|
 For
a general introduction to the work we do,
visit Dr. Baur's page on the Department of
Chemistry web site.
The main focus of our research effort is to develop the
Scanning Electrochemical Microscope (SECM)
for imaging dynamic, living biological systems. Our goals are to improve the resolution of the
technique, adapt the instrument for long-term imaging in cell growth media, and use the instrument to
investigate the biological processes occurring as neurons develop and make synaptic connections.
This work is truly interdisciplinary in nature, as it requires expertise (or at least an interest and a
willingness to learn) in such diverse areas as electronics/instrumentation, nanoscale and microscale
fabrication, and physiology. The complexity of the work and the variety of challenges presented make
this a fascinating and rewarding area of research for chemistry, biology, and biochemistry/molecular
biology majors alike.
Different aspects of our work is described below. For additional technical details, consult the
original publications.
 Instrument Improvement
The SECM has only recently become commercially available from a handful of vendors such as
CH Instruments,
Uniscan Instruments, and
HEKA, but none of
these off-the-shelf instruments is designed for biological applications. In our application, we have the
additional requirement that the SECM be placed on the stage of an inverted optical microscope so that the
cells may be independently monitored during SECM imaging. For these reasons, we must develop our own
hardware, software, and techniques in order to use the instrument for
high-resolution chemical and topographical imaging. In conjunction with
Dr. David O. Wipf, a collaborator at
Mississippi State University,
we have developed a method for imaging cultured model neurons directly in the growth media (see Figure
at right). This technique uses the impedance of the SECM tip (the sensor used for imaging) to maintain a constant distance between it
and the substrate (the sample being imaged). By recording the vertical displacement of the tip,
the true topography of substrate is obtained. Because this type of imaging does not require a redox
mediator, and because it can be done directly in the cell growth media, this advancement will enable long-term investigations of release during neuronal development.
However, it is still necessary to adapt the BioSECM so that it can operate under incubation conditions (i.e. 37°C,
constant humidity, and 5% CO2). Therefore, a current project is the development of an incubation chamber suitable for
maintaining the health of the cell cultures during long-term imaging experiments.
A second
instrument improvement project involves the incorporation of
fast-scan cyclic voltammetry into the BioSECM instrument.
Fast-scan cyclic voltammetry allows for imaging O2 and pH, both vitally important to cell function and development.
This is a fairly complex instrumentation project that involves both software and hardware development in
addition to the usual biology and chemistry aspects of our work.
 Sensor Development
Because the tip provides the specificity for chemical imaging and determines the spatial resolution of the technique,
a primary focus of our work is the development of very small and chemically selective
sensors. The smallest sensors we presently use, carbon ring nanosensors and microsensors (see image at
right), do not work well with the impedance-based imaging because the distance dependence of the
impedance signal is not detectable until the tip is within only a few hundred nanometers of the
substrate. As a result, frequent (and highly undesirable) collisions between the tip and the cell occur.
A current project is to modify these sensors to reduce the background impedance so the small impedance
change can be more easily detected. Success with this project will mean than we will be capable of imaging neurotransmitter release and cell morphology with
unprecedented resolution.
Another sensor development project involves the construction and characterization of multifunctional sensors.
These sensors incorporate more than one type of sensor into a single tip, and are therefore capable of imaging multiple species simultaneously.
The construction of these devices is relatively simple, but the necessity of having two or more independent sensors in
very close proximity
introduces the potentially serious problem of cross-talk. One important aspect of this project is to develop theoretical models for this
cross-talk so that conditions may be found that minimize this interaction. This project requires the use
of a sophisticated simulation package (FEMLAB) for solving systems
of partial differential equations that describe diffusional transport to the multifunctional sensors.
 Applications
Neurodevelopment and synaptogenesis. One distinct advantage of the BioSECM is its capability for imaging both
cell morphology and neurotransmitter release. Our initial test system for this work is the PC12
cell
line. These cells, which are derived from a rat adrenal cell tumor, express a neuronal phenotype
when exposed to nerve growth factor (NGF), and are therefore used as model systems for studying neuronal
growth and development.
The figure to the right shows the release of
neurotransmitter from an undifferentiated PC12 cell in response to depolarization by K+.
This release was detected by a 5 µm diameter carbon SECM tip set to a potential at which common
catecholamine neurotransmitters are oxidized. Each spike in
the signal corresponds to the release of neurotransmitter (dopamine and/or norepinephrine) from an
individual vesicle--about 200 zmol
(200x10-21 moles) or only about 100,000 molecules. We wish to investigate how this neurotransmitter release, both its time
course and spatial distribution, changes during neuronal growth and the formation of synapses with other cells, a
process known as synaptogenesis. By studying these processes, we can learn fundamental mechanisms involved in neuronal
development. Presently, work in this area involves cell culture and the application of new imaging
techniques to the cultured model neurons.
Neurodegeneration. The types of microsensors and nanosensors used for the
BioSECM are also capable of producing short-lived chemical species that are known exert oxidative stress on cells.
These reactive oxygen species (predominately superoxide, O2-, hydroxyl radical, •OH, and hydrogen peroxide, H2O2)
are suspected of playing a role in neurodegenerative diseases such as
Alzheimer’s Disease and
Parkinson’s Syndrome.
We plan to develop techniques for generating reactive oxygen species with the BioSECM. We can then
focally apply a variable dose of the reactive oxygen species and then study changes in morphology
and neurotransmitter release by subsequent imaging with the same sensor.
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