Chapter 4: Imaging the Unseen: Viewing Structures Smaller than Light Waves – NanoInnovation: What Every Manager Needs to Know

Imaging the Unseen: Viewing Structures Smaller than Light Waves

Only he who can see the invisible can do the impossible.

– Frank Gaines

When it comes to nanoimaging, seeing is achieving.

Nanoscale imaging – the ability to view structures and processes at the nanoscale – is a core innovation that drives virtually all other nanoinnovations. As a manager, you should have a basic understanding of how nanoimaging works because so much depends on our ability to view this unseen world. Nanoimaging is used to design semiconductors and car engines, to engineer new types of materials, and study the safety of nanoparticles. Nanoimaging helps confirm the quality and consistency of nanopores in fuel injectors and desalination filters. Nanoimages aid the study of bacteria and viruses, and help diagnose and treat disease at the cell level.

Nanoimaging – better known as nanoscale microscopy – is still evolving. Every form of microscopy, from optical and electron microscopes to magnetic resonance imaging (MRI), is advancing rapidly. If you want to understand what is happening in nanoinnovation, it helps to have a sense of the different types of nanoimaging systems, and what is involved in viewing and manipulating the incredibly tiny structures of the nanoworld.

4.1 What Nano Images Reveal

If you surf the Web, you'll see thousands of nanoscale images displayed in online galleries. Many of these images are truly remarkable. They show us what Nature and humans can engineer at the nanoscale, and provide clues to new types of materials that can be developed and mass-produced by nanoinnovators for commercial applications in applications ranging from medical biomarkers to thin-film solar cells.


The nano portraits of President Barack Obama known as “nanobama” were created by Prof. Jon Hart at the University of Michigan (Figure 4.1a and b). This is one of the most iconic of all the nanoimages produced in the first decade of the current millennium, and was the first nanoscale portrait of a world leader. Nanobama provides an impressive demonstration of how a forest of carbon nanotubes can be transformed into a precise structure – in this case, a presidential portrait.

Figure 4.1 (a) John Hart's “Nanobama” image has become an iconic example of nanotechnology and is probably the world's most widely known nanoscale image. Each nanoscale portrait of Barack Obama is comprised of ∼150 million carbon nanotubes, which is approximately the number of Americans who voted in the 2008 presidential election. (b) Showing how the images were made, described in detail on the Web site. This is a terrific example of the convergence of nanoart and nanoscience, which is discussed in Chapter 5 (images courtesy of A. John Hart, University of Michigan).

DNA Triangles

In Figure 4.2a you can see rows of tiny triangles made from strips of synthetic DNA that look like soldiers trying to find their place in a parade formation. These nanosize pyramids were created by a group of IBM scientists led by Greg Wallraff; using a technique invented by Prof. Paul Rothemund at the California Institute of Technology (Caltech). Much denser clusters have been packed into these channels, offering the potential for new semiconductor circuit designs. In Figure 4.2b, DNA origami triangles were self-assembled in a single step by Dr. Rothemund, who developed the DNA origami process. Each triangle consists of over 200 DNA strands, each incorporating 15 000 nucleotides. Each side of the triangle is ∼120 nm.

Figure 4.2 (a) DNA origami techniques pioneered by Paul Rothemund at Caltech have been used to create an array of triangles made from DNA strands. The triangles are in the process of binding to the wide channels that were lithographically etched in the substrate. This technology holds promise for developing new types of semiconductors (image courtesy of IBM). (b) A close-up image of more complex self-assembled DNA triangles created by Rothemund – each triangle is only about 120 nm on each side (image courtesy of Paul Rothemund, Caltech).

Carbon Onions

One of the best known nanostructures is the buckminsterfullerene, commonly called fullerenes or buckyballs – but what many people do not know is that buckyballs can be nested one inside the other, like Russian matryoshka dolls. The nested form of a buckyball is called a “carbon onion.” A carbon onion is a multishelled nanoparticle consisting of many concentric graphite layers. Carbon nanotubes and carbon onions were discovered by Sumio Iijima in 1980. The images of carbon onions in Figure 4.3a and b were provided by Naiqin Zhao, an award-winning scientist and Chair of the Department of Metallic Materials at Tianjin University in China. Professor Zhao's research group has developed a method for obtaining pure carbon onion nanoparticles and fabricating them in large quantities [1]. Research on commercial applications of carbon onions was previously limited by the inability to fabricate quantities of this unique form of carbon. In recent years, several fabrication methods have been developed, which is opening the door to more aggressive research on functional applications for carbon onions.

Figure 4.3 (a) The concentric shells formed by the multiple “skins” on this hollow carbon onion, which according to the scale measures only about 10 nm in diameter. (b) Showing even more nested layers on this larger carbon onion, which also shows how this nanoparticle is not perfectly spherical, but is typically distorted. This level of detail is revealed or verified only in nanoimages that allow us to actually see the particles (images courtesy of Professor Naiqin Zhao, School of Material Science and Engineering, Tianjin University).

