Each of the 100 trillion or so cells that make up your body is the ultimate molecular machine. Cells can take in nutrients, convert these nutrients into energy, carry out specialized functions, and reproduce as necessary. Each cell stores its own set of instructions for carrying out each of these activities. A cell, although measuring only 10 micrometers on average, is a hugely complex system. Researchers are not even coming close to being able to build artificial cells that duplicate their biological archetypes. That doesn't mean they are not trying. But before you can duplicate something you first need to understand how it works and what its components are. In the case of biological cells, that mostly means understanding the role of proteins which carry out the bulk of cellular activity, and the molecular networks in which they operate. To probe these processes, scientists have developed single-molecule imaging techniques that allow them to observe, measure, and manipulate some the key processes at the very core of life. Scanning probe techniques allow imaging of single molecules on surfaces, and specialized optical techniques enable their characterization in complex environments. Single molecule biomechanical studies have been used to manipulate individual molecules and to measure the force generated by molecular motors or covalent bonds. Understanding and manipulating cellular function at the level of individual molecules is within reach. One of the requirements of single molecule techniques is the ability to follow an individual molecule for sufficiently long times in solution. However, it is a challenge to cope with the effects of Brownian motion (the random motion of small particles suspended in a gas or liquid) on this time scale. To meet this challenge, more recently, biomolecules have been encapsulated inside lipid vesicles, which are themselves tethered to a surface. Now, a novel nanocontainer offers controlled permeability functionality which not only is desirable for single molecule imaging but also is a very important property for micro- and nanodevices and for delivery of drugs or imaging agents in vitro and in vivo.
Whenever you read an article about nano this or nano that, chances are you come across a large number of confusing three-letter acronyms - AFM, SFM, SEM, TEM, SPM, FIB, CNT and so on. It seems scientists earn extra kudos when they come up with a new three-letter combination. One of the most important acronyms in nanotechnology is AFM - Atomic Force Microscopy. This instrument has become the most widely used tool for imaging, measuring and manipulating matter at the nanoscale and in turn has inspired a variety of other scanning probe techniques. Originally the AFM was used to image the topography of surfaces, but by modifying the tip it is possible to measure other quantities (for example, electric and magnetic properties, chemical potentials, friction and so on), and also to perform various types of spectroscopy and analysis. Today we take a look at one of the instruments that has it all made possible. So far, over 20,000 AFM-related papers have been published; over 500 patents were issued related to various forms of scanning probe microscopes (SPM); several dozen companies are involved in manufacturing SPM and related instruments, with an annual worldwide turnover of $250-300 million, and approx. 10,000 commercial systems sold (not counting a significant number of home-built systems).
A little trivia you might not know about: The Beatles are responsible for computer tomography (CT). No, John, George, Ringo, and Paul weren't moonlighting scientists, but you could argue that their musical success was directly responsible for the invention of CT. The technology, which produces cross-sectional images, or slices, of internal body structures has become an extremely important diagnostic tool in the field of medicine. British engineer Godfrey Hounsfield conceived and developed the idea at Thorn EMI Central Research Laboratories in England in 1967. He was able to spend four years developing the scanner into a practical clinical tool with funding made possible by the huge profits EMI earned from the sale of the Beatles' records. The first clinical CT scanners, which were installed in the mid 1970s, were dedicated to head imaging only, but whole body systems became available in 1976. By 1980, CT became widely available. There are now about 30,000 CT scanners installed worldwide. The first CT scanner developed by Hounsfield took several hours to acquire the raw data for a single scan and took days to reconstruct a single image from this raw data. The latest multi-slice CT systems can collect up to four slices of data in about 350 ms and reconstruct a 512 x 512-matrix image from millions of data points in less than a second. An entire chest (forty 8 mm slices) can be scanned in five to ten seconds using the most advanced multi-slice CT system. Over the past three decades, CT has made significant improvements in speed, patient comfort, and resolution. Today, CT scans are used to image bone as well as soft tissues. Because the digital image is sharp, focused, and three-dimensional, many structures can be better differentiated than with standard X-rays. CT has also been widely used for non-medical purposes, such as archaeology, soil science, biology, aviation security and non-destructive materials testing. However, other fields, such as chemistry, have not yet taken advantage of tomographic techniques. But, say chemists, the advent of nanotomography is beginning to change that. With recent advances in tomography, it has become possible to achieve experimental resolution at the nanoscale, which has enabled scientists to determine the three- dimensional (3D) distribution of materials.
There is a significant and growing need across the research and medical communities for low-cost, high throughput DNA separation and quantification techniques. The isolation of DNA is a prerequisite step for many molecular biology techniques and experiments. Although single molecule techniques afford extremely high sensitivity, to date, such experiments have remained within the confines of academic and research laboratories. The primary reasons for this state of affairs relate to throughput, detection efficiencies and analysis times. For example, in a conventional solution-based single molecule detection experiment, one can only detect approximately 10,000 molecules per minute, or one molecule every 6 milliseconds. While this may sound a lot, consider that a small drop of water (ca. 5 ml) contains approx. 1.67 x 10e23 molecules, that is 1.67 followed by 23 zeros. At that speed you need over 100 trillion years to detect all the water molecules in this single drop. Using a novel nanopore array developed by researchers in the UK, expect to be able to detect up to 1 million molecules simultaneously in the same 6 millisecond time window, representing an improvement in throughput of over six orders of magnitude (and bringing the timeframe for analyzing the molecules in a single water drop down to some 60 billion years - about five to six times the estimated age of the universe).
