The advances in equipment design relating to pre-amplifier improvements and noise considerations of the voltage sensing architecture were reported in last year's update. Since then effort has been invested in gathering experimental results on a sufficiently large group of corn coleoptiles to be statistically valid and produce acceptable weighted averages of the changes in 3D voltage distributions around corn seedlings subject to non-symmetric illumination. Notable experimental milestones include: i) voltage calibration of the tip electrodes using a specially-made reference sample containing alternate rings of gold and aluminium at various line thickness on a cylindrical electrode, so as to determine the work function of each tip before and after the experiment. The reference samples were fabricated using low pressure plasma deposition of 20 nm pure gold on a aluminium pipe. These scans allow the geometrical offset of each probe in the array to be determine and ensure that, the operator can verify that the tips remain unchanged, e.g., by possible collision with the corn coleoptile during voltage imaging or initial set up. ii) The tracking routine was significant optimised by adding a 'dithering' procedure where each tip is displaced in a controlled fashion and the Kelvin signal is measured, so as to ensure that the centre of the probe is maintained at the normal to the plant circumference (within ( 30 (m, on a typical plant diameter of 2000 (m) at all times. This procedure is essential to verify that the multi-tip probe correctly tracks the coleoptile contour as it scans up and down the plant, forming a 3D image of the voltage distribution. We will need to utilise similar technology on all human tissue (non-flat surfaces), in order to clearly demonstrate that measurements made at different times, at the same location, are comparable, i.e., performed with the same sensing geometry. Otherwise difficult-to-quantify errors due to changes in the geometrical position of sensing electrode with a non-flat surface. iii)The displacement tracking, or feedback loop, was improved so as to operate at the mechanical limits of the head array support system ,i.e., 400 nm resolution. Due to the voltage gradients outside living tissue, voltage measurements, or voltage scans, must be performed at constant distance or else the results are unreliable. It is a notable feature of the Kelvin method that the voltage and tip-to-sample spacing are separable, independent parameters, unlike, for instance the general ion-probe case. iv) A video-capture routine was enabled allowing the operator to position the initially probe array and capture a visual image of each sample in a form of time lapse photography. This is a useful asset in confirmation the direction of plant displacement due to a photo-stimulus. v) The vibrating frequency of the tips was increased from 70 Hz to 250 Hz permitting a more rapid sample rate of sample voltage acquisition. The higher frequency places more stringent control of probe tip spacing (see iii above) due to the smaller amplitude of oscillation. vi) The controlling software was developed to have artificial intelligence, i.e., the tips 'know' their position with respect to one another and the plant, otherwise a control command issued by the host computer on one tip may result in contact by another tip. vii)The plant was surrounded by a matrix of blue, green, yellow and red LED's. The low-level green LED's were used for initial set-up only however the Blue and yellow LED's (corresponding to maxima in the plant's photo-tropic response) could be switched on and off automatically at pre-programmed intervals, on different sides of the plant. This optical arrangement was utilised to study the plant system for longer periods that illumination in one direction would allow, thus enabling repeatable results on one plant to be achieved, rather than one-measurement per plant as was previously the case. viii) The Host PC platform was upgraded to a Pentium II processor so as to allow an acceptable high rate of data acquisition and tip control. The physical displacement of the seedling with time can be upwards of 4 mm at the tip (although less down the shoot) over a 60 min stimulus . With the 400 nm spacing resolution of the tips they have to be constantly monitored, and spacing adjusted, to avoid physical contact. These improvements allowed the plant growth and photo-tropic response to be followed for a period of up to 5 days on any one plant. Additionally, the voltage patterns developed by the plants during dark cycles, almost certainly a geo-tropic response, could be clearly observed as waves of potential passing around and down the shoot. These results will be submitted for publication in two journals: a paper on the technical developments of the multi-tip, Scanning bio-Kelvin Probe is in preparation for Review of Scientific Instruments and the Zea mays bio-potential data will be submitted for publication, together with a colleague from BRC, Dr. M. Porterfield. These papers represent a significant step for the investigator moving out of the area of traditional surface science to that of bio-physics and bio-analytical equipment development and are considered necessary pre-cursors for both high-resolution aerial and in-vivo voltage/displacement sensors.
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