Nicole Olson

This year’s award recipient, Megan Thielges, is a pioneer in the use of vibrational probes, in conjunction with two-dimensional infrared spectroscopy, to investigate the structures and dynamics of proteins, with the aim of understanding biological systems.

 an associate professor of chemistry at Indiana University, has won the 2018 Emerging Leader in Molecular Spectroscopy Award, which is presented by 

 magazine. This annual award recognizes the achievements and aspirations of a talented young molecular spectroscopist, selected by an independent scientific committee. The award will be presented to Thielges at the SciX 2018 conference in October, where she will give a plenary lecture, and be honored in an award symposium.

	Thielges earned her B.S. in biochemistry at Arizona State University. She went on to earn her PhD in biophysics from The Scripps Research Institute in La Jolla, California, in 2009. After three years as a postdoctoral fellow at Stanford University, she obtained her current position as an associate professor of Chemistry at Indiana University. She has published 11 papers as a principle investigator, and 22 as a postdoctoral, graduate, or undergraduate researcher. She has given 45 presentations and received six previous awards, as well as nominations for more. In addition to her research, Thielges has served as a reviewer for numerous scientific journals and on panels for many major national departments, including the National Institutes of Health, the National Science Foundation, and the Department of Energy.

The Challenges of Studying Protein Dynamics 

	The focus of Thielges's work is the study of protein structure and dynamics. Specifically, she is a pioneer in the use of multisite vibrational probes in conjunction with two-dimensional (2D) infrared (IR) spectroscopy to investigate the structures and dynamics of proteins.

	The study of protein structure and function is a field of great interest. Proteins are the mediators of biological functions, and the structure of proteins is complex. The simplest level of protein structure is the primary structure, which consists of a sequence of amino acids joined to form a polypeptide chain. The order of the amino acids is what affects the structure and function of the protein; a single substitution can have life-threatening consequences. The secondary structure consists of a regular pattern of substructures that are formed because of the bonds between the various amino acids that compose the polypeptide chain. Proteins are not necessarily static in their conformations, however. In the field of protein dynamics, scientists look at the transitions a protein can make between different shapes that can occur on various scales of length or time, or both. These conformational changes have been shown to affect cellular functions, such as enzymatic activity and cellular signaling. These changes can happen on extremely fast timescales, making vibrational spectroscopy an excellent technique to observe them.

	Given the dense nature of protein structure and the large amounts of molecules present, however, measured signals are congested, with large amounts of spectral overlap, making it is difficult to resolve the signals. A strategy for alleviating this signal congestion is to use a vibrational probe, which involves replacing one or more molecules in the structure with another whose vibrational signature operates in the so-called 

, a region free from native protein signals (1). But developing effective vibrational probes is challenging. For a vibrational probe to be useful at a specific protein site, it must be strongly responsive to the physical property being investigated, be sensitive only to its immediate environment, cause little to no change to the original structure of interest, and be able to be incorporated into the system (2). This is where Thielges comes in.

Harnessing Vibrational Labels and 2D IR to Study Proteins

	A major aspect of Thielges's work is the development of new vibrational labeling strategies. This is the crucial element of her contributions, according to Andrei Tokmakoff, a professor at the University of Chicago. "Her work has resulted in a toolbox that is opening up new ways of using IR spectroscopy in protein chemistry and biophysics," he said. "By combining these new labels with ultrafast 2D IR, Megan has [developed] a new way of studying protein chemistry, where fast dynamical processes involved in catalysis or binding are directly observed rather than inferred."

	One example of this work was Thielges's use of vibrational labels to study cytochrome P450 enzymes, which play a role in the biological functions of molecular synthesis and metabolic processes. They are the major enzymes involved in the metabolism of medications, making their structure and function of particular interest to medical researchers. Thielges's work combining a new labeling techniques she developed (heme-bound CO, in this case) with IR and 2D IR spectroscopy has revealed different substrate binding configurations at the active site in these enzymes and enabled characterization of the fluctuations and exchange of these configurations. Her studies on P450 enzymes have illustrated the importance and power of 2D IR spectroscopy to measure protein dynamics occurring on very small timescales, as well as the technique's ability to gather information about the role of dynamics in biological activity (3). The National Institute of Health has awarded Thielges research grants for this work. By resolving different substrate binding configurations and the fast dynamics of conformational interchange, these experiments may one day enable understanding of the drug-degradation process undertaken by these enzymes.

	Another important application of Thielges's work is in the study of cellular signaling. The SH3 region in proteins is responsible for controlling certain signal transduction pathways, or the process through which a physical or a chemical signal is carried through a cell (cellular signaling). By introducing carbon-deuterium probes into the SH3 domain in yeast cells, Thielges was able to observe variations in the binding conformations of a proline-rich peptide (4). This study illustrated the power of IR spectroscopy to uncover rapidly interconverting states in a protein–peptide complex for the study of cellular signaling.


	Thielges with one of her students, Sashary Ramos, in the laser laboratory at Indiana University.

Enabling the Study of Proteins at the Nanosecond Timescale

	With 2D IR spectroscopy, it is possible to detect the signals from vibrational probes. However, the experimentally accessible timescale has typically been limited to the picosecond regime. This limitation has led to the need to develop 2D IR probes with longer vibrational lifetimes to extend the experimental timescale. Thielges's group has introduced and studied a novel IR probe, p-cyano-seleno-phenylalanine (CNSeF), for the study of protein dynamics by 2D IR spectroscopy, that is able to extend signal lifetime into the nanosecond range. The group demonstrates that this new probe could be detected on timescales 20–50 fold longer than could be achieved with the popular probe cyanophenylalanine (5).


	Thielges giving a talk at the International Conference on Coherent Multidimensional Spectroscopy (CDMS 2018) in Seoul, Korea, in June 2018.

