MIFE system requirements

MIFE system requirements - April 2001

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 vibration-isolated bench 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. This is standard in an electrophysiology lab.

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. Readily available.

Electrical test equipment: Voltmeter (0.1 mV sensitivity) and mV source. 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 desirable. 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 hydraulic micromanipulator (4 below). Thus, during flux measurements, it is the chamber that actually moves relative to the fixed 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 can be modified to have the chamber fixed (or on a manual manipulator) and the electrodes on their multiple mount mounted on the hydraulic 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. The electrodes (if >1 are used) are on the multiple electrode mount that is itself mounted on the hydraulic manipulator in the usual configuration as for patch clamping. Although the electrodes are at an angle of ~45^o to the horizontal, the mount can be mounted on a block to allow the hydraulic manipulator axes, and electrode tip movement, to be horizontal and vertical.

4 Hydraulic micromanipulator (A piezoelectric translator, with power supply, is a single axis alternative, but is less satisfactory.)

A single axis manipulator is the minimum. We are familiar with the 3-axis Narishige MHW-103, and the single axis MHW4. Other non-drifting manipulators may be suitable. It is adequate to have a 1:1 control ratio, not the finer 1:5 ratio of the MHW3 and MHW4, which may be better for protoplasts. Narishige Web site (24 Apr 01) gives:
US$5100 for MHW-103, US$6350 for MHW3, US$1900 for MHW4.

The hydraulic micromanipulator is mounted on a rigid support from the microscope body in configuration A; on the manual manipulator in configurations B and C. Your machine shop will mount the micromanipulator.

5 Stepper motors and their electronic drives (for the hydraulic micromanipulator)

The stepper motors and their drive electronics can be provided with the MIFE system. Our estimates of costs: -

Mounting the motors to control the Narishige micromanipulator is included in the cost. Single axis: A$3700*
Three axis: A$5100*

For a different manipulator, the mounting may need to be done by your machine shop.

6 MIFE multiple electrode mount

This 4-electrode mount (or something equivalent in the manipulator below) is needed if more that one ion is to be measured at one time. It is mounted on the 3-axis manipulator (7 below) or on the hydraulic manipulator as described above. Our cost estimate A$6000*

7 Manual manipulator for adjusting the electrode position

A manual 3-axis manipulator is needed for general positioning. It must have a long (>3 cm) axis parallel to the electrodes, to raise and lower them. We have used Narishige MM-3. The MLN-3 may also work. Other brands also will be suitable. See manipulator supplier’s web pages or catalogues.

The manipulator is mounted 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 running DOS

Any PC running under DOS, or in a DOS Window, will work. A 386 will be slow in drawing the screen. 100 MB of disk space for data storage is useful. A data file can easily be >1 MB. Archiving facility is necessary for effective data management. Your local supply.

9 Data acquisition card for the computer

The DAS08 card (or equivalent), for which the software is written, is from Computer Boards USA. It comes with its own set-up and test instructions. It is best for you to buy this locally. We can supply this at estimated cost A$1100*.

10 MIFE electronics hardware

This is the central part of the MIFE system and is controlled by the software (included in the cost). Our estimated cost A$15,200*

11 The CHART/MIFEFLUX software

This software is essential to control the hardware and to analyse the data. It runs under DOS. A handbook is provided. Included in the above cost for the hardware.

12 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

13 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. Consultants fee: to be negotiated; consultants travel: at cost; consultants accommodation and local expenses: at cost.

You may wish to visit the Biophysics laboratory 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 with Dr Newman in the Biophysics lab. Hobart bench time: negotiated; your travel, accommodation and local expenses: at cost;

14 Guarantee and maintenance

The hardware is guaranteed against faulty workmanship for 12 months from delivery. Circuit diagrams will be provided and it may be more efficient for your local electronics technician to test and make repairs. 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 Biophysics lab, so it will be well maintained. Bugs will be corrected as quickly as possible. Improvements are made from time to time. It appears to be Y2K compliant if the operating system is. If any problems are not solvable by studying the Handbook, please E-mail Dr Newman. Corrected and improved versions will be provided to all users without further charge, until 2003 at least.

* These items are made and supplied by the University of Tasmania. Prices do not include GST and may change without notice. Please contact Dr Ian Newman, in the first instance, for a quotation for your particular MIFE requirements.