Myriads of Architectures

Nanostructures come in all shapes and geometries. The gallery of images in Figure 4.4 was provided by Dr. Paul Alivisatos' research group at Berkeley, and shows several semiconductor nanocrystals created in their laboratory. These are only a very few of the many thousands of intriguing images produced by nanoimaging systems. Let's take a quick look at how these images were produced, and the technologies that make it possible to view these structures, which are smaller than visible light waves.

Figure 4.4 These TEM images of nanocrystals synthesized by Paul Alivisatos' research group illustrate just a few of the many types of structures and form factors that nanoinnovators are experimenting with, to learn their properties and apply them to commercial applications. Shown here are (a) quantum dot nanocrystals of cadmium selenide (CdSe), (b) CdSe nanorods, (c) CdSe tetrapods, (d) hollow nanospheres, (e) striped binary nanorods made of silver sulfide (AgS) –cadmium sulfide (CdS), (f) CdSe tetrapods with cadmium telluride (CdTe) dendrimer branches, (g) nested Pt@CoO yolk-shell particles, (h) gold-tipped CdS nanorods, and (i) bismuth selenide (Bi2Se3) nanoflowers (images courtesy of Paul Alivisatos, Berkeley National Labs/UC Berkeley).

4.2 Using Electrons Instead of Light to View Nanoscale Structures

Most scientists today use microscopes that capture electrons instead of light waves, because nanoparticles are actually smaller than light waves and can't be viewed with conventional optical microscopes. Electron microscopes produce images that are as much as 100 000 times more detailed than an optical microscope. Nanoscale imaging systems come in dozens of configurations and variations.

The first generation of electron imaging systems required the samples to be sliced ultrathin and held rigid in a vacuum, or coated with a material such as gold. This limited the types of samples that could be imaged. Today, high-performance nanoscopes can image a virus that is suspended in the pore of a carbon matrix. A probe sharpened to one atom at the tip can be used to reposition a single atom. With a small charge applied, the same probe can pluck a single nanowire from a forest of tangled nanowires. Some types of nanoscopes can gently move the tip of the probe across the surface of a soft and delicate sample such as a cell. Nanoscope operators can even use light probes – called optical tweezers – to move nanoparticles.

Compared with what we could do with microscopes a century ago, we live in an imaging renaissance – and yet, if we peer ahead and consider what we will be able to image in 10 or 20 years, we may find that we are actually living in the nanoscopic Dark Ages.

Today, most nanoscale microscopes are generally known as scanning probe microscopes (SPM) because the same tips used to image nanostructures can also be used to manipulate them, although scientists use more than a dozen major nanoscale imaging technologies. There is a veritable alphabet soup of nanoscale microscopes that can be confusing to someone who is not working in the industry – atomic force microscope (AFM), SEM, scanning tunneling microscope (STM), scanning transmission electron microscope (STEM), and transmission electron microscope (TEM) – so let's take a moment to sort out what you need to know, starting with a brief history.

4.3 A Short History of Nanoscale Imaging

The concept of using electrons instead of light waves to image nanoscale samples is not new. The first electron images were microscale, not nanoscale. When the first electron micrographs began to appear in magazines like Life or Scientific American, readers were captivated by the bizarre images of rhinoceros-horned dust mites that live in our homes and close-ups of insect eyes or convoluted grains of pollen. These intriguing and often astonishing images caused us to wonder, “What else is down there?”

The first TEM was developed in Germany by Ernst Ruska and Max Knoll in 1931 and the first commercial TEM was produced by Siemens in 1939. The first electron micrograph of an intact cell was published in 1945. After World War II, the electron microscope was refined into the current form used today.

After the electron microscope was developed, the next major advance was the STM, which was developed to address some of the limitations of the electron microscope, such as having to work with rigid samples in a vacuum.

During 1965–1971, Russell Young and his colleagues at the National Bureau of Standards created an instrument he called the “Topografiner,” which was a precursor to the STM. This led to the invention of the STM by Gerd Binnig and Heinrich Rohrer at IBM-Zurich in 1981. In 1986, Binnig and Rohrer shared the Nobel Prize in Physics with Ernst Ruska, who received half the Prize in recognition of his pioneering work on the electron microscope. Russell Young's contribution was acknowledged in the Nobel Prize citation.

By the late 1980s, large corporations were experimenting with electron microscopes. Robert Dombrowski, a materials scientist and expert in nanocharacterization, worked with the first electron microscopes at Colgate Palmolive. “Back then, atomic force microscopes were still considered Rube Goldberg contraptions. I remember when we first got started, there was so much vibration in the lab, we had to put the microscope on bungi cords. Our first scanning electron microscope cost $400,000 and had vacuum tubes that you had to change like a TV repairman.” Robert was involved in early nanotechnology research involving skin products, electrospinning of nanofibers, development of biodegradable starch-based polymers for packaging applications, and medical applications that included using liposomes to deliver drugs.

