In this interview, Dr. Claudia Conti, a senior researcher at the Institute of Heritage Science (ISPC) of the Italian National Research Council (CNR), talks about her work in microspacially offset Raman spectroscopy (micro-SORS). Micro-SORS is a new method for the chemical analysis, identification, and assessment of decay of cultural heritage objects. The ability to measure the chemistry of multilayered, micrometer-scale structures is providing the answers and knowledge base from which to catalog and construct artwork histories and the decay processes of priceless cultural treasures. Before the development of micro-SORS, subsurface molecular analysis of valuable objects was performed using an invasive and destructive cross section approach, which for obvious reasons is completely unsatisfactory. The use of micro-SORS provides a breakthrough in noninvasive and nondestructive analysis of the subsurface layers of artworks and is a key development in the understanding of conservation science for cultural heritage objects. Conti is the winner of the 2020 Craver Award presented by the Coblentz Society, to be given at the 2020 SciX conference for her Raman spectroscopy research. This interview is part of a series of interviews with winners of awards presented at SciX.

You have worked with SORS related research for several years and were able to demonstrate its utility for analysis paintings (1). Specifically, you were able to penetrate the topmost highly turbid layers of paintings to measure and analyze the pure Raman spectra of paint sublayers completely obscured by the paint overlayers. What prompted you to explore this approach to surface layer analysis? Since this work, do think that SORS has lived up to your expectations, or from your perspective are there still further instrumentation or method developments that are required to make this an optimized analysis method?

After my PhD in Materials Engineering at Milan Polytechnic (2010), I searched for new innovative solutions to face critical needs in conservation of cultural heritage. My supervisor, Professor Giuseppe Zerbi, suggested that I attend the ICORS conference in Boston (2010), where I became aware of a novel tool developed by Professor Pavel Matousek that enables Raman sensing deep inside turbid media on a macro-scale. Right from the beginning, I realized the strong impact that this method could have in conservation sciences, permitting the noninvasive molecular identification of pigments and decay and conservation products through the stratigraphy volume of paintings, frescoes, painted sculptures, and so forth. In fact, the knowledge of the subsurface composition of artifacts is critically important in conservation sciences: Beyond the surface there are a number of hidden compounds, selected by the artist or formed during the decay processes or applied over the centuries for conservation purposes. Scientists are required to provide the conservators and art historians with the elucidation of these puzzles to facilitate comprehensive knowledge of these objects to enable proper planning of the conservation actions; to reach this goal we need to develop noninvasive methods, one of the most vital research activities of the last decades in our field.

At the beginning of the collaboration with Professor Matousek, macroscale SORS did not live up our expectations because it did not work with our samples. Then we tried to find an alternative way to discriminate between painted layers, extending SORS to the microscale by combining SORS with microscopy. In fact, conventional SORS is only applicable to relatively thick layers (on the order of millimeters or greater), whereas the thickness of typical overlayers of the concealed substances in artworks is an order of magnitude or two lower. After almost six years of microSORS research, I would say that this method exceeded my expectations because we have found a wide range of successful applications both within as well as outside the field of cultural heritage. Based on these promising achievements, and also taking into consideration existing limitations, we are now focusing on further technical and methodological developments to extend the noninvasive molecular subsurface investigations to more complex situations, for example, the detection of the diffusion of conservation treatments into decayed substrates and also the micro-SORS measurements in situ with portable devices.

What are the real advantages of using micro-SORS for probing through thin painted layers compared to other analytical methods, including confocal Raman microscopy and conventional Raman spectroscopy?

In-depth probing using conventional backscattering confocal Raman microscopy is applicable only to semi-transparent materials, since in turbid media the diffusion of the light in the sample leads to subsurface Raman signal being often overwhelmed and obscured by surface Raman signals and the deep photons cannot be discriminated by conventional confocal optics. These deeper generated Raman photons are subject to a large number of scattering events and consequently end up spread further apart from each other as they reach the surface. Cultural heritage materials are mainly turbid; transparent varnishes are an exception to this, and these can be scanned with conventional confocal z-scanning. Pigments, decay products, and other turbid compounds, as well as tissues and powders, need to be scanned with micro-SORS, which is able to acquire and discriminate the contribution of the sublayer from that of the top layer, even when dealing with highly diffusive materials. This is achieved by enlarging the illumination and collection areas or by collecting the Raman signal from an area which is spatially offset from the laser illumination point. So, in other words, before the development of micro-SORS the only feasible way to study the stratigraphy of cultural heritage objects with Raman involved destructive cross-sectional analysis, which should be avoided in this context.

