stimulation

SomeSome results results

Assuming that the activation of the ClAssuming that the activation of the Cl- - channels, that cause the depolarization,channels, that cause the depolarization,

is the direct consequence of the change is the direct consequence of the change in [Cain [Ca2+2+]]c c , , the measurementsthe measurements

quantitavely agree well with the model.quantitavely agree well with the model.

Examples from the world of Examples from the world of plants:plants:

c) the injury induced ionic currents in c) the injury induced ionic currents in the plant organs - leaves in the the plant organs - leaves in the higher developed plant higher developed plant Vicia fabaVicia faba, , detected magnetically by the detected magnetically by the multichannel SQUID systemmultichannel SQUID system..

Measuring setupMeasuring setup

The position of injury (panel B cut)The position of injury (panel B cut)

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

Some resultsSome results

All measured injured All measured injured Vicia f. Vicia f. plants responded to 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 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 wasNo long-distance spreading of electrical activity was

generally observed.generally observed.

ConclusionsConclusions Magnetic measurements offer also in the world of Magnetic measurements offer also in the world of

single plant cells and plants valuable noninvasive single plant cells and plants valuable noninvasive information.information.

Both, Both, multichannel multichannel SQUID system and SQUID SQUID system and SQUID microscope can be applied. microscope can be applied. The last option offersThe last option offers good spatial resolutiongood spatial resolution..

Results from magnetic measurements can be Results from magnetic measurements can be considered as considered as complementary to the existing electric complementary to the existing electric measurements in plants.measurements in plants.

SQUID measurements provide direct information on SQUID measurements provide direct information on the behavior of ionic currents.the behavior of ionic currents.

Participating ResearchersParticipating Researchers

University of LjubljanaUniversity of Ljubljana::

Vojko Jazbinsek, Matjaz Slibar, Ales Stampfl, Vojko Jazbinsek, Matjaz Slibar, Ales Stampfl, Robert ZorecRobert Zorec

PTB Berlin:PTB Berlin:

Sergio Erné (now at Univ. Ulm), Wolfgang Mueller, Sergio Erné (now at Univ. Ulm), Wolfgang Mueller, Gerd WuebbelerGerd Wuebbeler

TU Darmstadt:TU Darmstadt:

Gerhard ThielGerhard Thiel, Michael Wacke, Michael Wacke

Vanderbilt University:Vanderbilt University:

Franz Baudenbacher, Luis Fong, Jenny Holzer, John Franz Baudenbacher, Luis Fong, Jenny Holzer, John WikswoWikswo