	Tokmakoff wrote enthusiastically in support of Thielges's nomination for this award. He described her as an "ambitious and creative interdisciplinary scientist" who conducts "fearless research." What makes her stand out from her colleagues, he said, is not only that she is highly trained in two different scientific fields that rarely cross-protein and enzyme biochemistry and biophysics, and ultrafast laser spectroscopy-but that she is able to overcome the challenges of combining them successfully. As Tokmakoff said, leading spectroscopists often work with model systems (rather than meaningful biological problems), and chemical and molecular biologists are often unwilling to work with the long development process of new physical methods. "Megan is really the first of her kind-one who can do it all," he said. "She is truly a first class experimental scientist in both areas."

	Michael Fayer, a professor of chemistry at Stanford University, where Thielges did postdoctoral research, agrees with Tokmakoff's appreciation of Thielges's combination of skills. "Megan is an amazing blend of a real biologist and a hard-core chemical physicist," he said. "This is her great strength that allows her to make important contributions to biology and biophysics."

	Minhaeng Cho, the director of the IBS Center for Molecular Spectroscopy and Dynamics and a professor of chemistry at Korea University, first met Thielges at a conference three years ago, and the two have since collaborated on multiple occasions. According to Cho, Thielges's contributions to molecular spectroscopy started while she was still in graduate school. While at the Scripps Institute, he said, Thielges "introduced an extremely small and non-perturbative vibrational probe into proteins in a site-specific manner." This was important because of the spectral congestion that made using the plentiful C-H bonds to study the shapes and motions of proteins impossible. By replacing the hydrogen with deuterium at specifically targeted sites, Thielges demonstrated that the C-D stretch band, which falls into proteins' silent IR region, has "line shapes [that] are highly sensitive to protein conformations and secondary structures." Cho believes Thielges's research will lead her to address deep questions in biology, such as how protein–protein interactions and recognition are related to fast local fluctuation dynamics at the interface between proteins.

	Floyd Romesberg, who was Thielges's graduate advisor at Scripps, says Thielges is a remarkable experimentalist. "She totally revolutionized our experimental approach to data collection, and her methods are still used by my group today," he said. "She also organized a study group for the students to teach themselves the theory underlying time-resolved non-linear spectroscopy."

	Carlos Baiz, an assistant professor of chemistry at the University of Texas at Austin, describes Thielges as "creative, resourceful, [and] charismatic." He believes she will continue to "push the envelope" when developing more complex probes and methods to further evaluate protein structure and dynamics. He believes her new techniques will be more widely adopted by the ultrafast community in the coming years.


	Thielges receiving an award for her work as a session chair at the International Conference on Coherent Multidimensional Spectroscopy in Seoul, Korea, in June 2018.

	Peter Hamm at the University of Zurich conducts research that is very closely related to that of Thielges. But even though he characterizes their relationship as "a friendly competition," he nominated her for an award last year. Hamm describes Thielges's research as "an absolute prerequisite" for 2D IR spectroscopy to become a more versatile tool to study the structure and dynamics of proteins. "Her work is courageous," he says. "It does not seek the low-hanging fruit."

	Megan Thielges's colleagues all believe she has a very bright and promising future ahead of her. Romesberg summarized this outlook well. "Megan will continue to combine sophisticated biology and spectroscopy," he said, "not only to explore protein dynamics, but to begin to elucidate how [they have] been tailored by evolution for biological function."

	Megan Thielges is a pioneer at the forefront of combining biophysics with spectroscopy. She has been "fearless" and has overcome many practical obstacles to advance these two fields. She is a top-notch experimentalist who takes on big challenges. From organizing study groups to teach herself the theory behind time-resolved non-linear spectroscopy to developing methods as a graduate student that her advisor and his current group still use today, it seems there is nothing Megan Thielges can't do if she puts her mind to it.

	(1) R. Adhikary, J. Zimmerman, and F.E. Romesberg, 

	(2) J. Ma, I.M. Pazos, W. Zhang, R.M. Culik, and F. Gai, 

	(3) E.J. Basom, J.W. Spearman, and M.C. Thielges, 

	(4) R.E. Horness, E.J. Basom, J.P. Mayer, and M.C. Thielges, 

	(5) S. Ramos, K.J. Scott, R.E. Horness, A.L. Le Sueur, and M.C. Thielges, 

is a freelance writer. Direct correspondence about this article to 

Articles

This year’s honoree, Megan Thielges, is a pioneer in the development of vibrational probes for use with 2D IR spectroscopy, to reveal new information about the structures and dynamics of proteins.

The 2018 Emerging Leader in Molecular Spectroscopy Award

Progress continues to develop in analytical spectroscopy as improvements are made to instrument sensitivity, limits of detection, and accessibility. This article discusses some of the advancements in techniques and modifications to technologies that are improving measurements and enabling well-established techniques to break into new and exciting fields of research across five spectroscopic disciplines: X-ray fluorescence spectroscopy, infrared spectroscopy, inductively coupled plasma–optical emission spectroscopy, Raman spectroscopy, and laser-induced breakdown spectroscopy. 

	Since their respective inceptions, spectroscopic techniques have been modified to adapt to new applications and to improve analytical performance. Recent development lies in instrumental fine tuning and slight alterations to allow new types of measurements. Advancements in the fields of biology and nanotechnology have created demand for new methods of measurement using instruments already accessible, pushing the boundaries of what was previously available. The following sections track some of these trends and more across five major techniques: X-ray fluorescence (XRF) spectroscopy, infrared (IR) spectroscopy, inductively coupled plasma–optical emission spectroscopy (ICP-OES), Raman spectroscopy, and laser-induced breakdown spectroscopy (LIBS).

