The equipment platform and software provide a multi-use data collection system for research and commercial assays. It utilizes a programmable 3D micro-positioning system, a real time data collection system and a video microscopy system. Variability of use, low cost, and user friendliness are the prime concerns in the development process. This platform is designed to grow with the user and remain useable for a long period of time. Other commercially available devices such as potentiostats, voltage or patch clamps can be integrated into the platform without software changes, although custom programming can be provided.
We have developed a computer-controlled, scanning micro-electrode measurement system for use in conductive solutions to measure electrical fields or ion and molecular fluxes non-invasively. Commercially available or lab-made micro-electrodes will work with the system hardware and software.
Our systems can accomodate virtually any voltametric or amperimetric sensor. We are currenty developing our new SMOT (Scanning Micro Optrode Technique) system that can measure dissolved oxygen with blue light. Eric Karplus of Science Wares, Inc. is the system designer and software developer. These are non-consumptive measurements of dissolved oxygen which is a major advance over polarographic reduction electrodes. Optrodes can be used simultaneously with other sensors and do not need a reference electrode since it is a light measurement.
The SVET (Scanning Vibrating Electrode Technique)
The SVET (Scanning Vibrating Electrode Technique) detects voltage gradients, in conductive solutions at a minimum speed of 50 milliseconds per measurement point. Voltage gradients are not disturbed by the probe’s vibrations which are typically in the range of 100 Hz to 1.0 kHz., because voltage is instantaneous, unlike ions or molecules. Voltage gradients are present whenever an electrical field is present in a conductive solution. Electrical fields are generated by corrosion activity or by living physiological specimens. Two dimensional vibration is accomplished by piezoelectric benders driven by sine wave oscillators at two different frequencies, one for each axis of vibration. Both vibration axes are driven at fixed frequencies. Scanning is done with a 3D micro-stepping motorized micro-manipulator (CMC-4). The SVET system is also capable of Electro-chemical Impedance Spectroscopy measurements. We call this SLEIS (Scanning Local Electro-chemical Impedance Spectroscopy). We are currently developing this technique to improve system capabilities.
Typical SVET/SLEIS System
The SLEIS (Scanning Localized Electrochemical Impedance Spectroscopy)
The SLEIS (Localized Electro-chemical Impedance Spectroscopy) can measure below the 1.0 kHz range (typically 10-100 Hz range). The micro-electrode remains stationary (non-vibrating) and the sample is stimulated with the oscillators in the PSD-2 amplifier, either directly, or via a potentiostat or frequency range amplifier. Another mode allows SVET and SLEIS data to be collected simultaneously while scanning a single micro-electrode over a metal surface under potentiostatic control. These methods of measurement provide the user with high sensitivity spatial resolution limited only by the electrode tip size, typically 5-50 µm diameter.
The SIET (Scanning Ion-selective Electrode Technique)
The SIET (Scanning Ion-selective Electrode Technique) can measure ion concentrations at picomolar levels, in aqueous media, but ions/anions must be measured slowly at around 0.5 to 1 second per point. This is mainly due to the mechanical disturbance of the gradient by the electrode movement, although the time constant of the LIX (Liquid Ion Exchanger) electrodes is also a factor. It takes a fraction of a second to reestablish the gradient again. LIX electrodes also have time constants in tenths of seconds (LIX dependent, see LIX specs). The electrode is stepped from one position to another in a defined sampling routine while being scanned with the 3D micro-stepper motor manipulator (CMC-4).
System mechanical setup is the same as described above except that an Ion head stage and/or a Polarographic head stage is substituted for the vibrator assembly (2DITL) and signals fed to an IPA-2 amplifier instead of the PSDA-2 amplifier. Note that SIET/SPET scans can be alternately done with SVET/SLEIS scans using a dual manipulator stage arrangement to hold the head stages and vibrator assembly.