Other analytical methods have been developed for the knowledge of the subsurface of cultural heritage materials by non-invasive means, such as multispectral, hyperspectral and Terahertz imaging, X-ray radiography and infrared reflectography, scanning macro and confocal X-ray fluorescence, and optical coherence tomography. The specificity of micro-SORS is the direct retrieval of the molecular composition of organic, inorganic, crystalline and amorphous compounds, not fully accomplished by these alternative techniques.

You have applied micro-SORS to artifacts of historical significance originating from Italy and dating from the medieval to contemporary periods (2,3). What discoveries you have made on rare artifacts using this Raman technique?

First of all, I would highlight that our research produced a methodological discovery, going beyond the specific results achieved on the analyzed artworks. We were able to “see” through the surface in a non-invasive way, and get information about the presence of hidden pigments, decay products, and preparation layers spread over different substrates such as paper, plaster, and stone. To name just a few examples of discoveries in artworks, we have analyzed prestigious Renaissance terracotta sculpture of the “Sacred Mounts” (UNESCO World Heritage sites), which have been repainted several times over the centuries, thus exhibiting a complex stratigraphy made of a number of superimposed micrometric layers. Conventional confocal Raman microscopy on the intact fragments identified only the pigments on the sample surface, and was prevented by the interfering surface signal from seeing the composition of individual layers below the surface; in contrast, a micro-SORS analysis on the same intact fragment allowed the presence of pigments as separate (deeper) layers to be ascertained. It is also important to be aware of the method limitations highlighted by the parallel cross-sectional analysis carried out to validate the micro-SORS results: A number of compounds may not be “visible” due to their weak scattering cross section or fluorescence, or due to their depth being deeper than the accessible depth of the technique. However, micro-SORS has demonstrated the possibility of recovering the pigment information from below the surface in a non-destructive way and thus reconstructing, even partially, the sample stratigraphy.

A second example concerns a publicity card from the 19th century that we placed directly under the Raman microscope objective without any sampling. We performed mapping of selected areas of the card, and, in all the micro-SORS sequences, the Raman signal of a pigment, white lead, which was not visible to the naked eye because it is covered by the external pigments, emerged from the subsurface. This outcome indicates the presence of a layer, on top of the card paper and below the external layer, applied for the preparation of the painting. Moreover, in a red part of the card, above the most internal white lead layer and below the external red layer made of cinnabar, we observed the presence of another, intermediate layer made of Prussian blue. This is important information for the understanding of artist’s technique and for conservation purposes and we achieved this in a noninvasive way.

You were able to expand the use of micro-SORS from cultural heritage objects to a variety of other sample types where detailed non-invasive analysis of diffusely scattering turbid layers were involved, including polymers, seeds, and paper (4). What was most surprising to you relating to technique or sampling requirements as you moved from cultural heritage objects to other materials? Are you able to image or create images of surfaces using micro-SORS?

One of the most exciting experiments outside cultural heritage was the investigation by micro-SORS of the structure of the wheat seed, research carried out in collaboration with the Norwegian Institute of Food. The identification of diseases at an early stage is very important for the grain producers, therefore the use of rapid and non-destructive methods for characterization of inner grain components is essential. The seed has an external micrometric envelope made of ferulic acid and an internal kernel containing starch. Micro-SORS experiments made it possible to unequivocally discriminate the Raman spectrum of the envelope from that of the starch, without touching the seed.