How a Scanning Tunneling Microscope Works

The STM captures images by moving a probe across the surface of the sample, close enough to allow electrons to jump from the surface, which allows them to be measured. The tip of the probe does not actually come into contact with the sample. As the probe detects electrons, it sends signals to a computer, which creates an image.

The STM mechanism is both elegant and complex. A sharpened tip only one atom thick at the point is attached to a movable arm, or cantilever. The probes are typically sharpened to one or two atoms at the tip and may be made from silicon, tungsten, diamond, or some other material. The tip of the probe is created by physically or chemically etching away the atoms until one or two atoms remain at the end of the probe. Some probes are tipped with a carbon nanotube.

The cantilever holding the probe is attached to a set of crystals that are each connected to a nanowire. When a minute current is passed through the nanowire, the crystals move. This movement is possible because of a phenomenon called the piezoelectric effect, which causes materials such as crystals or ceramics to move when electric current is applied. By sending electricity (electrons) to the crystals, the crystals can be made to move the cantilever back and forth along the XY-axis. By sending current to all the crystals, the cantilever can move up or down along the Z-axis. More detailed descriptions and diagrams are available online, but this is basically how it works.

4.4 Different Types of Nanoscale Microscopes

When you read about nanoinnovations, or if you work with colleagues involved in nanotechnology, you will come across references to a variety of nanoscale imaging systems. Here are brief descriptions of some of the most common imaging technologies.

The Atomic Force Microscope

In 1986, Gerd Binnig (IBM), Calvin Quate (Stanford University), and Christoph Gerber (IBM/University of Basel) developed the AFM. “Atomic force” refers to the microscope's ability to record forces between individual atoms on the surface and at the tip of the probe, such as the van der Waals force. A primary distinction between STM and AFM is that the STM probe is kept a short distance from the surface of the sample and measures the tunneling current but does not touch the surface, while the AFM probe can gently touch the surface of the sample, or can hover above the substrate sensing vibrations in the probe/cantilever. STMs maintain a constant tunneling electrical current and are limited to conducting materials. STMs offer very high resolution (0.1 nm) compared with AFMs, which offer slightly less resolution (2–10 nm), but can be used with more types of materials and surfaces. Systems with probes that come into contact with the sample have greater wear of the probe tips.

Scientists use scanning probe microscopes to determine the stiffness of nanomaterials, to detect defects in carbon nanotubes or nanopores, to determine the geometries of nanoparticles, and to process variations in electrical, magnetic, or mechanical signals that are continuing to reveal a wide variety of quantum properties and effects (Figure 4.5).

Figure 4.5 A researcher at the European Commission's Joint Research Centre (JRC) uses a scanning probe microscope to analyze nanoparticle samples. The JRC supports nanotechnology research and maintains an inventory of nanoparticles used in commercial products (photo courtesy of the European Commission, Joint Research Centre, Copyright 2010, European Union).

While scanning tunneling microscopes still provide the most detailed images at the nanoscale, there are several other methods for imaging nanoscale objects. Other forms of scanning probe microscopy include magnetic force microscopy (MFM), chemical force microscopy (CFM), Raman microscopy, scanning force microscopy (SFM), electrostatic or electric force microscopy (EFM), and total internal reflection fluorescence (TIRF).

TIRF is a type of microscope that uses an evanescent wave to image a thin region of a sample, usually <200 nm. TIRF was developed by Daniel Axelrod at the University of Michigan in the 1980s. With TIRF, the fluorescence of a single molecule can be observed, which is especially useful for imaging biological structures.

One of the most intriguing and advanced areas of research in nanoscale metrology involves the use of optical imaging systems to image objects that are smaller than light waves, that is, using optical microscopes to image suboptical structures. As paradoxical as this may sound, this approach is helping to overcome some lingering limitations in our ability to view the nanoworld, especially in biomedicine and organic chemistry where structures change shape and interact differently at different stages, and over time.

Measuring Neutrons

A neutron is a subatomic particle found with protons in the nucleus of the atom. Neutrons are part of the nuclear glue that helps stabilize the atom. Neutrons also play a role in nanoscale imaging. Neutron scattering is one of the most complex and interesting methods for analyzing nanoscale structures – especially effective for imaging and time-series analysis of biological structures such as proteins. A sample is bombarded with a beam of neutrons that moves through a series of mirrors to focus on the sample. The neutrons interact with the sample, then scatter and are read by a detector that captures the data. This system allows researchers to deduce the size and shape of the sample, including elements that are less than a nanometer in size. The most sophisticated systems can also produce time series showing the stages of development of biological structures associated with causing or curing a disease.