IR Spectroscopy: Measurements at the Nanoscale

	Standard IR spectroscopy shines infrared light on a material and measures the fraction of light that is absorbed at a particular wavelength. Light is absorbed when the vibrational frequency of a bond in a molecular structure is the same as that of the incoming light. IR spectroscopy is a valuable and mature technique, but challenges arise when trying to measure nanomaterials or very small amounts of samples. Typically, IR spectroscopy can measure down to 1 mg of sample; however, to be free from the interference effects of water, the sample must be completely dry-a preparation that can take days to complete (1). A new technique called 

) has emerged in recent years to measure engineered nanomaterials and very small amounts of sample. As nanotechnology continues to advance, this technique holds an immense amount of promise to assist in many applications, such as pharmaceutical quality control, precision diagnosis, and even drug delivery. In NAM-IR, nebulized samples are collected on nanomechanical resonators, such as strings or membranes, through diffusion and inertial impaction. The higher the velocity of the incoming particles, the more efficient the collection is. When irradiated with infrared light, the captive particles change the resonance vibrations of the resonator via photothermal heating. This resonance shift is directly proportional to the absorbed energy (1). Previously these NAM-IR measurements had to be performed in a specialized laboratory, but recently a group (2) has designed a NAM-IR system based on magnetic transduction. This instrument uses 10-µm gold electrodes in between two Halback arrays of neodymium magnets to increase the magnetic field over the sensor chip in which the resonator is embedded. In this case, the nanomechanical resonator used is a perforated membrane. An alternating current is applied to two electrodes to drive an initial vibration of known frequency through the resonator. To test their setup, indomethacin, a common oral anti-inflammatory drug, was analyzed. Approximately 100–250 pg of sample was calculated to have deposited onto the membrane, and the resultant measured spectra were in good agreement with those obtained from traditional Fourier transform infrared (FT-IR) spectroscopy.

	Another technique that has emerged recently to study objects at the nanoscale is a combination of atomic force microscopy and infrared spectroscopy (AFM-IR). In this approach (1), a pulsed, tunable laser causes a localized thermal expansion of the sample that displaces an AFM tip and cantilever. The resulting oscillation is measured with a second laser and is directly proportional to the amount of IR radiation absorbed by the sample. Because the displacement of the AFM tip is not limited by the diffraction limit of the IR wavelength (about 5 µm), the spatial resolution of this technique is substantially higher than that of standard IR spectroscopy. This method has proven especially valuable in the biological sciences by providing a wealth of information about the nanoscale chemical, structural, and mechanical properties of biological materials. AFM-IR has been used in attempts to understand the pathways of neurodegenerative diseases. These are thought to be caused by the misfolding of proteins in the brain that then trigger the formation of more misfolded proteins. The malformed proteins clump together, forming aggregates of nonfunctional brain tissue. Standard IR techniques are not able to elucidate heterogeneous tissue at a single species level and therefore can't provide a link between the individual component of an aggregate and its toxicity. Ruggeri and colleagues (3) used AFM-IR to determine that the ataxin-3 protein, which is responsible for spinocerebellar ataxia-3, misfolds during protein aggregation and not before as previously thought. Another group, also led by Ruggeri (4), showed that the quality of the intermolecular hydrogen bonds and the structural organization of an amyloid sheet structure are fundamental to determining toxicity in polyQ diseases such as Alzheimer's and Huntington's. Obtaining a better understanding of these pathways presents the possibility of human intervention to disrupt or delay the onset of such diseases.

XRF: Space Exploration and Cultural Heritage Analysis

	XRF is broadly used for elemental analysis. When excited by an external energy source, individual atoms emit X-ray photons of a characteristic energy. This technique is commonly used in astrophysics and planetary science to study the geologic composition of celestial bodies. Standard X-ray spectrometers pose a few problems when sent into space because of their large masses and volumes. Previously launched models intended to investigate objects in space have been based on simple collimators, which is fine if the bodies of interest are large enough to fill the instrument's field of view. However, the sky is full of X-ray sources that are often stronger than the X-ray output of nearby small bodies of interest and can easily interfere with measurements. Problems occur when the space and weight available to the instrument is considered. The primary factor in the design of multi-instrument probes is how much the instrument components weigh because of the enormous cost of launching each additional gram into space. If full advantage is to be taken of these instruments with respect to observing small bodies, a new lightweight design is necessary. A team at Harvard and the Smithsonian Astrophysical Observatory (5) has recently developed prototype XRF instruments based on focusing optics to better study small planetary bodies, such as asteroids. Named MiXO, short for miniature lightweight Wolter-I focusing X-ray optics, this instrument is based on hybrid X-ray mirrors consisting of metal and ceramic. The mirrors are crafted through a combination of electroformed nickel replication and a plasma thermal spray process; a ceramic layer provides stiffness, and a nickel-cobalt layer maintains the smoothness needed for X-ray reflection. In this prototype, much of the nickel has been replaced with alumina to reduce the weight by a factor of 30. A few example configurations are described that illustrate a threefold improvement in light-collection power, and at least 10 times improvement in angular resolution, detection sensitivity, and energy band coverage over the current leading system for observing the X-ray fluorescence of astronomical objects.

	Closer to home, XRF spectroscopy continues to expand into the analysis of historical and cultural heritage objects. The big advantages of using XRF in this field are that it is a nondestructive technique and can be used in situ, without having to physically move the object of interest to a laboratory. While the use of XRF continues to expand into this area, specialized modifications to the technique are being made. Much of the recent literature is focused not on analytical developments, but on new ways to gain insight into the origin of the materials, mostly though the use of multiple techniques. For example, to fully understand chemical changes that have occurred over time within a painting, X-ray techniques (such as XRF, X-ray diffraction, and X-ray absorption spectroscopy) are combined with FT-IR or Raman spectrometry (6). When measuring heritage objects, conventional XRF only gives a two-dimensional (2D) pigment distribution because of the depth to which the X-ray beam penetrates. A group in Japan (7) designed an instrument that combines a confocal XRF spectrometer and two polycapillary lenses to image multiple layers and generate elemental three-dimensional (3D) maps of artwork. A half lens sits in front of a silicon drift detector and a full lens is attached to a compact X-ray tube. The excitation and detection focusing spots are adjusted to be the same location, called the 

. Using a micro X-ray beam to irradiate the sample, the team successfully located a hidden black cat underneath multiple paint layers in a replica work of Daubigny's Garden originally by Vincent van Gogh. This system had a resolution depth of about 54 µm and can successfully be used to identify pigments in multiple layers.

