MIFETM system requirements - March 2012

The following are likely requirements for the MIFE system.
  Facility or component Source and cost
1 Electrophysiology laboratory with bench and Faraday cage Assumed present
There is need for general electrophysiological experience Assumed present or available
A bench that is sufficiently vibration-isolated can be made from a strong table. On top of the table, a slab of steel or aluminium-covered composite is supported on three small, inflated inner tubes. All these components can be bought locally
2 Microelectrode preparation and filling facilities
Electrode puller. A simple one is sufficient. Those with multiple pulls can give lower-resistance electrodes. It is useful to be able to make tips of 2 to 5 mm diameter.  This is standard in an electrophysiology lab. There are many suppliers.
Two simple (perhaps old) micromanipulators are needed for filling the electrodes. A background light is also needed. These are readily available or can be purchased.
A small oven, to 250 C, with gloves and electrode racks. It is in a well ventilated room or fume hood to disperse the silane safely. Readily available commercially.
Electrical test equipment: An adjustable mV source (0-50 mV, 0-500 mV).
Voltmeter (0.1 mV sensitivity) to measure the source voltage accurately.
Usually present in electrophysiology labs. We have a diagram for a mV source.
3 Microscope with vertical or horizontal optic axis, for one of three configurations: A, B, C.
A. A standard compound microscope, lying on its back, is convenient for measuring fluxes from roots or other tissue immersed in solution. This configuration is illustrated with an E. nitens seedling. Long-working distance objectives and condenser are necessary. Electrodes are mounted on a multiple electrode mount (6 below), which itself is mounted on a manual manipulator (7 below) fixed above the microscope. The E. nitens illustration is of a manual manipulator that incorporates its own three electrode mount. The microscope stage is removed to provide space for a Perspex chamber to hold the tissue. The chamber is mounted on the  driven micromanipulator (4 below). Thus, during flux measurements, it is the tissue chamber that actually moves relative to the electrodes. An existing microscope can be used.

Make the chamber to suit your tissue and microscope.

Your machine shop will do the mounting.

B. The configuration A could in principle be modified to have the chamber fixed (or on a manual manipulator) and the electrodes on their multiple mount mounted on the driven manipulator above the microscope. This configuration is generally more difficult to arrange mechanically. We have not used it.
C. An inverted microscope  is good for single cell studies or protoplasts, particularly when patch clamping the same cell. The electrodes (if >1 are used) are on the multiple electrode mount (6 below) that is itself mounted on the driven manipulator in the usual configuration, as for patch clamping. Although the electrodes are at an angle of 30o-45o to the horizontal, the mount can be mounted on a block to allow the driven manipulator axes, and electrode tip movement, to be horizontal and vertical. No multiple electrode mount yet exists that can be safely supported by the PatchMan NP2 (100 g load limit). Narishige manipulators must be used.
4 Driven micromanipulator  
MIFE custom-assembled Narishige manipulator system (SM-17, MHW-4, MX-2), with a MIFE stepper motor drive.
or:
Eppendorf   PatchMan NP2.
Obtain a quote from UTas for the Narishige system or from your Eppendorf supplier for the NP2.. 
Estimate ~A$18,000 in each case.
The driven micromanipulator is mounted on a solid stand (obtainable from the supplier) or on a rigid support from the microscope body. Your machine shop may be needed to mount the micromanipulator.
5 Electrode holders and Ag/AgCl half cells 
Commercial holders are available in various styles, with silver wires or with pellets. They must have a side hole for pressure relief as the prepared microelectrode is inserted. They should have a 1 mm hole for the electrical connector to the MIFE preamplifier.
WPI, Clark  and others can supply them. See their catalogues. 
For our own use, we have made our own holders.
6 Multiple electrode mount
A commercial 2, 3 or 4-electrode mount is needed if more that one ion is to be measured at one time. It is mounted on the driven manipulator as described above or on the 3-axis manual manipulator (7 below), of which it may be part. It must be able to hold the 6 mm diameter electrode holders, and have a sufficient angle ( ~ 22 ) between them so that the electrode tips can be adjusted together without their shanks touching.
7 Manual manipulator for adjusting the electrode position
A manual 3-axis manipulator is needed for general electrode positioning. It must have a long (>3 cm) axis parallel to the electrodes, to raise and lower them. We have used Narishige MMT-5. The MLN-3 may also work. Other brands also will be suitable.  See manipulator supplier’s web pages or catalogues. Median cost ~ A$3000.
The manipulator is mounted on a rigid stand or onto the body of the microscope, for optimum rigidity of support, in a way that depends on the configuration chosen. Mounting done by your machine shop.
8 PC computer
A PC  running Windows 98 or ME with one ISA-bus slot and a spare slot beside it. Windows XP or later will not allow acccess to the DAS08 card. One data file can easily be >1 MB, so an archiving facility (USB port or network connection) is needed for effective data management.   Your local supply.
9 MIFE electronics hardware*
This is the central part of the MIFE system and is controlled by the software (which is included in the cost). Please ask for a quote. Our estimated cost ~ AU$30,000*
10 The CHART/MIFEFLUX software*
This software is essential to control the hardware and to analyse the data. A handbook is provided. . Included in the cost for the hardware.
11 Spreadsheet software
General-purpose spreadsheet software is used to display and print the ASCII text files produced by CHART and MIFEFLUX. Most labs will have this already 
12 Consultancy, training and other MIFE set-up costs 
The entire system to hold and view tissue, to prepare and set up electrodes, to adjust them close to the tissue and to move them to and fro, is technically complex. Freight and insurance: at cost. We can provide suitable consultancy services. 
You may wish to have a consultant from Tasmania come to set up the MIFE system and give basic training in its use. The time required is likely to be about two weeks, but should be arranged by negotiation with the University of Tasmania Research Office Commercialisation Unit.  Please contact Prof. John Dickey. Consultants fee: to be negotiated; consultants travel: at cost; consultants accommodation and local expenses: at cost.
You may wish to visit the MIFE Centre for Membrane Transport at the University of Tasmania, in which the MIFE system has been developed, to be trained in its use and setting up. Time required is likely to be about two weeks. Suitable arrangements should be negotiated initially with Professor Sergey Shabala. Hobart bench time: negotiated; your travel, accommodation and local expenses: at cost;
13 Guarantee and maintenance
The hardware is guaranteed against faulty workmanship for 12 months from delivery.  You pay the freight to Hobart; the hardware will be repaired or replaced. We will pay the return freight.
No guarantee is given that the software will perform as expected. It is working well and is in regular use in the MIFE Centre, so it will be well maintained. Bugs will be corrected as quickly as possible. Improvements are made from time to time. If any problems are not solvable by studying the Handbook, please E-mail Ian Newman. or Professor Sergey Shabala. Corrected and improved versions will be available for MIFE users.

* These items are made and supplied by the University of Tasmania . Specifications of the MIFE System are available. Prices will not include Australian GSTax, and may change without notice. Please contact Prof. John Dickey., in the first instance, for a quotation for your particular MIFE requirements.

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Maintained by Ian Newman. Date . University of Tasmania.