To Biophysics home page.

	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 vibration-isolated bench 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.	This is standard in an electrophysiology lab.
	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.	Readily available.
	Electrical test equipment: Voltmeter (0.1 mV sensitivity) and mV source.	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 desirable. 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 hydraulic micromanipulator (4 below). Thus, during flux measurements, it is the chamber that actually moves relative to the fixed 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 can be modified to have the chamber fixed (or on a manual manipulator) and the electrodes on their multiple mount mounted on the hydraulic 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. The electrodes (if >1 are used) are on the multiple electrode mount that is itself mounted on the hydraulic manipulator in the usual configuration as for patch clamping. Although the electrodes are at an angle of ~45^o to the horizontal, the mount can be mounted on a block to allow the hydraulic manipulator axes, and electrode tip movement, to be horizontal and vertical.
4	Hydraulic micromanipulator (A piezoelectric translator, with power supply, is a single axis alternative, but is less satisfactory.)
	A single axis manipulator is the minimum. We are familiar with the 3-axis Narishige MHW-103, and the single axis MHW4. Other non-drifting manipulators may be suitable. It is adequate to have a 1:1 control ratio, not the finer 1:5 ratio of the MHW3 and MHW4, which may be better for protoplasts.	Narishige Web site (24 Apr 01) gives: US$5100 for MHW-103, US$6350 for MHW3, US$1900 for MHW4.
	The hydraulic micromanipulator is mounted on a rigid support from the microscope body in configuration A; on the manual manipulator in configurations B and C.	Your machine shop will mount the micromanipulator.
5	Stepper motors and their electronic drives (for the hydraulic micromanipulator)
	The stepper motors and their drive electronics can be provided with the MIFE system.	Our estimates of costs: -
	Mounting the motors to control the Narishige micromanipulator is included in the cost.	Single axis: A$3700* Three axis: A$5100*
	For a different manipulator, the mounting may need to be done by your machine shop.
6	MIFE multiple electrode mount
6	This 4-electrode mount (or something equivalent in the manipulator below) is needed if more that one ion is to be measured at one time. It is mounted on the 3-axis manipulator (7 below) or on the hydraulic manipulator as described above.	Our cost estimate A$6000*
7	Manual manipulator for adjusting the electrode position
	A manual 3-axis manipulator is needed for general positioning. It must have a long (>3 cm) axis parallel to the electrodes, to raise and lower them. We have used Narishige MM-3. The MLN-3 may also work. Other brands also will be suitable.	See manipulator supplier’s web pages or catalogues.
	The manipulator is mounted 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 running DOS
8	Any PC running under DOS, or in a DOS Window, will work. A 386 will be slow in drawing the screen. 100 MB of disk space for data storage is useful. A data file can easily be >1 MB. Archiving facility is necessary for effective data management.	Your local supply.
9	Data acquisition card for the computer
9	The DAS08 card (or equivalent), for which the software is written, is from Computer Boards USA. It comes with its own set-up and test instructions.	It is best for you to buy this locally. We can supply this at estimated cost A$1100*.
10	MIFE electronics hardware
10	This is the central part of the MIFE system and is controlled by the software (included in the cost).	Our estimated cost A$15,200*
11	The CHART/MIFEFLUX software
11	This software is essential to control the hardware and to analyse the data. It runs under DOS. A handbook is provided.	Included in the above cost for the hardware.
12	Spreadsheet software
12	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
13	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.	Consultants fee: to be negotiated; consultants travel: at cost; consultants accommodation and local expenses: at cost.
	You may wish to visit the Biophysics laboratory 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 with Dr Newman in the Biophysics lab.	Hobart bench time: negotiated; your travel, accommodation and local expenses: at cost;
14	Guarantee and maintenance
	The hardware is guaranteed against faulty workmanship for 12 months from delivery. Circuit diagrams will be provided and it may be more efficient for your local electronics technician to test and make repairs.	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 Biophysics lab, so it will be well maintained. Bugs will be corrected as quickly as possible. Improvements are made from time to time. It appears to be Y2K compliant if the operating system is. If any problems are not solvable by studying the Handbook, please E-mail Dr Newman.	Corrected and improved versions will be provided to all users without further charge, until 2003 at least.