ICP-OES: Sample Introduction and Cost of Operation

	ICP-OES ignites argon to create a plasma that ionizes nebulized samples to determine elemental composition. Most of the recent advances in ICP-OES have been improvements to its analytical capabilities, primarily through increasing the efficiency of sample introduction. Most samples that are analyzed by ICP-OES are aqueous solutions, which presents a few drawbacks when it comes to instrument sensitivity: The sample introduction depends on the nebulization efficiency, less energy is available for sample atomization because of the vaporization cost of the solvent, and the introduction of matrix effects (8). Much work has been done in recent years to find different ways of introducing samples into ICP-OES instruments as gaseous species rather than aqueous ones. Electrothermal vaporization (ETV) has been used as a sample introduction method for many years now, but only recently has the number of reports using ETV in conjunction with ICP-OES increased (8). A major advantage of this sample introduction technique is that it can be used to directly analyze solid material, which increases sample throughput and decreases both the time and possibility of contamination during sample preparation. It also significantly improves the transport efficiency versus a traditional pneumatic nebulizer from <5% to at least 60% (8). A significant limitation of this technique, however, has been relatively slow data transfer that results from a slight incompatibility between the signals produced during sample vaporization and the detection system of the ICP-OES. Luckily, researchers are finding ways to overcome this signal lag. Chaves and colleagues (9) have written a MATLAB program to increase data transfer time. This program can generate 3D spectra while simultaneously performing fast background correction and signal integration for as many as eight analytes.

All necessary calculations are performed offline, further reducing the time it takes to process the data.

	The use of chemical modifier gases during vaporization has been investigated to improve sensitivity. Hassler and colleagues (10) investigated the detection of trace elements in high-purity copper samples using fluorinated gases or halogenation reactions for most elements. They had a high rate of success using this method and were able to elucidate 22 elements present in the samples.

	Standard ICP-OES using the current Fassel-type torch design uses around 12–20 L/min of argon, making it expensive to operate (8). In recent years, a couple of new designs have been proposed and tested to reduce the costs of argon consumption. A redesigned plasma torch was introduced (11) in 2005 called the 

), consisting of a quartz tube with a spherical excitation zone and an external air cooling system. This design significantly reduces argon consumption to around 0.05–1.00 L/min. Though first proposed more than 10 years ago, recent studies utilizing the new torch for comparison with standard ICP-OES are still ongoing. Nowak and colleagues (12) studied the arsenic content in three kinds of fish and had an argon flow rate of 0.66 L/min, compared with the same test using 14 L/min with a conventional ICP torch. Another approach (13) to reducing argon consumption focused instead on the amount of argon used to purge the instrument of molecular oxygen and water vapor before testing. In this sealed optical system, argon is recirculated through a small purifier cartridge. Though this approach saves a much smaller amount of argon compared to that necessary during torch operation, no refill is required-once a system has argon inside for purging, no more is needed. This sealed design was also found to improve analytical performance in the vacuum-ultraviolet (UV) region (less than 190 nm) and potentially allows for fewer matrix effects than the SHIP torch system.

Raman Spectroscopy: Measurements in Biology and at the Nanoscale

	Raman spectroscopy is based on the inelastic scattering of monochromatic light to reveal the molecular composition of a sample. This technique has two inherent disadvantages that prevent it from being used unmodified for high-resolution measurements: low sensitivity and diffraction-limited spatial resolution. Surface-enhanced Raman spectroscopy (SERS) is often used to overcome the low sensitivity. Substrates for analysis usually consist of nanostructures or a rough metal surface. When the adsorbed sample particles are irradiated with particular wavelengths of light, the Raman scattering is enhanced by this surface. Though able to overcome low sensitivity problems, SERS alone does not provide spatial resolution on a nanometer scale. Tip-enhanced Raman spectroscopy (TERS), however, does. TERS exploits a resonance between metallic nanostructures at the apex of a probe and an incident light to generate a strong, localized electric field that enhances Raman scattering.

	The expansion of TERS into new application areas, specifically the biological sciences, has made it necessary to find a way to prevent sample damage because of the Joule heating that occurs during electric field generation. Using thiophenol molecules with known thermal desorption profiles, Mochizuki and colleagues (14) were able to monitor desorption and accordingly adjust the incident laser intensity during testing to prevent a damaging increase in probe temperature. This group also looked at the dissipation of generated heat into the surrounding medium through probe oscillation during measurement. For temperatures up to the thermal desorption of thiophenols (60–100 °C), the combination of these two methods have proven to effectively prevent damage to heat-sensitive samples such as organics and biomolecules.

	For the first time, small molecules inside a cell have been mapped using TERS. Previously, the best-known technique to map small biomolecules nondestructively was super-resolution fluorescence microscopy. However, this relies on the attachment of fluorophores to molecules, which can have an impact on their behavior and function. Additionally, only those molecules of interest that have been tagged can be seen with this technique-all others are invisible. Using excised cultured mouse pre-adipocyte cells, Kumar and colleagues (15) mapped the growth of newly synthesized phospholipid accumulation. Deuterated stearate ions were metabolized by the cells to provide a fatty membrane tail that could be imaged because of the carbon–deuterium bonds visible in the Raman spectra. The resolution obtained was less than 20 nm, effectively demonstrating another analytical tool for nano-imaging that the authors hope will help pave the way for better design of therapeutic drugs that need to target specific cell areas.

	Side-illumination TERS is a variant in which the incident laser light comes in at an angle from the side, providing very efficient near-field enhancement when paired with a metal substrate. However, this method presents two challenges. First, the focal spot projected on the sample tends to be quite large, leading to a high level of background noise. Second, the use of a low numerical aperture objective lens limits the collection efficiency. To overcome these obstacles, Shen and colleagues (16) proposed a new probe design to increase the signal-to-noise ratio and collection efficiency. The latter criteria is essential for a fast acquisition time to reduce potential image distortion. The proposed design is theoretical and was mathematically demonstrated to increase the localized electric field by 25 times when compared to a regular tip. The presence of concentric grooves on the theoretical tip surface are shown to strongly enhance the field intensity without affecting its spatial resolution. Two asymmetrical nanorods at the tip assist in unidirectional emissions. The researchers note that the nanofabrication of the two nanorods would be very difficult, but can be done. Overall, these two modifications allow more light to be directed toward and collected by the collection objective. This design has the potential to be very useful for single molecule measurements in a variety of sample types.

