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Innovations in Imaging and Microscopy

Nongjian Tao, PhD, director of the Center for Bioelectronics and Biosensors at Arizona State University’s Biodesign Institute and professor in the School of Electrical, Computer, and Energy Engineering, talks to contributing editor Tanuja Koppal, PhD, about a new technique called plasmonic-based electrochemical microscopy (P-ECM) developed in his lab for imaging localized chemical reactions from single nanoparticles. He talks about the advantages of this technique when compared to conventional optical microscopy and scanning electrochemical microscopy (SECM) and its potential uses in diverse areas.

by Tanuja Koppal, PhD
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Q: Can you give us some details about your institution and your laboratory?

A: The Biodesign Institute at Arizona State University is focused on use-inspired research. It has 12 centers, each covering a very broad spectrum of research areas, from infectious diseases to developing new detection technologies. My lab is in the Center for Bioelectronics and Biosensors, which focuses on developing new detection technologies.

We have nearly 40 researchers—who include 15 PhDs who are faculty, research professionals, and postdoctoral fellows—and about 25 graduate students. All of them come from very diverse academic backgrounds. I have a background in physics and electrical engineering, and others come from chemical engineering, bioengineering, mechanical engineering, material science, computer science, and chemistry. It’s all cross-disciplinary research, and so we need people with different academic backgrounds to solve a particular problem. In order to build a new detection technology, we need concepts from physics, we need to build the device with help from mechanical and electrical engineers, we need computer science people who can write the software code and chemists to modify the surfaces for detection, and we need people with a biomedical background to help us with the applications.

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Q: How do the new detection technologies that you are developing help fill existing gaps?

A: We are very interested in imaging technology because it tells us not only that something is happening but also where it is happening. It gives you the spatial location that is extremely important, especially in biology, when you are studying cells and tissues. Just like with DNA and protein microarrays, where the homogenous surface can be divided into individual elements, with imaging you can measure each individual element on the sample surface simultaneously to get a highthroughput (HT) detection technology.

Multifunctional plasmonic imaging.  Experimental setup. Incident light is directed onto a glass slidesupported metal film via a high numerical objective(21) or a prism (not shown here), and the reflected beam is detected with a CCD camera. At a proper incident angle, surface plasmons, or collective motion of free electrons in the metal film, are excited, which is referred to as surface plasmon resonance (SPR), creating an evanesce wave (near field) near the surface of the metal film. The interaction of the near field and sample on the surface creates a plasmonic image. The sensitive dependence of the plasmonic image on the local refractive index near the metal film and surface charge density of the metal film has led us to develop morphology, chemical reaction and electrical impedance imaging capabilities. In addition, the setup is compatible with conventional bright field and fluorescence imaging, allowing one to combine strengths of different imaging principles into a single setup. Right: Bright field (a), plasmonic (b), and electrical impedance (c) images of 3 viruses. Note that the contrast of the bright field image is too low to reveal the viruses.

We are particularly interested in developing techniques that can give chemical information such as chemical reactivity and charge, which are different from a conventional imaging approach. Most of the traditional imaging and microscopy techniques provide information about the morphology of samples. Optical microscopy, for instance, provides very limited chemical information and does not tell you what it is that you can see. Fluorescence microscopy is also a very powerful technique, but using labels has its own problems, since labels can change the properties of a molecule. Moreover, fluorescence emission tends to be weak, and so you need to accumulate a large amount of signal, which takes time. Often with fluorescence you cannot get a fast dynamic detection process that takes just microseconds. Hence, we are focusing on developing label-free detection technologies that are faster and noninvasive for the study of biological samples.

Q: Can you talk about this label-free approach and where it can be used?

A: We have developed techniques to detect electrochemical reactions. The best-known example of an electrochemical detector is the glucose sensor (glucometer). However, traditional electrochemical methods do not tell us the location of the current generated. To get around this problem, Professor Allen Bard at University of Texas in Austin invented the scanning electrochemical microscope (SECM). He used a microelectrode to scan the entire surface of the sample to map the localized electrochemical current.

What we have created is a different approach called plasmonic-based electrochemical microscopy, or PECM, where we use visible light instead of a microelectrode to scan the sample surface. However, visible light is not so sensitive to most electrochemical reactions. So we combined it with surface plasmon resonance (SPR), which is very sensitive to electrochemical reactions. We use light to excite SPR on the surface of an electrode (we use gold-coated electrodes), and that gives rise to a large SPR signal. We image this signal on the electrode surface, and that gives us information about the localized electrochemical reaction taking place in the sample. (See accompanying figure and caption.) The conventional SECM approach scans the surface line by line using a microelectrode, whereas with light you can look at the entire surface almost simultaneously. This enables us to perform measurements in a microsecond time scale. Also, using light instead of a mechanical device makes the technique noninvasive and does not destroy the sample surface.

Q: What are the current limitations of P-ECM? What improvements are you working on?

A: We have demonstrated how we can image localized electrochemical reactions using this technique. We are now looking to expand it to plasmonic-based electrochemical impedance microscopy (P-EIM). Impedance is another quantity that is very useful to characterize samples. It measures how different parts of a sample show charge response to changes in the electrical field. We have used this to study ion channel activity in cells.

In summary, what we are providing is a technology platform, and people can certainly use it for different applications. It’s a new imaging approach that provides capabilities to look at localized chemical reactions and charge distribution, and it can also be used to study localized molecular binding processes. For instance, we have studied membrane protein binding activity while the proteins are still in the lipid environment, in the cell. Traditionally you have to extract membrane proteins from the lipid environment in the cell in order to study them. This takes a lot of work and effort, and, more important, when you extract the protein its structure and function can change. With PECM we can image the cell response and look at the localized binding events at the organelle level, such as in the mitochondria. We are looking to achieve higher sensitivity and higher spatial resolution using this technique. We are also coupling this technique with fluorescence, bright-field, and atomic force microscopy and looking to commercialize it as well.

Q: What do you need in terms of samples and expertise to work with this technique?

A: It’s very similar to conventional optical microscopy. The only difference is that we coat the microscope cover slide with a thin layer of gold film because we need to excite the free electrons from the surface, using light to give rise to a SPR signal. If you are familiar with optical microscopy, then you can work with this technique too. The gold-coated slides can be purchased from commercial vendors, or if you have an evaporator you can coat them yourself. You need standard cell culture protocols for working with cell lines and primary cells; [you] load the cells onto the gold film, and that’s all there is. You will need to load new software for image processing, and that may take some time to learn.

Q: What are some of the applications beyond life sciences?

A: This is a wonderful technique to characterize the catalytic reactions and chemical properties of nanomaterials. We have used this technique to detect trace amounts of chemicals, specifically explosive chemicals that collect on a surface. We were able to image the chemical reactions of a few particles of trinitrotoluene (TNT) on a fingertip using this technique, although we could not see those particles with an optical microscope. We are trying to make this technique faster, better, and more accurate to look at other diverse applications.

Dr. Nongjian Tao is the director of the Center for Bioelectronics and Biosensors at the Biodesign Institute at Arizona State University. He joined the ASU faculty as a professor of electrical engineering and an affiliated professor of chemistry and biochemistry in August 2001. Before that, he worked as an assistant and an associate professor at Florida International University. He has patents, has published more than 200 refereed journal articles, and has given more than 200 invited and keynote talks worldwide. He is an elected fellow of AAAS and the American Physical Society. His current research interest includes mobile health devices, chemical and biosensors, molecular electronics, and nanoelectronics.