A New Spectrum
Raman spectroscopy has long been a powerful tool for biological research. The addition of atomic force microscopy is adding an important new dimension.
In 2012, a team of researchers in London imaged, for the first time, the structure of the DNA double helix. James Watson and Francis Crick discovered DNA 60 years ago by laboriously studying x-ray diffraction images of millions of DNA molecules. However, Dr. Bart Hoogenboom and Dr. Carl Leung used atomic force microscopy (AFM) to directly “feel” the molecule’s structure in a fraction of the time.
Since then, the team has taken AFM further, conducting a single-molecule analysis of the secondary structure of a biomolecule. This ordinarily requires crystallization, but was, in this case, done with a conventional sample at room temperature. Their tools included Multimode 8 and FastScan Bio AFM systems from Bruker Nano Surfaces, Santa Barbara, Calif. They also used a Cypher microscope from Asylum Research, Santa Barbara, Calif., equipped with biolever mini-cantilevers from Olympus America, Center Valley, Penn., to conduct amplitude-modulation experiments. With these tools in hand, they accurately reproduced the dimensions of an important—and extremely small—DNA crystal structure.
This leap in accuracy is the result of a marriage between AFM and Raman spectroscopy. These techniques have been used together in materials science for a number of years to map surfaces and acquire chemical information. More recently, scientists working in bio-related fields are recognizing its potential. And instrument developers are responding by introducing new capabilities for imaging biological systems in unprecedented detail.
Raman enters the nanoscale realm
For many years after its discovery, Raman spectroscopy was held back by the diffraction limit of light, which limits vibrational spectroscopic acquisition, and the chemical information this data contains, to 250 nm. Instead biologists looked to other methods, such as electron microscopy and near-field optical microscopy (NSOM), a form of scanning probe microscopy (SPM) that combines optics with a probe that reads evanescent waves. In recent years, fluorescence-based techniques have allowed vendors to market super-resolution optical microscopes that peer deep into the nanoscale. And in the Raman space, a technique called surface-enhanced Raman scattering (SERS) takes advantage of small-scale electromagnetic effects to “boost” the Raman scale by a factor of 10 or more, helping Raman surpass the diffraction limit.
But SERS isn’t capable of localization. To understand precisely the position and activity of individual nanoscale biological features, namely cells and proteins, researchers need the ability to map, or scan, extremely small regions. Tip-enhanced Raman spectroscopy (TERS) provides this capability by shrinking the sample to the size of a cantilever tip.
“With Raman, you have imaging capability down to 250 nm, which is about 200 times less resolution than with AFM. At this point TERS comes into play. The typical approach is to use aperture probes to beam lasers through fiber or a hollow cantilever to get a small spot size on a sample,” says Emmanuel LeRoy, AFM-Raman product manager at Edison, N.J.-based Horiba Scientific. “This doesn’t work well with Raman because the scattering is weak. Only one in a million photons are scattered back and, because the volume is so small, no Raman signal is returned.”
The probe itself supplies the enhancement, which is localized at the bottom of the probe, in an area smaller than the tip. With TERS probe tips, the tip size is 20 to 30 nm, providing a localization of just 5 to 10 nm. This sort of capability has attracted the attention of biophysicist Volker Deckert, a professor at Jena Univ. in Germany who has successfully used Horiba Scientific’s TERS-capable instruments to conduct studies of biological constructs such as DNA origami.
“TERS allows structural investigations with an impressive lateral resolution,” says Deckert. “The benefit is two-fold. First, the driving force behind the development of this technique was, and still is, our general interest in molecular structures below the diffraction. Second, AFM-based vibrational spectroscopy techniques, and TERS in particular, are best suited when the use of additional labels is prohibitive or simply impossible.”
In addition, adds Deckert, it’s always desirable to perform work on “real” systems. Acquisition of chemical data from living systems can be done with high lateral resolution, in real time and label-free. TERS has allowed him to study complex samples such as amyloid fibrils, cellular proteins and even crystal structures produced by the malaria parasite. “A vast number of scientific questions can now be addressed,” says Deckert.
TERS isn’t perfect for biology, however. Like any SPM-based technique, it’s limited to surface analysis. Also, LeRoy says, the method is comparatively slow: Only a handful of molecules are studied at any one time, which reduces Raman signal. Plus, biological samples have a tendency to “stick” to the probe tip, which can complicate some soft tissue analysis. Finally, the lack of highly developed probes held back research because a broken or damaged tip couldn’t easily be replaced. This is being rapidly corrected, however, as scientists like Deckert are developing more efficient and reproducible tips in the laboratory.
Horiba Scientific, in turn, is working closely with SPM specialist AIST-NT, Novato, Calif., to increase the capabilities of its TERS-ready microscopes, which include the compact, automated XplorRA nano, broad wavelength HR Evo Nano and bioresearch-ready CombiScope XploRA. Each are equipped with AIST-NT’s SmartSPM for AFM-Raman studies. Horiba has added a side port to its microscopes, along with high 0.7 NA objectives, to allow customers to use polarized lasers to achieve maximum Raman enhancement.