Most textbooks, articles, and online Web sites depict a protein as an artist's rendering of a spaghetti-like tangle of fibrils. To understand protein behavior – such as how a mutant protein can cause or cure a disease – it is important to see the actual development of the protein from inception to maturation. The only way to capture this is to view actual images of the protein during formation. In May 2011, researchers at the Department of Energy's Oak Ridge Laboratory and the University of Tennessee Medical Center used a neutron-scattering instrument called Bio-SANS (biological small-angle neutron scattering instrument) to image and study the formation of the Huntingtin protein, a spaghetti-shaped mutant protein that causes Huntington's disease. The team's groundbreaking research produced the first images of the protein at the various stages of its formation (see Figure 4.6) [2]. This image clearly shows the spaghetti-like fibrils that are as tiny as one or two-tenths of a nanometer in diameter. More details are included in Chapter 9.

Figure 4.6 A mutant form of the Huntingtin protein thought to cause Huntington's disease. The individual fibrils of the spaghetti-shaped protein are clearly visible in this image captured by the Bio-SANS neutron-scattering system at Oak Ridge National Laboratory in Oak Ridge, Tennessee. This is a rare detailed image showing a protein and its subnanoscale fibrils (image courtesy of Christopher Stanley, Tatiana Perevozchikova, and Valerie Berthelier, University of Tennessee).

Raman Scanning and Microscopy

The Raman microscope is a popular type of nanoscale microscope that measures the Raman effect. It is named after the distinguished Indian scientist Sir C.V. Raman who won the Nobel Prize in Physics in 1930 for his discovery that a change in the wavelength of light occurs when a light beam is deflected by molecules. When you aim a beam of light at an object, light scatters in a pattern that is unique to each molecule, which is useful in identifying the chemical composition of a substance. Each molecule has a unique Raman pattern. Raman testing can be used to measure the vibration, temperature, orientation, and composition of a sample.

The first Raman microscope was invented in 1955 by Harvard graduate Marvin Minsky. Minsky was frustrated by the blurred images that resulted in optical microscopes, caused by a large amount of scattered light. The solution was to use pinhole apertures (nanopores) to focus the light and eliminate the scattering without reducing the brightness. The development of economical lasers in the 1980s facilitated commercial use of Raman technology, which uses lasers to focus the light and amplify the signal.

Raman technology has a wide array of applications in addition to nanoscale spectroscopy. Today, drug enforcement agencies use Raman scanners to detect more than 100 types of drugs. Raman technology is used to detect trace elements of bacteria, chemical pollutants, and explosives. It is possible that one day Raman scanning could analyze the molecular components in human blood, which could make traditional blood tests obsolete [3].

To target diseased cells, scientists seek to identify biomarkers that are unique to those cells. Biomarkers can identify the diseased cells and can also provide an “address,” so a nanocarrier knows where to deliver a drug, protein, therapeutic gene, or other cell-level solution. Biomarkers have been identified for many diseases and offer a promising solution for detecting very tiny early-stage cancer tumors and free-roaming cancer cells that could cause a cancer to metastasize. Some of these biomarkers are being engineered, so they can be identified using Raman technology.

In November 2012, researchers at Washington University announced the development of nanoscale probes that bind to biomarkers at disease sites and light up when hit by an infrared laser to reveal their location. These probes, called BRIGHTs (bilayered Raman-intense gold nanostructures with hidden tags), are made of 20 nm gold nanoparticles covered with “Raman reporter” molecules, and wrapped in a thin gold shell. The BRIGHTs form into polyhedron shapes (with 12 uniform pentagonal faces). The Raman reporter molecules scatter light at specific wavelengths when hit with a laser, and the outer shell and core create an electromagnetic hotspot that concentrates energy in the gap between the gold nanoparticle core and the outer gold shell. This increases the emission from the Raman reporter molecules by a factor of nearly a trillion [4].

4.5 Bringing Biological Nanostructures into Focus

In the past, bioscientists were forced to view only dead biomolecules, immobilized and coated with a material to facilitate viewing. Today we can view and study biomolecules that are still alive and functioning, in their natural environments.

It is easier to treat a disease if we can actually see how a disease is formed, at the nanoscale. Nanomedicine is benefiting from our ability to view and record biological processes in action. Many of the bioscientists interviewed for this book commented on how much has been revealed, including surprise discoveries, as a result of new capabilities to image proteins, viruses, DNA strands, and other structures.

The benefits of this most basic capability are truly profound. For example, researchers on Dr. David Eckmann's team at the University of Pennsylvania Health System use scanning electron microscopes to study how components of the blood accumulate and adhere to the surfaces of materials used in stents and other medical implants. Their goal is to identify better surfaces and coatings for medical implants. One of their goals is to identify materials with the smoothest surfaces, for use in stents where the smooth continuous flow of blood is vital. Nancy Tomczyk, who has been working on Dr. Eckmann's research team since 2008, explained that even the smoothest surfaces used in stents and other medical implants appear to be rough and irregular when viewed under an atomic force microscope. These surfaces have the ability to snag and accumulate plaque and other blood components. “Our goal is to modify the surfaces to make them more biocompatible,” she said. “To do this, we need nanoscale images to see what's really happening.”

