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

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-From Human Health to Mars

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.

IR-Observing Complex Biological Systems

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

ICP-New Techniques and Applications

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 TiO₂ 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 TiO₂ 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.

Conclusion

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.

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Nicole Olson is a freelance science writer. She works in research and development in St. Paul, MN. Direct correspondence to nicolerose5482@gmail.com.