Ultrastable Atomic Force Microscopy and Other Research Tools
The Thomas Perkins group develops innovative precision-measurement technologies as part of its quest to understand the physics of single biomolecules. According to Perkins, precision measurement remains the grand scientific challenge of biophysics.
The group’s work includes development of an ultrastable atomic force microscope (AFM), continual improvements to a stabilized optical-trapping microscope, and refinements to micron-sized optical traps made from a single tightly focused laser beam (optical tweezers). These tools are helping the scientists investigate the mechanical properties of DNA, dye-DNA interactions, DNA-based molecular motors, single-molecule biophysics, and biologically derived force standards.
After years of effort, the Perkins group has an AFM setup that leads the world in force precision and force stability. It is poised to watch individual proteins fold and unfold in minutes, a feat that is a 25-fold improvement over what was possible before. Recently, for example, the researchers modified a short AFM cantilever to be much less noisy and much more stable. These two changes allowed them to resolve the motion of single proteins! This long-sought capability was made possible by the merger of nanofabrication and biophysics.
The three key modifications of the short AFM cantilever were (1) the use of a focused ion beam to carefully cut out a large rectangular hole at the base of the cantilever, increasing its sensitivity and responsiveness; (2) the addition of a transparent protective patch over the cantilever’s gold coating, preserving the cantilever’s high reflectivity; and (3) the removal of the remaining-unprotected gold coating from cantilever and the probe tip, enhancing stability. These improvements resulted in dramatically improved cantilever performance.
To show that the new cantilevers worked with real proteins, Bull and his colleagues mechanically unfolded, then studied a collection of proteins connected head to toe. The new enhanced cantilevers performed well, as shown by the green signal in the picture. The relatively noisy red signal came from the group’s previous favorite cantilever. The new cantilevers were 50 times better at detecting protein motions in real time, with no loss of stability. The enhanced stability was even preserved when the researchers pulled on a protein anchored to a surface.They're are also easy to make, easy to use, and relatively inexpensive.
Previous improvements included an optically based detection method eliminates the unmeasured, environmentally induced drift that occurs in other scanning-probe instruments and the removal of gold coating from the AFM’s cantilevers, which enhanced the instrument’s force precision in liquid by an order of magnitude.
The group continues to work on improving AFM technology to make it an even better tool for research on single biomolecules. Better instruments that can track folding and unfolding over seconds rather than minutes will allow researchers to probe deeper into the complex behavior of molecules that make up living organisms.
High Resolution Optical Trapping Microscope
The Perkins group has also developed a high-resolution optical-trapping microscope that uses stabilized-laser beams to reduce instrument noise via improved pointing stability and differential back-focal plane detection. The latter suppresses unwanted common-mode noise. The microscope is capable of measuring subnanometer motion of biological systems in 3D. With this microscope, the group can monitor an enzyme stepping along DNA with one base-pair resolution (approx. 0.4 nm). The microscope can also detect the unfolding of RNA and DNA structures in real time.
The group has also refined an optical-trapping assay (using optical tweezers) to exhibit Ånstrom-scale scalability and resolution in 3D. To achieve this sensitivity, the group minimized various kinds of laser noise such as pointing, mode, polarization, and intensity. The group also stabilizes samples in 3D for surface-coupled assays using optical tweezers. Not surprisingly, the group plans to pursue further advances in optical-trapping technology.
Multidimensional Fourier-Transform Spectroscopy
The Steve Cundiff group is developing multidimensional Fourier-transform spectroscopies for the study of quantum effects in semiconductor nanostructures. Currently, the group routinely uses two- and three-dimensional (2D and 3D) Fourier-transform spectroscopy. Both 2D and 3D experiments employ two custom-built high-precision JILA Multidimensional Optical Nonlinear SpecTRometers, or JILA MONSTRS.
A MONSTR is a compact, ultrastable platform machined out of two cast aluminum plates. Sandwiched between the plates are cascaded and folded interferometers. Together, these interferometers split an incoming laser pulse into four identical pulses. The time travel of each pulse through the MONSTR can be exactly set to allow a well-defined sequence of three pulses to interact with a sample. Researchers can then generate a signal that can be recorded as a function of one, two, or, possibly, three time dimensions. A Fourier Transform (mathematical manipulation) converts sets of time data into multidimensional frequency spectra. These spectra are much easier to interpret that those produced by other methods.
