Sergey Shabala, Lana Shabala and Ian Newman
This protocol outlines how to quantify kinetics of net ion fluxes from plant roots using non-invasive ion-selective microelectrodes, following the MIFE system.
If an ion is taken up by root cells, its concentration in the proximity of the root surface will be lower than that further away. Vice versa, if the ion is extruded across epidermal root cells, there will be a pronounced electrochemical potential gradient directed away from the root surface. The principle of the method is to measure this electrochemical potential gradient by slow, square-wave movement of ion-selective microelectrode probes between two positions, close to (position 1), and distant from (position 2) the sample surface (Fig. 1). At each position, electrode voltage is recorded and then converted into approximate concentration using the calibrated Nernst slope of the electrode. It is assumed that convection and water uptake are negligibly small and unstirred layer conditions are met. More details on the theory of non-invasive MIFE ion flux measurements are available in Newman (2001) and Shabala et al (2006).
Fig. 1. The principle of the MIFE ion flux measurements (modified from Wherrett 2006)
- MIFETM hardware (see below)
- MIFECHART and MIFEFLUX software
- Inverted microscope with long-working distance objectives (100x or 200x)
- Anti-vibration table
- Faraday cage
- Electrode puller
- Borosilicate glass capillaries
- Specific ion selective resins and salinising agent (all from Fluka)
- Electrode holders (e.g. E45W-F15PH; Warner Instruments, USA)
- Non-metallic syringe needle for filling micropipettes (MF34G-5, 0.1mm ID; WPI, Sarasota, FL, USA)
- Electrode filling station consisting of two simple micromanipulators and a stereo microscope
- A small oven, to 250∘C, with gloves and metal electrode racks
- A fume cabinet
- Measuring chambers to immobilise plant roots
MIFETM hardware consists of (Fig. 2):
- MIFE main amplifier, preamplifier and controller
- Multi-manipulator providing 3- dimensional positioning (e.g. MMT-5, Narishige, Japan)
- One- or three-dimensional hydraulic micromanipulator (e.g. Narishige MHW-1 or MHW-3 models)
- Stepper motor and power supply to drive hydraulic manipulator
- Mechanical micromanipulator (e.g. Narishige MX-1) for positioning assembled MMT-5/MHW-3 ensemble.
- PC for system control and data acquisition
- CIO-DAS08 card for analogue to digital conversion
Units, terms, definitions
LIX – liquid ion exchanger;
OD– outer diameter
Step 1- pulling out electrode blanks
- Insert non-filamentous borosilicate glass capillaries (GC150-10, OD = 1.5 mm; Harvard Apparatus Ltd, Kent, UK)) into a vertical pipette puller (e.g. PP 830, Narishige, Japan)
- Pull the blanks to <1 μm diameter tips
- Store pulled electrodes in a stainless steel or aluminium rack in a vertical position
Step 2 – baking and salinising the blanks
- Place electrode blanks tips in upright, base down position, in a rack
- Oven dry at 220∘C overnight
- Ten to fifteen minutes before silanisation, cover electrodes by a steel lid to create a closed container with the blanks
- Add two drops (approximately 50 L) of tributylchlorosilane (90796, Fluka Chemicals) under the lid using yellow tip pipettor
- Ten minutes later, remove the lid and bake electrode blanks for the further 30 min
- Switch the oven off and let electrodes to cool down
- Take electrodes from the oven and keep them in an enclosed container
Step 3 – filling the electrodes with an appropriate liquid ion exchanger
- Prepare a LIX-containing tube by dipping a broken-tip glass mirocapillary (tip diameter approximately 50 μm) into the LIX, taking up a column of cocktail of approximately 1 mm
- Mount the pulled microelectrode blank horizontally on a three-dimensional micromanipulator
- Flatten the blank tip to achieve external tip diameter of 2-3 μm by placing it against a flat glass surface under a stereo microscope
- Position the blank co-axially with the LIX-containing tube at 100 to 200 μm distance
- Fill the blank with appropriate back-filling solutions (see Shabala et al 2006) using a syringe with a non-metallic needle
- Front-fill the blank tip with LIX by putting it into a brief contact with the LIX-containing tube to achieve the column length between 100 and 150 μm
- Store electrodes having their tips immersed in solution until use (up to 8-10 hours)
- Mount electrodes in the holder
- Fabricate a reference electrode by inserting a chlorided silver wire (galvanised in 0.25 N HCl for 15 min) into a broken glass micro-capillary (ca 50 μm tip diameter) filled with 1 M KCl in 2 % agar and sealing it with a parafilm. Alternatively use a commercial reference electrode.
- Prepare appropriate set of at least three standards covering the expected range of the ion in question
- Calibrate electrodes using the MIFE CHART calibration routine (see Other Resources)
- Halt the calibration by pressing ALT-H or the <H> option in the <M>ain Menu
- Produce AVC file by using <A>veraging routine in <E>lectrometer menu
- Check calibration results and discard electrodes with responses less than 50 mV per decade for monovalent ions and 25 mV per decade for divalent cations, and with correlation coefficients less than 0.999 (for the three-point calibration)
- Mount roots in the measuring chamber filled with an appropriate solution
- Under the microscope, position microelectrode tips in one plane, separated laterally by 1-2 μm, 20 to 30 μm away from the root surface.