In the early 1870s, the German physicist Ernst Karl Abbé formulated a rigorous criterion for being able to resolve two objects in a light microscope. According to his equation, the best resolution achievable with visible light is about 200 nanometers. This theoretical resolution limit of conventional optical imaging methodology was the primary factor motivating the development of recent higher-resolution scanning probe techniques. The interaction of light with an object results in the generation of what is called 'near-field' and 'far-field' light components. The far-field light propagates through space in an unconfined manner and is the visible light utilized in conventional light microscopy. The near-field (or evanescent) light consists of a nonpropagating field that exists near the surface of an object at distances less than a single wavelength of light. So called near-field microscopy beats light's diffraction limit by moving the source very close to the subject to be imaged. When the first theoretical work on a new technique called "scanning near-field optical microscopy" (SNOM or NSOM) appeared in the 1980's, Abbé's classical diffraction limit was overcome, and resolution even down to single molecule level became feasible. However, light microscopy is still the only way to observe the interior of whole, or even living, cells. The use of fluorescent dyes makes it possible to selectively obtain images of individual cell components, for example, proteins. Today, the wavelength dogma has been overcome with the development of the stimulated emission depletion (STED) microscope. Now, the German team that developed STED is reporting layer-by-layer light microscopic nanoscale images of cells and without having to prepare thin sections with a technique called optical 3D far-field microscopy. They use a chemical marker for fluorescence nanoscopy that relies on single-molecule photoswitching.
"Children begin to learn by seeing, hearing, tasting and, above all, by touching. In a very similar approach, we are currently learning to orient ourselves in the nanoworld by 'feeling' materials - not with our fingers, but with microscopes that allow us to probe these materials with atomic resolution." (Robert W. Stark, LMU Munich in "Getting a feeling for the nanoworld"). Researchers' ability to engineer materials and achieve superior electronic, thermal, magnetic, and mechanical properties depends on tools that can identify and characterize material components and their spatial arrangement at the nanoscale. Equally important, understanding structure and function relationships in biological systems also demands tools that can probe structural properties with molecular resolution. Atomic force microscopes (AFM) are the most widely used tools to image matter at the nanoscale. Due to its mechanical operation, the AFM can in principle also perform nanomechanical measurements. This aspect of the AFM has been explored by researchers over the past two decades. However, current state-of-the-art techniques are very slow (it takes about one second for the AFM tip to approach, push into and retract from the surface of a material) and they apply rather large forces during the measurement process that damage the tip and the sample. Researchers at Harvard and Stanford universities have developed a specially designed AFM cantilever tip, the torsional harmonic cantilever (THC), which offers orders of magnitude improvements in temporal resolution, spatial resolution, indentation and mechanical loading compared to conventional tools. With high operating speed, increased force sensitivity and excellent lateral resolution, this tool facilitates practical mapping of nanomechanical properties.
You might remember our Spotlight from a few months ago (25 years of scanning probe microscopy and no standards yet) where we gave an overview of how scanning probe microscopy has flourished over the past 25 years. The most versatile implementation of the scanned probe principle is the atomic force microscope (AFM). It has become one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale. The essential part of an AFM is a microscale cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law. A multi-segment photodiode measures the deflection via a laser beam, which is reflected on the cantilever surface. Because there are so many promising areas in nanotechnology and biophysics which can be examined by AFM (force spectroscopy on DNA, muscle protein titin, polymers or more complex structures like bacteria flagella, 3-D imaging, etc. ) the availability of instruments is crucial, especially for new groups and young scientists with limited funds. The price tag of AFMs runs in the hundreds of thousand s of dollars, though. Until now, AFM heads are made of metal materials by conventional milling, which restricts the design and increases the costs. German researchers have shown that rapid prototyping can be a quicker and less costly alternative to conventional manufacturing.
Cells are the smallest 'brick' in life's building structures. Every living organism is made of cells. Individual cells carry their own DNA and have their own life cycle. Considering that larger organisms, such as humans, are basically huge, organized cell cooperatives, the study of individual live cells is a hugely important scientific task. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated. Especially if scientists want to look into cellular processes that occur in cells in their natural state and that cannot be observed by traditional cytological methods. It is well known that cells move, grow, duplicate, and move from point A to point B. Up to now people studied these mechanical properties with optical microscopes because it is the most common and simple method, very efficient, a very well developed and advanced technology. However, with optical microscopes detection is limited to objects no smaller than the wavelengths of the visible region of light, roughly between 400 and 700 nanometers. Distances or movement smaller than this range cannot be seen with these instruments. Researchers in Kyoto, Japan have applied a near-field optical approach to measure cell mechanics and were able to show intriguing data of nanoscale cell membrane dynamics associated with different phenomena of the cell's life, such as cell cycle and cell death.