As for the second question, we carried out an exciting experiment concerned the reconstruction of images hidden by a turbid overlayer. We simulated in a laboratory environment real situations encountered in cultural heritage that deal, for example, with hidden paintings vandalized with graffiti or covered by superimposed painted layers or whitewash. One of the mock-up specimens was prepared by painting the letter “F” using phthalocyanine blue over a whitewashed background; two pieces of paper colored with pink ink were used as the top layer. Conventional mapping showed only the Raman signal distribution of the pink ink, whereas, with the chemical image obtained with micro-SORS mapping, we were able to “read” through this turbid overlayer and reconstruct the graphic symbol of the letter “F.”

In one research application of micro-SORS, you analyzed medieval polychrome sculptures from the Parma baptistery and Ferrara cathedral (5). What specifically was learned from these objects?

In a sculpture of the Ferrara cathedral portal, painted with a red hue, we discovered that, under the external red layer made of cinnabar, a second hidden red layer is present and made of minium (a bright orange red pigment made of red lead), indicating that the external cinnabar layer is most likely a repainting. In fact, it is well known that the portals of European cathedrals could have been subject to more than one decorative phase, probably whenever the color started to look shabby due to the outdoor environment and natural degradation, and with micro-SORS we had direct analytical evidence of this historical information. Moreover, in the same sample we have found the presence of two salts, related to decay processes; micro-SORS sequences and the related spectral subtractions allowed us to ascertain that they are mainly diffused on the surface and within the cinnabar layer. This is an outstanding outcome, since it informs us about the migration and the re-crystallization processes of soluble salts within the layers, with a consequent effect on the mechanical stress of the entire system.

What were some of the key challenges you encountered during your research? How did you overcome them?

The first major challenge emerged after the development of the first micro-SORS setup, called defocusing. This is the most basic and easily deployable micro-SORS variant, but it is not as effective as a full micro-SORS setup because there is not a real separation between laser illumination and Raman collection areas. We realized that a more sophisticated variant could be necessary to investigate complex and heterogeneous materials as encountered in cultural heritage. With limited funds, we focused our research for more than one year on searching for the best technical solution; these research efforts ultimately led to the successful modification of a conventional micro-Raman, able to separate the laser and Raman paths. In this way, we fully extended the macro-SORS to the microscale.

One of the major obstacles in Raman spectroscopy is the fluorescence emission from the sample, especially when dealing with cultural heritage materials. We have faced a critical situation when the top layer is fluorescent and we needed to recover chemical information from the subsurface. It was tricky because it is well known that fluorescence signal can be much more intense than Raman, and, furthermore, in our case the target compound was covered by a turbid overlayer. The challenge was very high but we applied the analytical capability of micro-SORS in the most advanced variant and it worked; at the imaged position the Raman spectrum was overwhelmed by the fluorescence of the top layer, whereas at a certain value of spatial offset the signal of the subsurface kept increasing, emerging clearly from the fluorescence background.

What would you consider to be the most meaningful contributions of your research work? What discovery has most impressed you?

Recently, we obtained an outstanding result: We analyzed with micro-SORS two 16th century panel paintings by Marco d’Oggiono, using a modified portable instrument directly in situ. This is one of key goals of our research since the beginning, namely the development of a portable device able to carry out micro-SORS without any restrictions on the dimensions of the artwork or its location. In fact, cultural heritage objects cannot be easily moved and transferred to our laboratory for analytical purposes; we have to find a way to analyze them where they are (museum, churches, historical buildings, and so forth). In this context portable micro-SORS can be considered a very important development, providing a further step toward establishing a more powerful multianalytical approach, essential when dealing with complex materials as encountered in the cultural heritage field. Portable micro-SORS could be relevant also outside the art field, as in forensic science and solar cells analysis. Further optimizations of our current portable device are still required to improve its stability and the sensitivity; this work is in progress.

Would you share with our readers your philosophy of how to work in a research environment?