It is impossible to overstate the importance of nanoscale imaging to bionanotechnology and nanomedicine in particular. As we delve deeper into the cell-level causes of a disease, we are discovering the impact of very subtle nuances that can only be fully understood by observing them in motion or over time. Many disease-related factors become clear only when directly observed. In this sense, bionanoimaging is a critical gateway to next-generation treatments and cures.

Until the mid-2000s, most bionanotechnology structures and processes had to be visualized using artist's concepts and computer simulations because scanning probe microscopes were limited in their ability to image “soft” or “morphing” structures in real time. Some samples needed to be hardened with a coating, or imaged in a vacuum. However, most biological processes occur over time (requiring time-lapse images or videos to fully understand). It is difficult to selectively follow the dynamic behaviors and interactions of structures inside a cell, especially when those structures are constantly moving and changing shape. For example, a protein can change shape in a few billionths of a second. Nanoscale structures such as viruses and DNA molecules are also difficult to isolate individually. Thanks to nanoinnovations, scientists are rapidly moving from theory and simulation to direct observation.

In 2012, a group of UK scientists captured the most detailed image to date of the DNA molecule – the iconic double-helix shaped molecule that contains the code that defines us as humans (Figure 4.7). Although scientists had previously theorized that the ladder-shaped molecule could twist clockwise or counterclockwise as a “right-handed” or “left-handed” molecule, this was visually confirmed by the DNA images captured by the research team. To capture the image, the team adjusted the vibration of the cantilever in the scanning probe microscope to extract a greater level of detail. They used a JPG Nanowizard® AFM system, and “homemade” variations that allowed them to couple the system to inverted optical systems that extend the capabilities of the AFM instruments. This allowed the team to study and manipulate single biomolecules at a spatial resolution of ∼1 nm, which enabled them to study the function and behavior of biomolecules and processes in their natural (aqueous) environment.

Figure 4.7 These images captured in 2012 are the most detailed images produced to-date of a DNA molecule. The images show two “grooves,” which are seen as major and minor grooves. The major groove is the space between the twisting curves of the ladder and the smaller groove is the space between the two strands of DNA that make up the DNA ladder (image courtesy of Carl Leung).

This bioimaging breakthrough was achieved by Dr. Bart Hoogenboom, Dr. Carl Leung, and 10 research colleagues at the London Nanotechnology Centre at University College London [5]. This is exactly the kind of innovative thinking that is needed to open the window to biological processes that were formerly hidden from view. It is a marvelous example of how faculty and student researchers are pushing the boundaries of bioimaging to reveal those parts of the nanoscale landscape that are still hidden from view.

4.6 Using Optical Imaging Systems to View Nanoscale Structures

Most nanoscale microscopes measure electrons instead of light because nanoscale objects are smaller than the bandwidth of light waves. While you may have read that it is impossible to optically image a nanoscale object, scientists are working on methods for producing suboptical images of nanoscale objects that are smaller than light waves. Typically, the smallest scale that can be optically imaged is ∼200 nm, which is the lower focus limit of visible light waves. However, there are some ingenious techniques being developed that have improved the resolution of optical microscopes to view objects that are 50 nm or less.

A traditional optical microscope (which you probably used in a school biology class) uses a tube with two or more glass lenses to magnify an illuminated sample for viewing or imaging. The sample is fixed on a glass slide or sandwiched between two thin glass plates. The sample may be stained to reveal its features – especially if it is clear or transparent, like a human cell. It may be suspended in liquid, especially if it is a cell or bacterium that lives in an aqueous environment. It may be coated in a material to keep it rigid.

You have never seen a nanoparticle with your naked eye because nanoscale objects are smaller than visible light waves and smaller than the eye can perceive. To view nanoscale samples, we need to be able to see things smaller than 100 nm, while the lowest limit of visible light is about 400 nm and the best resolution of an optical microscope is around 200 nm. That would seem to rule out optical microscopes for nanoscale imaging.

Actually, there are ways to make nanoparticles optically visible, using some very clever tricks. One method is to use a “superlens” or “hyperlens” made of metamaterials to capture images of objects below the lower limit of visible light waves. A superlens is a lens that can view features smaller than the diffraction limit of light. Several types of metamaterials including gold have been used to magnify nanoscale objects to capture an optical image. Scientists have used surface plasmons and a superlens to capture a type of light wave called evanescent waves that are normally scattered and lost in conventional optical microscopes.