- Start MIFECHART software.
- Setup parameters for the run using the function key F9.
- Set or check the electrometer offsets using the <S>et Offsets option in the <E>lectrometer menu.
- Start data acquisition using ALT-S or the <S> option in the <M>ain Menu. A small window opens to let you set a filename that encodes date and time. It is easiest to accept the default by typing S.
- Then a window opens to let you set the starting time, duration of measurements and other values.
- Accept all the defaults by typing G.
- A one-line window opens in which you should type your name and other identifying information about the experiment you have planned. Press <ENTER> to close it and begin the data acquisition.
- Navigate the Chart Display screen, using the “buttons” at the bottom of the screen, to view any or all of the data as desired (more instructions are available in the MIFE Handbook)
- Stop data acquisition by pressing ALT-H or the <H> option in the <M>ain Menu.
- If you are not analysing the run that you have just made, load and display the required data file using the MIFECHART software (use <P>lot routine under <F>iling menu)
- Produce AVM file using <A>veraging routine in the <E>lectrometer menu (see Averaging Routine in Other Resources for more details)
- Quit the CHART program by pressing ALT-Q
- Start the MIFEFLUX program and follow the routine prompt (see Other Resources)
- Once calculations are completed, the resultant FLX file (ASCII format) can be opened by any spreadsheet and evaluated in a conventional way.
Newman, I. A. (2001). Ion transport in roots: measurements of fluxes using ion-selective microelectrodes to characterize transporter function. Plant, Cell and Environment 24, 1-14
Shabala, L., Ross, T., McMeekin, T., and Shabala, S. (2006). Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment. FEMS Microbiology Reviews 30, 472-486
Shabala, S. N., Newman, I. A., and Morris, J. (1997). Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Physiology 113, 111-118
Shabala, S. (2006) Non-invasive microelectrode ion flux measurements in plant stress physiology. In: Plant Electrophysiology – Theory and Methods (ed. A. Volkov). Springer, Heidelberg. pp. 35-71
Health, safety & hazardous waste disposal considerations
The silane used to make the electrodes hydrophobic is a toxic chemical, so the silaning should be done in a fume cabinet.
Notes and troubleshooting tips
MIFE Calibration Routine
To make a calibration, you will need to measure a range of standard solutions for each ion used. The range should cover the concentrations you expect to find. Optimum accuracy in linear fitting is obtained from equal factors change in standard concentrations chosen. Put electrodes into your first standard solution, close the Faraday cage and start data acquisition. Press the function key F7. In the window that opens, correct the temperature, type the names of all the ions to be used and press <ENTER>. When the data for the ion of the first solution is stable and in range, press F7 again and enter the concentration for that ion. After you press <ENTER> the data recorded will be used in the calibration calculation until a third press of F7, after about 20 seconds, allows you to terminates that set, either keeping it or rejecting it. Details are given under F7 in the Handbook file THECHART.DOC.
Repeat the process for the other concentrations and the other ions. When all needed data have been recorded type ALT-H to stop data acquisition. From the <E>lectrometer menu, choose the <A>verage option and the <C>alibration average. CHART will do the least squares fit to the data for each ion, create the .AVC file and display in a window the parameters of the electrodes. If you are unhappy with the calibrations, you can start data acquisition again to repeat as many ions as you wish. No need to quit CHART. Eventually, in running MIFEFLUX, you will have the option of selecting just those calibrations you want from the .AVC file(s) you have made.
Data averaging routine
You are required to specify a “Valid Time” at the end of each stage of manipulator movement, for which the actual “Stage Time” is provided. Pressing ENTER moves the highlight to the next stage. Pressing UPARROW moves up to the previous one to allow you to correct an error. Finally you must press ENTER to accept the order of the curve (“Kind of <F>it”) to fit the data (typing F to cycle through the orders). Linear is probably what you need. As the averaging is done, the chirp and the Line Count indicate progress. An AVM file (for “AVerage Manipulator”) is created with the same name as the DAT and LOG files of the data. The AVM file is read by the MIFEFLUX program to calculate the fluxes, but you can import it into a spreadsheet or view it in a text editor.
The MIFEFLUX program is run under DOS in your current directory, which contains the AVC and AVM files for the flux calculations. The batch file MFLUX.BAT, which is in the C:MIFE directory, will do this, just by typing MFLUX in your current directory. The program first asks you for the AVC file(s) you want, then for the AVM file of your averaged flux data, and finally for the kind of analysis. After calculation is done you can calculate more fluxes using the same calibrations. The most often used defaults are indicated by and are obtained just by pressing <ENTER>.
Links to resources and suppliers
- MIFETM hardware and software: www.mife.com
- Narishige manipulators: http://www.narishige.co.jp/english/
- Ionophore cocktails for the ion selective electrodes: http://www.sigmaaldrich.com/
- Microelectrode holders: http://www.warneronline.com/