From my experience, I learned that curiosity triggers the research. This may seem trivial, but, in practical terms, I noticed that it is not easy to keep our minds open to new inputs, accepting the risk of going beyond our comfort zone and far from what we already know. Making curiosity prevail over habit is one of the most prolific exercises we have to perform every day; how does it help me? To find researchers with whom to share passion for knowledge and innovation, someone to learn from, and create a research team available to overcome limitations and barriers. I will never forget when my PhD supervisor, Professor Zerbi, pushed me to broaden my horizons presenting my analytical results at one of the most important spectroscopy conferences in the United States when I still was a PhD student. I felt inadequate for that, but I went to the conference, and I discovered that curiosity and boldness were an integral part of my work. The power of our micro-SORS research is the fruitful and unceasing collaboration between CNR Italian and UK teams, providing the necessary incentive to face the numerous challenges of the research.

1. C. Conti, C. Colombo, M. Realini, G. Zerbi, and P. Matousek, 

2. C. Conti, C. Colombo, M. Realini, and P. Matousek, 

3. A. Botteon, C. Colombo, M. Realini, S. Bracci, D. Magrini, P. Matousek, and C. Conti, 

4. C. Conti, M. Realini, C. Colombo, K. Sowoidnich, N.K. Afseth, M. Bertasa, 

5. C. Conti, A. Botteon, C. Colombo, D. Pinna, M. Realini, and P. Matousek, 

Claudia Conti is a permanent researcher at the CNR-ISPC (Institute of Heritage Science), unit of Milan. Conti obtained a degree in geological sciences at the University of Perugia in the field of archaeometric problems, and a PhD in materials engineering at the Politecnico di Milano working on the analysis of chemical stability of calcium oxalate in conservation and in historical films. Conti has established expertise in the area of advanced applications of vibrational spectroscopy to material analysis, particularly in the field of cultural heritage. In 2012, she established a major collaboration with the Rutherford Appleton Laboratory (UK) to explore a novel tool, Spatially Offset Raman Spectroscopy (SORS) that enables Raman sensing deep inside turbid media on macroscale. This collaboration lead to the successful transformation of macro-SORS conceptually to the area of cultural heritage and the demonstration of a new SORS variant, micro-SORS, capable of resolving micrometer thick layers of paint for the first time. Outside cultural heritage, the most relevant impact of her research on micro-SORS concerns the biomedical area, polymer, paper, and food fields.

Articles

Spectroscopy Magazine spoke with Claudia Conti about her work in micro-SORS.

Craver Award: Micro-Spatially Offset Raman Spectroscopy (Micro-SORS) for Research in the Conservation of Cultural Heritage 

Have you used Raman spectroscopy? If not, you’re really missing out, and now is a great time to try. Raman spectroscopy has come a long way since its discovery in the 1920s. Named after the Indian scientist C. V. Raman, who won the 1930 Nobel Prize in physics, Raman spectroscopy had remained for a long time mostly a specialized tool only accessible to trained scientists. Since the early 2000s, however, advances in laser technology, fiber optics, and grating technology have come together to break down technology barriers and revolutionized and democratized Raman spectroscopy technology.

Most modern Raman spectroscopy instruments focus a laser beam to a tight focal point where Raman signals are generated. The back-scattered signals are then collected and dispersed using a grating onto a charge-coupled device (CCD) camera to generate Raman spectra. Such vibrational spectra contain information similar and complementary to that provided by infrared spectroscopy. The sampling mode in Raman spectroscopy is highly versatile, and can be achieved using microscopes or probes in both contact and non-contact fashion, and even in a remote-sensing setup. The development of these versatile sampling capabilities has enabled users to carry out many unique types of analysis not previously possible, such as the non-invasive analysis of historical paintings or archaeological artifacts.

Advances in chemometrics, big data, and artificial intelligence are now enabling maximum extraction of the rich information embedded in Raman spectra. Compared to separations-based analytical technologies such as gas chromatography (GC) and liquid chromatography (LC), chemical resolution by Raman spectroscopy is achieved through analyte spectral separation, rather than through physical separation of analytes via the use of separation columns. Analysis of convoluted spectra has often been a bottleneck in Raman spectroscopy, but this challenge is being addressed through the use of the abovementioned computational tools.