The MONSTRs are colorful creatures. Cundiff’s two MONSTRs are fed with high-intensity infrared laser light that is bright enough to detect visually. The Jimenez MONSTR features bright green laser pulses. The MONSTRs also get some of their internal color from a red helium-neon (HeNe) laser beam. The HeNe beam traces the same path as the IR or visible pulses and is used as a ruler to set the length of each pulse’s time steps in the three interferometers. Beautiful red-anodized plates stabilize the MONSTRs’ guts and form their exoskeletons.
A JILA MONSTR uses a sequence of coherent laser pulses to excite particular frequencies of the electronic polarizations inside a material. The process is like hitting a bell with a hammer several times in succession while changing the spacing between the hammer strikes in near perfect increments. The hammer (i.e., laser) pattern sets up coherent interference patterns that deaden some tones in the bell’s ring (sample response) and amplify others. The resulting ring-tone pattern contains information about the structure and behavior of the bell (sample). Similarly, laser-induced frequency patterns created in physical systems ranging from atoms and molecules to proteins and semiconductor materials make it possible for scientists to deduce the structural and dynamical behavior of electrons at the nanoscale.
The Cundiff group uses MONSTRs to perform 2D Fourier-transform spectroscopic studies on single quantum dots and simultaneously investigates hundreds of thousands of quantum dots in one sample. The 2D spectroscopy technique is an extension of conventional transient four-wave mixing techniques. It is the optical analogue of multidimensional nuclear magnetic resonance spectroscopy, known popularly as magnetic resonance imaging, or MRI.
The Raschke group specializes in using novel optical near-field microscopies to investigate plasmonic, molecular, and solid-state materials. Techniques include linear and nonlinear optical spectroscopies (including ultrafast) employed over a wavelength range from the near ultraviolet to the far infrared. Important research areas include scanning near-field optical microscopy and tip-enhanced Raman scattering.
Scanning Near-Field Optical Microscopy (s-SNOM)
Scattering scanning near field optical microscope (s-SNOM) makes use of the optical antenna properties of metallic nanostructure to concentrate and enhance optical fields with nanometer spatial and femtosecond temporal resolution. s-SNOM provides a label-free method to probe the chemical composition of a material, easily distinguishing proteins from inorganic chemicals as well as other biochemical constituents.
The technique is used for a range of applications, including the study of (1) transition metal oxides and other quantum systems with correlated electron systems, (2) ultrafast electron dynamics in metals and the control of coupling and nano-focusing of light using plasmonic and optical antennas, and (3) investigations of supermolecular, biomolecular, and copolymer nanostructures, including the manipulation of optical molecular swithces and tuning of optical coupling in molecular plasmonics.
In biophysics experiments, s-SNOM is used with the tip of an atomic force microscope (AFM), which acts like an antenna for the IR light, focusing it onto the sample. The AFM-tip antenna also helps capture the signal emitted by the sample, sending it back to a detector for identification and location. The Raschke group uses a similar technique for chemical analysis with spatial resolution greater than 10 nm and sensitivity down to 10-20 mole, or 103 functional groups.
The group is developing a unique IR light source for its s-SNOM microscopy with its new synchrotron infrared (IR) nanospectroscopy, or SINS. SINS is an innovative way to peer inside superconductors, new materials for solar cells, or even a single cell and identify the inner workings of these complex systems. The new method is able to determine where the different chemical constituents are located and how their spatial distribution determines their function.
SINS combines scanning probe microscopy with IR synchrotron radiation, which is intense, directional, coherent, and spans a broad range of IR wavelengths ranging from 2 to 15 µm. This range of wavelengths is critical for detecting and identifying electronic and vibrational states in semi- and superconductors, polymers, biomolecules and other materials.
The first SINS experiment started with a synchrotron, but the Raschke group was able to focus its infrared power down to the nanoscale with the help of a tiny needle that concentrated the synchrotron light into a tiny region.
Plasmonic Scanning Probe Tips
The Raschke group has developed plasmonic metal nanostructures as molecular sensors and nanophotonic devices. Research with these tips explores the correlation between nanoparticle size and shape, nanoparticle spectral response, and a better understanding of the details of the local field distribution.
The use of these tips with s-SNOM allows researchers in the Raschke group to map strong field variations around plasmonic nanostructures and optical antenna geometries from the visible to the mid-infrared.
A special application of plasmonic probe tips is Tip-Enhanced Raman Spectroscopy (TERS), a nanoanalytic tool. It has applications in material and surface science as well as analytical chemistry. This technique capitalizes on the enhancement and confinement of an electric field caused by plasmonic coupling between a scanning probe tip and a metallic sample. It results in Raman signal enhancement of up to 109 and all-optical resolution down to 10 nm. Consequently, TERS has single molecule sensitivity.