LIBS: 3D Mapping, Molecular Signatures, and Instrument Sensitivity

	LIBS measures atomic and ionic compositions by using short pulses of laser light to create a dense, high-temperature plasma. Discrete spectral peaks are emitted when excited atoms fall back down into their ground states. This is a technique for measuring the atomic and ionic signatures of a material in the UV to near infrared (NIR) spectral range. Recently, it has been demonstrated that it is possible to also obtain intact molecular information by analyzing the spectra in the mid- and long-IR wavelength range (LWIR-LIBS) (17). Current detection systems are comprised of mercury-cadmium-telluride (MCT) arrays that can acquire a full LWIR spectrum (5.6–10 µm) in under 5 s (18). One group (17) that has done extensive work with this new technique tested a setup to acquire both LWIR and UV–NIR LIBS spectra simultaneously by simply connecting the two different detection systems into a linear array. The test was successful, and the known ingredients of two major over the counter medications were easily identified.

	As previously noted in this article, nanoparticles have received a lot of attention in the medical field lately as a vector for drug delivery and immunotherapy. Gimenez and colleagues (19) recently developed a technique to image manufactured nanoparticles consisting of gadolinium, calcium, or gold using LIBS in biological tissues. Using these nanoparticles, developed specifically for image-guided radiotherapy and which are smaller than 5 nm in diameter, they successfully created a 3D image of excised rodent renal tissue. Traditional methods of tissue imaging rely on the use of labels to mark the introduced nanoparticles so they can be distinguished, but this approach can have a drastic influence on the shape, size, or charge of the particle and affect its distribution, or even detach from the nanoparticle entirely. LIBS circumvents this challenge because it relies solely on the excitation emissions of the nanoparticles themselves. LIBS may not be as sensitive or as high-resolution as other spectroscopic techniques (though it is still excellent), but it has the advantage of an acquisition rate up to 100 times higher, making the creation of 3D maps of large samples (that is, entire organs) a relatively quick process. This study used nanosecond infrared laser pulses of 500 µJ focused through a 15x microscope objective at epoxy-encased tissue to obtain a 2D map. Two approaches to creating a 3D map were studied and found to be complementary. The first was to slice the sample into many 200-µm-thick layers, image them all independently, and then construct a 3D map from the individual results. The second was to repeatedly scan the same ablation area, adjusting the focus for the new depth between each scan. The latter approach obtained a spatial resolution of about 10 µm in all directions. Because of the (relatively) destructive nature of the laser-ablation process, its use is limited to excised tissue and could only be used for imaging removed samples, such as during biopsies.

	Improving the sensitivity of LIBS continues to be a popular topic of investigation. Valjanen and colleagues (20) have shown that by coupling a 2.45-GHz microwave with the generated plasma, the signal lifetime is greatly increased from just a few microseconds to hundreds of microseconds. This allows for a longer signal integration time and a significant enhancement of the signal. In particular, the copper content of a copper–alumina solid was analyzed and the limit of detection found to be 8.1 ppm-an improvement of 93 times over conventional, single-pulse LIBS. The effects of self-absorption were also mitigated, which could be promising for measuring high concentration elements. Wang and colleagues (21) investigated sustaining the LIBS-generated plasma in a different way. They have shown that increasing the diameter of the ablation crater increases the persistence of the Cu(I) line, possibly because of shock waves from the ablation itself compressing the plasma and increasing its temperature enough to cause further atomic excitation. They note, however, that the depth does not seem to have the same effect on the Cu(I) line.

	The full potential of analytical spectroscopy has yet to be realized. As technology advances and its utility continues to grow, old laboratory staples are being improved upon, refined, and used in new ways to helps us understand the world around us.

 (Wiley Online Library, 2017), pp. 1–26.

	(17) C. Chaffee, "LIBS Continues to Evolve," 

https://www.osa-opn.org/home/articles/volume_28/may_2017/features/libs_continues_to_evolve/

 is a freelance science writer. She works in research and development in St. Paul, Minnesota. Direct correspondence to 

This article discusses advances in techniques and modifications to technologies that are improving measurements and enabling well-established techniques to break into new and exciting fields of research.We survey developments in infrared spectroscopy, Raman spectroscopy, X-ray fluorescence spectroscopy, inductively coupled plasma–optical emission spectroscopy, and laser-induced breakdown spectroscopy


Current Trends in Analytical Spectroscopy: Technique and Instrument Modifications for both New and Improved Measurements

Analytical spectroscopy is a mature field, but advances in instrumentation, measurement, and 

sample preparation techniques continue to be made. Much recent development has seen a heavy emphasis toward techniques and instrumentation that allow for noninvasive, in situ, and in vivo procedures while still retaining a high degree of sensitivity. This article will review some of this progress over the past two to three years across five major spectroscopic fields: Raman, laser-induced breakdown spectroscopy, X-ray fluorescence, infrared, and inductively coupled plasma techniques.

	Spectroscopy is used in almost every scientific research discipline, yet continuous improvements and refinements to the field are still enabling expansion into new industries. Perhaps the strongest trend of the past few years, as can be seen across numerous scientific fields and types of spectroscopy, is the push toward smaller, more efficient instrumentation that can perform measurements in situ, in vivo, and with minimal invasion. A primary goal of any analytical science is to acquire reliable, precise, and reproducible data as quickly, cheaply, and easily as possible. As technology improves, spectroscopic instrumentation continues to become increasingly smaller and more portable. Much of the development lies in methods that enhance the capabilities of existing techniques through sample preparation and experimental design, external factors such as magnetic fields, and the coupling of different spectroscopic methods. These new methodologies aim to increase performance, spectral clarity, and instrument sensitivity. One of the most exciting fields that has seen recent, rapid advancement is the healthcare industry. The use of spectroscopic technologies for use in diagnosis and treatment has grown exponentially in recent years. Diseases such as diabetes and cancer can now be monitored using spectroscopic techniques. From the exploration of our deep seas and extraterrestrial neighbors to healthcare and the arts, the following topics are examples of current research that illustrate the recent in situ, in vivo, and minimally invasive techniques and innovations that are evolving analytical spectroscopy. The five ensuing sections track this trend, as well as new applications and expansion into new industries, across five spectroscopic fields.