Raman spectroscopy has found its way into numerous application fields, a number of them shown in Figure 1. The reason behind such a wide range of successful applications stems from the robustness and versatility of modern Raman spectrometers, as well as the rich information available from the Raman spectra. Robust and miniaturized Raman instruments enable users to deploy Raman spectroscopy not only in everyday life, such as for the analysis of microplastics and the identification of unknown substances by law enforcement, but also in remote reaches of science, such as deployment on the Mars rover to study the geology of the red planet.

Numerous commercial options for portable and handheld Raman spectrometers are available. This range of options makes it a “buyers” market for users interested in exploring Raman spectroscopy for their own unique applications, but may also make it a daunting task to select the right tool. Interested readers are recommended to find expert opinions on such topics from professional organizations such as the Society for Applied Spectroscopy (https:// www.s-a-s.org/index.html) and the Coblentz Society (http://www.coblentz.org/).

Raman spectroscopy is a maturing technology, and there are many areas under active academic and industrial investigation. For example, surface enhanced Raman spectroscopy (SERS) enables orders-of-magnitude improvements in detection limits. Spatially offset Raman scattering (SORS) allows chemical analysis of objects beneath opaque packages. Tip-enhanced Raman spectroscopy (TERS) overcomes the diffraction limit and offers chemical information with a spatial resolution on the order of tens of nanometers. From the nanoscale to microscopy to applications of handheld and portable instruments to analyzing industrial-scale reactions, Raman spectroscopy is a widely used and valuable analytical technique.

Raman Spectroscopy

Working at the frontiers of biotechnology, fiberoptics, lasers technique, and molecular spectroscopy, Tuan Vo-Dinh of Duke University has developed multiple sensor technologies useful for medical research and diagnostics. In this interview, he talks about his work in spectroscopy and photonics. Vo-Dinh is the winner of the 2019 Royal Society of Chemistry (UK) Sir George Stokes Award and is scheduled to give a plenary lecture at the 2020 SciX conference. This interview is part of an ongoing series with the winners of awards that are presented at SciX.

Working at the frontiers of biotechnology, fiberoptics, lasers technique, and molecular spectroscopy, Tuan Vo-Dinh of Duke University has developed multiple sensor technologies useful for medical research and diagnostics. His contributions include the first antibody-based fiberoptics fluoro-immunosensor (FIS), the first antibody-based nanoprobe for monitoring biochemical species in a single living human cell, the first paper on the analytical use of surface-enhanced Raman spectroscopy (SERS) for chemical analysis, the development of a laser-induced fluorescence (LIF) technique for non-invasive optical biopsy, and plasmonic nanoprobe assays for early cancer diagnostics. Throughout this work, Vo-Dinh and his research colleagues have brought spectroscopy to biomedical applications. In this interview, Vo-Dinh talks about his work in spectroscopy and photonics. Vo-Dinh is the winner of the 2019 Royal Society of Chemistry (UK) Sir George Stokes Award and is scheduled to give a plenary lecture at the 2020 SciX conference, which takes place October 11–16 in Sparks, Nevada. This interview is part of an ongoing series with the winners of awards that are presented at SciX.

For many years, you have worked in the development of analytical capabilities for antibody-based fiberoptics biosensor probes using the concept of the fluoro-immunosensor (FIS) (1). In early work on FIS, you had proposed that these sensing probes would prove useful in detection of various analytes for cancer research, environmental monitoring, and other health applications. How did you become interested in this specific analytical approach, and how have these tools proved effective or useful in clinical applications?

Throughout my research career, I have always been interested in working at the intersection of multiple scientific disciplines. I strongly believe that the integration of different areas of science and technology is needed to provide solutions to complex problems of global importance. More than three decades ago, I collaborated with a team of chemists, spectroscopists, and biologists from Oak Ridge National Laboratory (ORNL) and the University of Tennessee to integrate molecular spectroscopy, fiberoptics technology, laser techniques, and molecular biology; together, we developed the first antibody-based fiberoptics fluoro-immunosensor (FIS) device. The FIS is a fiberoptics biosensing device that combines antibodies designed to recognize and trap target molecules of interest, laser excitation to induce fluorescence emission from the targets, and ultrasensitive optical detection. The FIS concept was quite a “novelty item” at that time. This technology has now opened new possibilities for a wide spectrum of chemical, environmental, and biomedical clinical applications, such as the assessment of an individual’s exposure to chemical carcinogens, monitoring response to drug therapy, and identification of toxic substances in the environment. For instance, we demonstrated that the FIS is capable of detecting DNA-adduct biomarkers of human exposure to carcinogens in human clinical samples. The FIS technology opened new horizons for a fundamental technology of the “smart catheter/sensor” for in vivo biomedical analysis. Today, the usefulness of fluoro-immunobiosensors as powerful bioanalytical tools is well recognized worldwide.