Research in this field is fairly recent. In 2003, Sir John Pendry, whose seminal writings and research provided much of the foundational work in this field, proposed a design using a curved cylindrical lens to achieve magnification at the nanoscale. The concept was demonstrated by research teams in 2007. In the past few years, various types of superlenses have been developed, including a metamaterial superlens made from an array of nanowires. Several teams have also used an array of nanoholes to concentrate the light so as to improve the resolution.

In January 2013, a team of researchers at UCLA created a method for viewing viruses and other nanoscale objects by using tiny liquid lenses that self-assemble around microscopic objects [6]. The team was led by Aydogan Ozcan, Associate Professor of Electrical Engineering and Bioengineering, who is also on the faculty of the surgery department at UCLAs David Geffen School of Medicine. The team was able to detect and image nanoparticles including viruses and viral particles on a glass substrate. Their system used an LED to illuminate a nanolens object assembly and incorporated a silicon-based sensor array, which is used in cell phone cameras. The system produced holograms of the nanoparticles, which were rapidly interpreted using a personal computer. The prototype was not of as high resolution as an electron microscope, but the field of view was >20 mm2, which is useful in detecting and imaging nanoparticles that are sparsely distributed. This is only one example of the creative efforts underway to image nanoscale objects using new types of optical microscopy.

Failure to accept the obvious is a hallmark of any innovator. It is a testament to the core values of nanoinnovators that a handful of scientists refused to accept the logic that optical microscopes could not be used to image nanoscale structures because the structures are smaller than conventional light waves. This is the type of ingenuity that makes nanoinnovation such an exciting and promising field.

4.7 Probing the Future

One of the most exciting consequences of being able to view and image the nanoscale world is the ability to manipulate atoms, molecules, and other nanoscale structures. Most nanoscale imaging systems today are called SPM because they include the ability to probe and manipulate structures, as well as image them.

The most famous and earliest demonstration of this capability was the creation of the IBM logo using individual atoms on November 11, 1989, when Don Eigler and his colleagues at IBM's Almaden Research Center positioned 35 Xenon atoms to form the IBM logo (Figure 4.8).

Figure 4.8 (a) This IBM logo was created by Don Eigler in 1989, using a custom-built microscope to precisely place 35 xenon atoms on a nickel substrate. (b) In 1993, Dr. Eigler's group arranged a circle of 48 iron atoms to form a “quantum corral” on a copper substrate – the progression is shown here. Quantum corrals are structures built atom by atom on an atomically clean metallic surface that result in “electron waves” that can be imaged as shown in this figure (images courtesy of IBM).

Progress in this field has been remarkable, considering that we have only been able to manipulate atoms and molecules for two decades. Today, nanoscientists routinely use a variety of probes for nanoscale manipulation. Most of these are point-to-point probes tipped with a few atoms – as small as one atom at the tip of the probe. To manipulate atoms, a force is applied to the probe, which attracts or repels or otherwise transforms the sample.

Using Light to Manipulate Particles

Another type of probe is called a nanotweezer – similar to cosmetic tweezers used to shape eyebrows, although these tweezers are 100 000 times smaller than a human hair. Nanotweezers can be made from carbon nanotubes and nanowires, tungsten, single-crystal silicon, or other materials.

One form, called “optical tweezers,” actually uses light as a force to manipulate objects. In 1997, the Nobel Prize in Physics was awarded to Steven Chu, Claude Cohen-Tannoudji, and William D. Phillips for their seminal research on optical tweezers, including the “development of methods to cool and trap atoms with laser light,” which derived from earlier work by Bell Labs scientist Arthur Ashkin.1)

Dr. Heike Noethe, who edited this book at Wiley-VCH, used optical tweezers earlier in her career to position live cells in certain patterns on surfaces. She recalls, “It felt more like a trap than a tweezers to me because when the laser surface approaches a cell, the cell is pulled into the focus and is ‘trapped’ there.”

Dr. Noethe describes an optical tweezer as “a highly focused beam of light that is able to trap and manipulate individual atoms, molecules, viruses, bacteria and other particles at the micrometer, nanometer and sub-nanometer scales.” This type of probe is particularly useful for manipulating and imaging biostructures and measuring events such as DNA replication and RNA transcription.

Developing Better Nanoprobes

More robust probes are needed to extend the life and lower the operating costs of scanning probe microscopes. Better methods are also needed to prevent the probes from getting contaminated or coated with atoms from the materials being probed.

Many of these challenges are being actively researched at the Nanoprobe Lab in the Nano/Bio Interface Center (NBIC) directed by Dawn Bonnell at the University of Pennsylvania. Dawn is Trustee Professor of Materials Science and Engineering and Vice Provost-Research at UPENN. The Nano/Bio Interface Center facility is working with industry and academic partners to develop next-generation probes, and makes these innovations available to the research community before they are commercialized.