The SPET (Scanning Polarographic Electrode Technique)
The SPET (Scanning Polarographic Electrode Technique) can measure molecular gradients in aqueous media down to a fraction of a percent of concentration in a conductive solution. The electrode is polarized to create a current (I) on a metal or carbon fiber micro-electrode tip to either reduce or evolve molecules present in the experimental solution. The system is programmed to do automatic polarization plots of the electrode at different voltages to determine the best operating voltage. This system is capable of detecting less than a 0.01% change in dissolved oxygen over a 10 micrometer excursion using a user-programmable repetitive positioning algorithm as with the SIET. Virtually any kind of polarized electrode can be used with the system. Cyclic voltammetry control are also programmed in the software. Note: A polarized electrode has an effect on local concentrations at the electrode tip as a function of the reduction/evolution reaction at the electrode tip/media interface. A polarized electrode creates an electrical field. If using SPET with another technique distance between sensors (40 micrometers or more) is necessary to avoid sensor cross-talk.
ASET-LV4 is written for the LABVIEW software environment and utilizes National Instruments, Corp. PC hardware for motion, video and signal processing. Custom programming is available via www.sciencewares.com
Science Wares' ASET-LV4 software was programmed to utilize specifically use Applicable Electronics components, but the software is mostly hardware independent. Most common lab equipment can also be used with the system as long as it has an output in the range of +/- 10 VDC. This system is capable of automated voltage (V) current (I) or optrical measurements in aqueous media utilizing micro scale sensors, a 3D scanning system and a video microscope to analyze metal surfaces for corrosion studies or biological specimens in physiology studies.
The microelectrode is repeatedly moved in a programmable move-wait-measure routine (1,2 or 3D) and also grid or vector scanned or positioned with a 3D motorized, micro-stepping micro-manipulator. (CMC-4 system)
Essentially, the electrode-sampling scheme is either controlled by hardware, as in the case of the SVET or by software as in the case of the SIET (Ion), SPET (Polarographic), and SMOT (Optrode) measurements. The micro-electrode measures two points, one near the sample the other point a fixed distance away from the sample, over a small space (5-50 µm) in a repeatable manner. This software-controlled electrode sampling scheme is positioned at different points on the sample by a scan routine preammed by the user. Programmable grid and vector scans are available as well as single point observations. For the SIET, SPET, SMOT micro-gradients and absolute concentrations are recorded simultaneously.
The system also works as an ion concentration measurement system.
3D stepper motor manipulator can be programmed to step in discrete, vertical increments, at different locations in mud sediments or bio-films with sub-micrometer accuracy.
The major limitation when measuring extra-cellular micro-gradients is the time domain of data collection.
The SIET/SPET technique works with computer control so there is no need for continuous human interaction unless your specimen will move or grow. You can manually map or track growing specimens such as plant roots, root hairs or pollen tubes. Programmable scan sequences provide for an automated scanning system.
The ASET software is the heart of the system.
This software is the culmination of over 30 years of program development. ASET-LV4 is a hardware independent platform, though it is focused on the hardware designs of Applicable Electronics. The programming makes the system flexible and easily configurable. It is written for the WINDOWS OS environment using LABVIEW (National Instruments) software. Collected data is transportable to most commercially available spreadsheet and plotting programs. Video frames are also stored (JPEG, TIFF, BMP, MPEG) for transport to common photo programs. Movies and time lapse functions are also provided in the ASET-LV4 software.
A micro-stepping motor positioning system has also been designed.
Advances in stepper motor technology have allowed us to utilize low cost, commercially available units to precisely position micro-electrodes. Stepper motors have two major disadvantages over linear motor positioning systems. One is that current is applied to the stepper motor when at rest, which produces an electromagnetic field around the stepper motors. The other problem is the stepping (staccato) action of the motor. Software minimizes these effects to a point where they are not a real concern since these are move-wait-measure techniques. Shielding prevents inductive noise pickup from the stepper motors. The motion control system is very versatile and can stand-alone for other micro-positioning needs.
Video images with vector overlays or surface plots over video images provide great visualization of data.
Video is extremely valuable for visualizing the experiment. It also provides a wealth of information synchronous with data collection. Since these techniques are non-invasive the distance between the microelectrode and the surface of what is being measured is critical to quantify data collected near the sample or specimen.