In the 1990s, we expanded the biosensing concept in a single-fiber approach to a multi-sensing platform, the multifunctional biochip by combining microelectronics, molecular biology, and biotechnology. The device was one of the first chip-based 2D array biosensor systems combining molecular bioreceptors with CMOS phototransistors integrated circuits. It was capable of performing simultaneous detection of multiple disease endpoints on a single platform by using different bioreceptors such as antibody and gene probes. The biochip could be used to diagnose genetic susceptibility and diseases, monitor exposure to bioactive environmental samples, and provide an important tool for medical diagnostics at the point-of-care and homeland defense applications.

You have been involved in a clinical study with the development of a laser-induced fluorescence (LIF) technique for non-invasive optical biopsy (2). How is this approach different from the traditional biopsy procedures currently used for cancer diagnostics? Would you discuss this technology and its potential applications?

In situ, rapid, and minimally invasive procedures for tissue diagnosis are important for early detection and timely treatment of cancer. Traditionally, endoscopy is used to detect abnormal tissues in the esophagus. Once an abnormality is found, biopsies are taken for determination of histopathology. The laboratory results are generally not available for several days. There was an urgent need for a more practical technique that would allow real-time in vivo classification of the tissue type and provide a less invasive tool for cancer diagnosis at lower cost. In this clinical work, I collaborated with clinical colleagues at the Thompson Cancer Survival Center, in Knoxville, Tennessee, to develop a laser-induced fluorescence (LIF) technique that rapidly provided effective indices to diagnose cancer in the esophagus. LIF measurements were conducted during routine gastrointestinal endoscopic examinations of patients using a fiberoptic probe inserted into the biopsy channel of an endoscope. This LIF diagnostic information could be available in real time without the need of surgical biopsy. The LIF measurement was completed in approximately 0.6 seconds for each tissue site. The results of this optical approach were compared with histopathology results of the biopsy samples and indicated excellent agreement in the classification of normal tissue and malignant tumors. The LIF method was successfully tested with over 100 patients, with nearly 100% accuracy. The LIF method could lead to the development of a rapid, minimally invasive, and cost-effective technique for cancer diagnosis. Such a technique that is minimally invasive, requires no physical biopsy, and provides instantaneous results could revolutionize cancer diagnostics as we know it.

You have also worked for many years on the development and improvement of SERS (3,4). What were some of the most valuable or surprising discoveries that you encountered during your research in SERS? Has this technique proven to be a useful tool for trace organic analysis and other applications? What comments would you have regarding the analytical figures of merit and limitations of the SERS technique?

The SERS effect was discovered in 1977 by R. Van Duyne, J.A. Creighton, and coworkers, who reported an enormous intensity enhancement of Raman signals for some chemicals adsorbed on roughened electrode surfaces. In 1984, my team reported the first demonstration of the analytical potential of the SERS effect. Until our work, the reproducibility and quantification of SERS-active media (mainly electrodes, silver sols) had been a major difficulty for SERS to be used in chemical analysis. An additional limitation in the early development of SERS was that the Raman enhancement had been observed for only a few highly polarizable small molecules, such as pyridine, benzoic acid, and their derivatives. Even the general applicability of the SERS effect was still questioned in the early 1980s. Our work on SERS analysis of several polycyclic compounds contributed to firmly establish the general applicability of the SERS phenomenon. We introduced a new type of SERS-active substrate based on a silver metal film on arrays of 30-nm nanoparticles. In a sense, more than three decades ago and long before the hype about nanotechnology, we were already adopting “nano” when nano wasn’t “cool.” The kind of SERS-active substrates we developed, also referred to as nanowaves or metal film on nanoparticles (MFON), has led to substantially improved reproducibility and quantification. Since then, they have been widely adopted and used by other research groups.