Professor Bonnell described some of the innovations being researched at the Nano/Bio Interface Center that involve next-generation probes and related technologies. The Bonnell group recently characterized the properties of a single layer of protein on an electrode, information that is critical to the development of transformational biochemical sensors. Another project provided new insights into the ferroelectric properties of 50 nm thin-film heterostructures, unique materials that may enable new devices for energy harvesting. Yale Goldman and his team combined total internal reflectance and microfluidics to map how individual proteins move in 3D. Dennis Discher and his colleagues combined atomic force microscopy and fluorescence microscopy to simultaneously probe mechanical properties and chemostructural properties of biomolecules – proteins and ribosomes. Another Nano/Bio Interface Center team including the Savan, DeGrado, and Therien groups developed a new family of artificial proteins that respond to light. Research groups directed by A.T. Charlie Johnson and Dawn Bonnell are contributing to this research. In collaboration with the Johnson and Bonnell groups, these new proteins are being developed into chemical sensors. Along the way, a new mechanism for turning light into electricity (that involved surface plasmons) was discovered. These are only a few examples of research underway at the Nano/Bio Interface Center, which is now benefiting from the completion in 2013 of the University of Pennsylvania's Krishna P. Singh Nanotechnology Center.

“The development of probes that let us ‘see’ atoms has already transformed our view of the nanoscale world and led to new technologies. Future probes will be multi-function probes capable of detecting and measuring multiple parameters in samples,” Bonnell predicted. “Instead of a specialized microscope for each condition we want to measure, we will have multi-function microscopes. Just as nanoscale imaging advanced the field of nanotechnology research, new capabilities in nanoscale probes will result in a similar transformation, taking our scientific understanding and our ability to manipulate matter to a new level. Our research at the Singh Nanotechnology Center will contribute to this transformation.”

4.8 Nanoscopes on Mars

If we focus our gaze on the future, one of the most fascinating applications for nanoimaging systems involves space exploration. NASA and its academic and corporate partners have produced some fascinating innovations for conducting nanoscale analysis – on other planets!

In 2007, the Phoenix Mission to Mars carried an Atomic Force Microscope to Mars as part of an instrument package that helped confirm the existence of water on Mars. According to William Boynton, lead scientist for the Thermal and Evolved-Gas Analyzer (TEGA), this was the first time Martian water was “touched and tasted.” Water on Mars was previously observed and detected, but this was the first evidence from direct soil sample analysis. This finding was hugely important because one of the goals of Mars exploration was to determine whether surface water ice is available in a form that can be used as a source of power (in fuel cells) and drinking water, which is essential to a human Mars mission.

The AFM used in the Phoenix was contained in an instrument package called MECA (microscopy, electrochemistry, and conductivity analyzer) (Figure 4.9). MECA included four wet chemical reaction cells, an optical microscope, and an atomic force microscope, and was designed to analyze soil samples from a Mars crater thought to contain water ice. In addition to the nanoscale analysis, stereo photos taken by Phoenix revealed an “ice-dominated terrain as far as the eye can see,” according to Mark Lemmon, lead scientist for the surface imaging camera [7]. The ability to analyze soil and water on other planets using both optical and atomic force microscopes is amazing, considering that half a century ago, the best we could do is peer at other planets through telescopes – and now we are sending robots to take pictures and analyze the environment down to the nanoscale!

Figure 4.9 (a) This image showing the eight tips of the AFM on the Phoenix Mars Lander's MECA system was taken before the space probe was launched. These probes mapped soil samples from the surface of Mars in three dimensions. (b) This image shows the scoop filled with dirt preparing to drop it onto the exposed sample wheel directly below – the wheel rotates to the position of the AFM probe that will analyze the sample (images courtesy of the Jet Propulsion Laboratory, NASA).

The AFM used in the Phoenix Mars mission was designed by a Swiss Consortium that developed the first AFM for the Mars Surveyor 2001 mission. The AFM had a resolution of 10 nm and an overall image range of 10 µm. Most space probes are equipped with redundant systems. The Mars AFM included an AFM chip with eight adjustable sensors and cantilevers on one chip. This gave the Phoenix team on Earth eight sensors, so if one became broken or dirty it could be broken off to access the next sensor on the chip. The Mars AFM system for analyzing Martian soil used a robot arm to scoop and load soil onto a sample plate on a wheel. The wheel was rotated to the scan range of the AFM, which scanned the sample with the selected cantilever on the AFM chip. The images were stored and transmitted to Earth. This was one of the first attempts to capture AFM images from another planet. Though everything did not work perfectly, the effort should be vigorously applauded for its ingenuity.