	Raman spectroscopy relies on the inelastic scattering of monochromatic light to determine what molecules are present in a sample. Since it is a nondestructive technique, it is an ideal tool to measure samples whose original composition is important and for use in vivo in patients.

	In the past few years, Raman spectroscopy has become an emergent healthcare tool. The spectra acquired from this powerful analytical technique can illuminate biochemical changes in cells, tissues, and biofluids that can indicate the presence of disease (1). In particular, Raman spectroscopy has been widely investigated as a technique for cancer diagnosis. The advanced optical technologies, high chemical specificity, and lack of sample preparation give Raman the potential to be a minimally invasive, in vivo approach that can be used for the diagnosis of cancer and that is an aid in the treatment of cancer. Raman spectra have been shown to provide a clear distinction between cancerous and normal cells, and Raman spectroscopy’s ability to distinguish between different cancerous cell lines has been demonstrated (2). Currently, to determine if a tumor is cancerous or not, a biopsy is required. This approach is an invasive procedure that requires internal tissues to be removed from a patient and sent for an analysis by a highly trained professional, which is a process that can take weeks and can involve a painful recovery for the patient. One approach to overcome this time-consuming and highly invasive procedure is to pair fiber-optics with a hypodermic needle that can be inserted directly into a suspected tumor. Coupled with Raman spectroscopy, this technique could provide instant diagnostic results and eliminate unnecessary biopsies, and ensure that treatment can begin as soon as possible. In the past three years, Petterson and colleagues have tested a prototype model (3) and an improved successor that greatly reduced the signal-to-noise ratio and increased the sample area with an average accuracy of 97.5% (4). In addition to diagnosis, this probe could be used during surgery to guide and ensure complete tumor removal. No testing has yet been performed on patients, only on excised tissue samples. One study has investigated the detection of circulating tumor cells, released into the blood stream by malignant tumors in extremely small quantities, to develop prognoses or monitor therapy progression (5). Another research group used Raman spectroscopy to investigate the response of tumor cells to radiation, with the aim of monitoring progression and predicting whether or not the patient is likely to respond to radiation treatment (6).

	Cancer diagnostics is not the only healthcare field looking to further integrate Raman spectroscopy. In late 2016, researchers developed a contact lens that uses Raman to monitor glucose in tears (7). Though not the first lens designed to monitor glucose levels in type I diabetics, it is the first that uses a system of vertically stacked plasmonic nanostructures that act as a surface for surface enhanced Raman spectroscopy (SERS). Using solvent-assisted nanotransfer printing, gold nanowires are stacked on top of a gold film to create extremely dense and regular hot spot arrays. Standard monitoring of glucose levels in diabetics is very invasive and requires drawing blood or having a continuous monitor inserted underneath the skin. Before it can become a marketable, noninvasive device, however, a relationship that shows correlation between glucose levels in tears with those in blood must first be demonstrated.

	Healthcare has not been the only industry to benefit from recent advances in Raman technology. In 2020, NASA plans to send a new rover to Mars. One of its constituent instruments, SHERLOC, is a Raman spectrometer designed to search for signs of past or present life on Earth’s nearest neighbor (8). A new interpretation of Raman spectra might make this search easier. Researchers at MIT have developed a new technique for analyzing acquired Raman spectra that gives more information about the chemical makeup of the sample. So far, there has been no way to determine if a carbon signature is biological in origin or if some other chemical process created it. This information is exactly what this new analysis will be able to determine, by allowing researchers to estimate the ratio of carbon to hydrogen atoms by looking at the substructures in Raman spectra. When laser light hits organic matter, the resulting spectrum consists of two main peaks. The wide peak, also called the D (disordered) band, is a function of the carbon atoms that have a “disordered makeup” and are bound to many other elements. The narrow peak, or the G (graphite) band, corresponds to less disorganized structures. Researcher Nicola Ferralis and colleagues (9) found that substructures within the wide D band directly relate to the amount of hydrogen a sample contains. When more hydrogen is present, the sub-peaks are higher, indicating that the original chemical composition is relatively intact. By zeroing in on these samples of rock and soil that have changed little with time, the chances of determining if life ever existed on Mars or not increase. Though neither chosen nor designed with this new analysis in mind, the technique can be applied to spectra acquired from SHERLOC and help it home in on which samples to target for closer analysis.

	LIBS-In Situ Developments for Harsh Environments

	Laser-induced breakdown spectroscopy (LIBS), a type of atomic emission spectroscopy, is already a quick and portable in situ technology. It has the unique advantages of being able to take rapid data with minimal sample preparation and to operate in harsh environments. However, its use is limited by its primary constraint-sensitivity. This lack of sensitivity makes detecting trace elements difficult. The most common trend this field is seeing is various methods designed to enhance signal clarity and intensity during use in situ.

	Researchers at China’s Dalian University of Technology recently studied the effect containment in a magnetic field has on the signal intensity of LIBS (10). Their experiments show a direct correlation in the intensity of spectra (they looked at aluminum and lithium) with the intensity of an external magnetic field. As the field strength increases, the laser-induced plasma is confined to smaller and smaller areas, increasing the temperature and density inside and, consequently, the number of electron and ion collisions. These extra collisions result in a greater number of excited atoms that radiate light when their electrons transition between energy levels. The intensity of the signal acquired is therefore stronger. Though it may not be practical for every application, or even most applications, this particular study was designed for in situ monitoring of fuel retention and impurity deposition in tokomak fusion reactors.

	A group at the University of South Carolina is looking to apply LIBS to the study of underwater hydrothermal vents (11). Typically found near mid-ocean ridges 800–3600 m below the surface (at pressures of 80–360 bar), these vents expel hot water rich in minerals at temperatures as high as 400 °C. Such extreme conditions make chemical analyses of these vents and their products difficult, especially considering that irreversible changes are caused when samples are removed from their original environment. The goal is to be able to deploy a LIBS instrument to study these phenomena at the source. The group has shown single-pulse LIBS to be an acceptable analysis of ions in aqueous solution at pressures as high as 280 bar. Double-pulse LIBS would increase the sensitivity of these measurements, though it is so far unable to obtain results at as high pressures as the single-pulse technique can. Possible ways to remove this barrier for underwater double-pulse LIBS include changing the laser configuration and optimizing the optics, as suggested by the group working on this project.