AFM modes contribute their part
Many major microscope vendors have launched systems intended for combined AFM-Raman nanoscale research. They include Thermo Fisher Scientific, JPK Instruments, Asylum Research and Nanonics.
One vendor, WITec GmbH, Ulm, Germany, has based much of its product line on confocal Raman microscopy, and has added AFM to many of its instruments. The approach, which helped earn the company a 2008 R&D 100 Award, reflects an increasingly modular approach to instrument construction and implementation.
“The advantage of the WITec systems is that all imaging techniques are truly integrated in a single instrument with one controller and software,” says Dr. Sonja Breuninger, technical marketing director for WITec. “The setup is highly flexible and can be upgraded or complemented when required. Imaging techniques such as NSOM or scanning electron microscopy (SEM) can be easily added.”
A WITec microscope can be as basic as the alpha 300 M for single Raman spectrum measurements or as complex as the alpha 300 RAS, equipped with combined Raman, AFM, NSOM, fluorescence and luminescence imaging techniques. This adjustability, says Breuninger, pays dividends in biological research where analysis conditions change frequently. Software is a particularly important addition in this field, she adds, because it can generate depth profiles and 3-D images, even of soft tissue samples.
At Bruker Nano Surfaces, developers have streamlined AFM functionality directly into an existing inverted research microscope. According to Andrea Slade, AFM applications specialist at Bruker Nano Surfaces, the introduction of Bruker’s Integrated Raman Spectroscopy System (IRIS) TERS probes in 2010 have generated more than 330 scientific papers. Of these, 110 were directed at biological or bio-related research, followed closely by detailed study of polymers.
“In chemistry and biology, many techniques complement each other. Raman spectroscopy complements direct vibrational spectroscopy very well,” says Slade, which is why Bruker launched the IRIS technology. “There’s an increasing interest in AFM activities, or modes, in conjunction with Raman and fluorescence. A lot of this work is physics oriented, but biologists have experimented and developed bio-applications that work.”
It’s still a challenging technique, she adds, and requires more finesse. “It involves having the right tips and probes, and having to deal with sample restrictions,” she continues.
To facilitate the addition of scanning probe analysis, IRIS-ready instruments have been designed with open optical heads to avoid interference. AFM mode development is proceeding quickly at Bruker, which has pioneered several new techniques in recent years. Arguably the most influential new mode is Peak Force Mapping (PFM), which allows researchers to perform studies on biological tissues such as amyloid fibrils, which can have deleterious effects on brain cells. Using PFM, researchers can accurately determine a cell’s modulus and what type of force allows the fibril to pass through a cell membrane. Hoogenboom and Leung relied on PFM for their recent DNA research.
A new variant of Bruker’s PFM, called Peak Force Mapping QNM (Quantitative Nanomechanical Property Mapping), permits quantitative mechanical mapping of material properties while also imaging sample topography at high resolution. QNM works by oscillating the probe at resonant frequencies of 1 or 2 kHz to allow imaging of soft samples. In tapping mode, researchers can use the AFM tip to “push” on samples. The system addresses an existing problem with using resonance to image fluids. In tapping mode, the user must continuously tune the probe to achieve the resonant peak. This can’t easily be done for liquid samples. QNM instead feeds back on the applied force between the tip and the surface, and that force is always precisely known. When used with Bruker’s Scan-Asyst technology, the set point self-optimizes, allowing more consistent results on the sample.
“QNM is being used now to look at glioblastomas, and whether they are stiffer than healthy counterparts, or are less stiff and more deformable. This information is important because it gives researchers an idea of how cancer cells metastasize,” says Slade.
In the case of Hoogenboom’s DNA analysis work at the London Centre of Nanotechnology, the AFM probe tip on the FastScan Bio can determine minute changes in the topography of DNA molecules. By looking at the force-distance curves generated by the AFM and comparing them to the Raman spectra collected by the microscope, the researchers can build a database of relational data that can help inform scientific studies.
Combined systems to aid “mechano-biology”
Only 12 years old, TERS as a laboratory imaging technique is still in its infancy. Some scientists have eagerly explored the new limits the technique offers, but it remains out of reach for most. Major global research efforts like the BRAIN Initiative will continue to rely on targeted fluorescence-based techniques for discovery. But Slade says that Bruker’s customers now generally recognize the advantage of AFM as a complementary microscopy technique, and that an understanding of “mechano-biology” will be important going forward.
The possibilities are substantial: In 2013, a team led by Zhenchao Dong at the Univ. of Science and Technology in China combined AFM-Raman with a scanning tunneling microscope to successfully map an individual molecule at a resolution of less than 3 nm.