When we think of the difficulties and risks involved, it was a miracle that the AFM made it to Mars intact. This groundbreaking – or rather, ground analyzing effort – was an incredibly complex undertaking. An important purpose of this mission was to prove that an AFM could survive the space trip and deliver an image, which was in fact demonstrated (a fuzzy image of a particle of dust was captured). The AFM was not expected to produce a gallery of nanoimages, but hopefully will do so on a future mission. The primary value of the AFM on the Phoenix mission was to demonstrate proof of concept. The instruments did not produce a gallery of nanoscale images, but it did produce a test image and imaged a grain of Martian dust, not spectacular but nevertheless remarkable considering the obstacles involved.

Consider what was needed to deliver the nanoscale microscope to Mars. First, the AFM had to survive the vibration and shocks involved in the rocket launch from Earth, and then the impact of the Mars landing. The system had to account for the fact that the Martian atmosphere is mainly carbon dioxide, which can cause direct atmospheric discharges, so all electrical voltages had to be limited to a maximum of 15 V. Furthermore, the Martian temperature ranges between –90 °C (−194 °F) and 10 °C (50 °F). To process the signals, digital-to-analog converters and amplifiers had to be incorporated. During the trip to Mars, solar radiation and bombardment by heavy ions could damage or destroy the CMOS chips used on the spacecraft's circuit boards, including the AFM system. These are just a few of the daunting challenges the Mars Phoenix team had to anticipate and compensate for, in order to deliver the AFM imaging system to the surface of the Red Planet. The excellent writer Andrea Thompson wrote a great series of articles on the Phoenix mission for, which includes images from the mission. You can find these on More information on the AFM instrument package can be found online at (note: FAMARS stands for first atomic force microscope on MARS).

Of course, the real quest on Mars involves the search for life and nanoimaging is bound to play a role as nanospace explorers look for Martian bacteria, viruses, and possibly alien nano-life forms.

4.9 The Future of Nanoscale Imaging

In the decade ahead, we can expect to see improved techniques for nanoscale imaging, manipulation, and fabrication. The results will include better functionality such as sharper resolution and control, new techniques for imaging across the electromagnetic spectrum, and more affordable systems that are needed to mainstream nanotechnology, especially in schools.

There are many scientific goals and innovations that require better nanoimaging to move forward. For example, one of the areas where we can expect to see some dramatic innovation involves nanovideo.

One promising technology involves a technology called 4D electron microscopy, which was developed by Ahmed Zewail and his colleagues at Caltech in the mid-2000s (note: the “fourth dimension” referred to in 4D is time). Dr. Zewail won the 1999 Nobel Prize in Chemistry for pioneering the science of femtochemistry – the use of ultrashort laser flashes to observe fundamental chemical reactions. These laser bursts can occur at one millionth of a billionth of a second. Capturing a series of femtosecond images provides sequenced visualizations of atomic oscillations that are the equivalent of nanoscale motion pictures. This imaging approach can capture images of atoms forming or breaking apart from molecules, changes over time in physical and biological matter, oscillations of individual atoms and materials, and so on. A patent based on this framework was granted to Caltech in 2006.

Dr. Zewail2) and biology professor Grant Jensen [8] are leading research programs that are expanding this research to include biological imaging within cells. There are some key challenges that still need to be overcome such as preserving samples in the vacuum of the electron microscope, extracting three-dimensional information from the images, and recording information before samples are destroyed by the high-energy electron beam.3)

As imaging systems are improved and refined, more nanoinnovations will result. There is an indisputable connection between nanoinnovation and our ability to image the nanoscale. It is logical to assume that increased investments in nanoimaging research will pay significant dividends in nanoinnovation across all areas of science.

As much as we've achieved in our ability to image and manipulate the nanoscale world, we're just getting started. There is much more to come – and lots of nanoscopic challenges ahead. The most intriguing question in the field of nanoimaging is: What haven't we seen yet?



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  2. 2. Oak Ridge National Laboratory (2011) Neutrons provide first sub-nanoscale snapshots of Huntington's disease protein. Press Release No. mr20110518-00, May 18.
  3. 3. Aiyar, S.A. (2011) Raman effect: fingerprinting the universe. India Times, May 9.
  4. 4. Gandra, N. and Singamaneni, S. (2012) Bilayered Raman-intense gold nanostructures with hidden tags (BRIGHTs) for high-resolution bioimaging. Advanced Materials, 25, 1022–1027.
  5. 5. Leung, C., Bestembayeva, A., Thorogate, R., Stinson, J., Pyne, A., Marcovich, C., Yang, J., Drechsler, U., Despont, M., Jankowski, T., Tschöpe, M., and Hoogenboom, B.W. (2012) Atomic force microscopy with nanoscale cantilevers resolves different structural conformations of the DNA double helix. Nano Letters, 12, 3846–3850.
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  7. 7. NASA Mars Lander (2008) NASA Spacecraft Confirms Martian Water, Mission Extended. NASA webpage (July 31, 2008).
  8. 8. Caltech Media Relations (2008) Caltech 4D microscope revolutionizes the way we look at the nano world. Caltech News Release, November 20.