	As of mid-2015, the metallurgical industry was relying on solidified metal samples that needed to be transported and tested after production for quality assurance. Researchers at the Chinese Academy of Sciences have found a way to analyze elemental components of molten steel, allowing for in situ quality testing concurrent with production (12). The ability of LIBS to take measurements under harsh conditions with no sample preparation made it an obvious candidate considering the high temperatures used in steel manufacturing. Techniques and systems to apply LIBS analysis to this process have been created and developed since the early 1990s. The greatest challenge to implementing any of these techniques has been the harsh environment and the toll it takes on the equipment. Approaches that have immersed probes into molten metal have issues with the long-term protection of the probe and the optics it contains. When using an open-path approach to analyze the surface of the melt, problems arise because of slag, a by-product, which sits on the surface of the target. A new approach encloses the optical and electrical equipment away from the high-temperature melt. Connected to the optics is a hollow steel tube tipped with a refractory lance that can be inserted under the melt surface. Using this double-pulse LIBS setup, the content of carbon, manganese, and silicon was measured in good agreement with more time consuming, traditional analysis methods.

	XRF-Improving the Study of Cultural Heritage Objects

	X-ray fluorescence (XRF) has long been used to study the pigmentation in painted works of art. It is a nondestructive technique with a wide range of small, portable instruments commercially available that can provide vital information on how to properly store or restore a painting, as well as valuable historical insight. The main limitation of this technique results from the effects of interlayer absorption. Recently, three major advances in this technology have made analysis of cultural objects easier and more effective.

	First, a new technique has emerged that couples an electron beam with a laser-based X-ray source (13). When the combined laser hits the target at a high intensity, the beginning of the pulse causes the target matter to be rapidly ionized. The tail end of the pulse interacts with the expanding plasma on the target surface with sufficient intensity to extract, accelerate, and then re-inject electrons. This interaction produces Bremsstrahlung radiation and the characteristic X-ray emission of the target. Using a collimator and a pair of magnets placed between the sample and the source, the electrons can be deflected back toward the sample.

	Compared to that of an electron beam, the wavelength of a X-ray is considerably longer and able to penetrate into multiple layers of the work. This can be of some importance, as old works have sometimes been found to have been painted on recycled canvas, complete with an older, separate painting underneath. Whereas X-ray–based sources provide information about both the surface and layers underneath, electrons are stopped in the first layer of paint and provide information about the pigment composition of the visible work only. The combination of information gained from both sources can provide easy, valuable insight into the structure of any painting.

	Second, since it produces optimal spectra, many of the portable, in situ XRF instruments that are commercially available today rely on polychromatic excitation. A team in Krakow recently described a theoretical basis showing that the correction for monochromatic absorption can be extended to polychromatic as well (14). Using a method based on the zeroing of the correlation coefficient, interlayer absorption effects can be taken into account and removed from spectra acquired using existing instruments and traditional X-ray methods during data analysis.

	Third, portable XRF systems are widely used to study cultural heritage objects that cannot be moved-the only data that can be acquired using the fluorescence provide information about the elemental composition of the piece. A few portable X-ray diffraction (XRD) instruments have been produced in the last 15 years, but these have left much room for improvement. Typically based on angle-dispersive X-ray diffraction (ADXRF), these instruments use low-energy X-rays that don’t penetrate many layers of an object and take a very long time to acquire the spectra. The Multidisciplinary Laboratory at the Abdus Salam International Center for Theoretical Physics has developed two new instruments using energy-dispersive X-ray diffraction (EDXRD) that have many advantages over the ADXRD ones (15). An unfiltered polychromatic X-ray beam allows for faster acquisition times and diffraction lines are collected simultaneously without mechanical movement of the detector or the source. Both prototypes combine X-ray fluorescence with X-ray diffraction based on EDXRD. Initial trials showed good analytical performance in the identification of elements, minerals, inert materials, and crystalline phases. The systems are compact and portable, using only one detector for both XRF and XRD measurements.

	A major advantage of infrared spectroscopy is its ability to discern functional groups in compounds that exist in any state of matter. It is nondestructive and only needs a clear sight line to analyze samples, making it ideal for in situ use with minimal invasion.

	Infrared spectroscopy is a label-free method commonly used to analyze biological samples in solution. The standard technique for this type of observation is Fourier transform infrared (FT-IR) spectroscopy, which works well if acquisition time is of no concern. To investigate dynamic systems in real time, however, a new approach is required. Researchers at the Kirchhoff Institute for Physics in Germany have coupled a quantum cascade laser (QCL) with an FT-IR microscope to monitor three different living systems (16). QCLs emit mid- to far-infrared radiation and allow for short acquisition times at discrete, midrange spectral frequencies as well as over a broad spectral range. Fermentation, a slow moving amoeba, and a fast moving nematode were observed in situ on a real-time scale, as defined by each respective process. The movements of the nematode were tracked using discrete frequencies and resulted in clear, non-blurry images. Substructures within the amoeba were identifiable in a slow moving specimen, but required a shorter acquisition time when a faster moving sample was observed. Overall, QCL-coupled FT-IR microspectroscopy has resulted in clear images of dynamic living processes at high magnification and high spectral resolution with an acceptable signal-to-noise ratio.

	FT-IR is already being used to identify microbes, but it has only recently been used to determine the species of fungi. Fungal infections in humans are difficult to treat, and the correct species must be identified before the correct treatment can begin. A team in Brazil (17) has successfully distinguished between two fungi from the 

 genus using attenuated total reflection FT-IR (ATF-FTIR) and multivariate analysis of the functional groups from each fungus. These two species, 

, cause infection of the central nervous system and the lungs when their spores are inhaled. This approach is faster and less labor intensive than traditional methods, and could be a cheap alternative to routine diagnostic analysis.

	Fungi not only infect humans directly, but can also cause infection via food contaminated with mycotoxins, which are a group of toxic chemicals they produce. Some of these contaminants are carcinogenic or immunosuppressive, while others cause acute infections. Compliance with the regulatory mycotoxin limits that have been imposed both nationally and internationally has raised the economic burden placed on food producers at all levels. Current mycotoxin detection methods are sensitive, including high performance liquid chromatography, gas spectrometry, thin-layer chromatography, and immunoassays. However, these methods are destructive, time consuming, costly, and often labor intensive. The destructiveness of these methods presents a problem-the number of samples measured has to be large enough to ensure the safety of the whole batch, but not large enough destroy a significant portion of it. The past few years have seen increasing research into the potential of IR spectroscopy as a nondestructive, quick, and cost-effective method for the detection of mycotoxins. One study (18) found IR spectra of functional groups analyzed using a bagged decision tree analysis to be 80–85% accurate in assessing contamination of corn and peanuts with multiple mycotoxin varieties. An important disadvantage of IR is that it cannot collect spatial information. Because of this disadvantage, another avenue being explored is near-infrared hyperspectral imaging (NIR-HSI), a technique that collects both physical and spectral data from a sample (19). Using two types of NIR-HSI instruments, correct mycotoxin contamination has been measured with over 90% accuracy. These easy IR methods have the potential to replace costly and destructive testing at all levels of food production, storage, and transport.

	Inductively coupled plasmas (ICPs) are created through electromagnetic induction. These plasmas are often used as energy sources for spectroscopic techniques. A sample must be converted into an aerosol and injected into the plasma to be analyzed. This requirement makes these techniques destructive and not ideal for analysis in situ or in vivo. However, these are still important techniques that can measure extremely low element concentrations and that continue to push into new industries.

	Single-particle inductively coupled  plasma–mass spectrometry (ICP-MS) has been around for over a decade, but has only just begun to attract serious interest. In general, it is used to characterize nanoparticles in aqueous solutions that are so dilute that statistically only one nanoparticle enters the plasma at a time. Mean size, size distribution, number concentration, and mass concentration in a solution can all be determined using this method. Any standard ICP-MS instrument will work when analyzing single nanoparticles if the sample is correctly prepared before intake, but the data that is acquired requires heavy reduction and laborious analysis to remove background noise. Data programs are being developed to help remove this analysis burden, as well as instruments specifically designed for single-particle ICP-MS that will eliminate the need for the extra reduction. The past few years have mostly seen theoretical studies trying to work out the optimal operating conditions, but most recently the emphasis has shifted to actual application-based studies (20). A new method called “the fast acquisition speed technique” that used a 0.1-ms dwell time was combined with a new algorithm to decrease the sensitivity threshold when measuring particle diameter from 10 nm down to 6.4 nm (21). With more improvement in instrument design and data analysis techniques, single-particle ICP-MS is a promising technology for the analysis of water in treatment plants (22), detection of nanoparticles in food (23), and the analysis of soil samples (24).

	Raman and IR have not been the only spectroscopic technologies applied to the healthcare industry. ICP-MS has recently been investigated as a diagnostic tool. Researchers in Poland looked for a correlation between the concentration of metals in the blood and saliva of patients and periodontal disease (25). These metals can be present in low concentrations and thus require a highly sensitive analytical technique, such as ICP-MS or ICP-optical emission spectrometry (OES). The group found a correlation between the disease and the elevated presence of three elements (Cu, Mg, Mn) in saliva. Using ICP-MS, all three elements can be detected simultaneously with no painful, costly blood draw required.

	New applications continue to be found across all fields of spectroscopy, particularly when that technique is faster and can easily replace old methods. ICP-OES has found a new use in measuring the content of TiO

 in pigments (26). This simple method consists of first dissolving the target sample in sulfuric acid and then feeding the solution into the ICP-OES instrument. Previous methods, including titrations, atomic absorption spectroscopy, and the Jones reductor method (27), were time consuming, nonstandard, or required more-volatile components. The ICP-OES method is a fast and simple way to quantitatively measure TiO

 concentrations in paints. This technique has already been adopted into standard practice at the Institute of Technology of Parana in Brazil, the home institution of the group who developed this application.

	As the techniques of analytical spectroscopy evolve, researchers continue to find new and innovative ways to capitalize on their strengths and improve upon their weaknesses. Much of this development in recent years has been seen in experimental design, data analysis, and the coupling of multiple instruments. This innovation has allowed for new methods and applications that facilitate minimally invasive, in situ, and in vivo approaches to spectroscopic analysis.

		L.M. Fullwood et al., Proc. SPIE 9704, 

Biomedical Vibrational Spectroscopy 2016: Advances in Research and Industry,

 97040G (March 7, 2016); doi:10.1117/12.2230005.

		G. Webster, “SHERLOC to Micro-Map Mars Minerals and Carbon Rings,” 

http://mars.nasa.gov/mars2020/news/whatsnew/index.cfm?FuseAction=ShowNews&NewsID=1680 

Biomedical Vibrational Spectroscopy2016: Advances in Research and Industry,

 97040J (March 7, 2016); doi: 10.1117/12.2213239

		Standard Test Methods for Chemical Analysis of White Titanium Pigments (ASTM), D 1394–76, USA, 2003, 246.

 is a freelance science writer. She works in research and development in St. Paul, MN. Direct correspondence to 

Analytical spectroscopy is a mature field, but advances in instrumentation, measurement, and sample preparation techniques continue to be made. Much recent development has seen a heavy emphasis towards techniques and instrumentation that allow for non-invasive, in-situ, and in-vivo procedures while still retaining a high degree of sensitivity. This article will review some of this progress over the past two to three years across five major spectroscopic fields: Raman, LIBS, XRF, IR, and ICP techniques.

Nicole Olson

Articles

The 2018 Emerging Leader in Molecular Spectroscopy Award

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Recent Trends in Analytical Spectroscopy: A Push Toward In Situ, In Vivo, and Minimally Invasive Techniques