My lab has also developed a wide variety of other types of SERS-active platforms, such as nanorods, nanodots, nanowires, nanostars, and nanorattles. Our SERS nanoplatforms have multiple applications in environmental sensing, bioenergy research, biomedical diagnostics, and molecular imaging. In 1994, we introduced a novel type of nonradioactive SERS-based DNA probe that could be used for genomic analysis, biomedical diagnostics, and pathogen detection. A decade later, we developed a novel type of SERS-based nanoprobe, referred to as molecular sentinels (MS) and inverse molecular sentinels (iMS), that can be used for the detection of early biomarkers of diseases. The iMS homogeneous assay format requires no washing steps and no sample amplification, due to the intense signal amplification of SERS. We used the iMS assay to monitor a class of molecular targets known as microRNAs. MicroRNAs have demonstrated great promise as important biomarkers for early detection of various cancers, yet these small molecules have not been adopted into early diagnostics for clinical practice because of challenging analytical aspects in the lab. Microarray-based technology, northern blotting, sequencing, and quantitative real time polymerase chain reaction (qRTPCR) analyses are often employed, but these lab-based techniques involve elaborate, time-consuming, and expensive equipment. Our team demonstrated successful detection of microRNA biomarkers for esophageal adenocarcinoma within patient tissue samples without requiring sample amplification (no PCR needed). Our studies have demonstrated the potential of the SERS-based nanoprobes for the detection of cancers (gastrointestinal cancer, and head and neck cancer) and infectious diseases (HIV, dengue, malaria). Our SERS nanoprobe approach could lead to an accessible strategy for early diagnosis, which is a major unmet need in screening, and will have transformative potential for medical research as well as diagnostic applications at the point of care.

I am also fascinated by the contribution of science and technology to the sustainability of our world. In this line of research, I worked with a team of bioengineers and plant biologists at Duke and physicists at Argonne National Laboratory to further develop the SERS iMS biosensing technology for renewable bioenergy research. Knowing when and which genes turn on inside a plant would be enormously useful to scientists, because they could potentially select plants with preferred growth patterns or control plant growth for optimizing biofuel production. We used our technology for sensing and imaging specific microRNAs that regulate flowering and vegetative growth in plants. We demonstrated the possibility to detect and image genomic biomarkers in living plant leaves under ambient conditions without requiring sample extraction and lab-based analysis. This research sets the basis for functional in vivo imaging of genomic biomarkers in plants, a much-needed tool for in vivo plant biology research and biofuel development.

In your work you have synthesized gold nanostars for use in SERS (5). Are you able to tune these nanostars for specific applications? What is the theory and modeling approach that can be used to tune these? For example, you have reported that variations in star size resulted in observed shifts of the long plasmon band in the near-infrared (NIR) region.

Among various kinds of metallic nanoparticles, gold nanostars (GNS) are of particular interest, as they offer optical tunability by engineering subtle changes in their geometry. Incident light (for example, a laser beam) irradiating metallic nanoparticles or nanostructures induces oscillations in the metal conduction electrons. These oscillating electrons, which are called surface plasmons—hence the term “plasmonic nanoparticles”— produce secondary intense electric fields concentrated at high curvature points on nanoparticles. The multiple sharp branches of GNS are ideal structures to create the so-called “lightning rod” effect that enhances the local electromagnetic field dramatically, thus producing intense SERS signals from molecules that are on or near the branch tips, in very good agreement with our theoretical calculations. Recognizing this unique property, my lab first proposed GNS as a platform for SERS sensing.

With the goal of developing biocompatible nanoparticles for in vivo biomedical applications, we first introduced a new surfactant-free synthesis of GNS that does not require toxic surfactant chemicals often used in nanoparticle synthesis. We demonstrated that GNS optical properties could be engineered by making subtle changes in their synthesis chemistry that resulted in geometry changes. For biomedical applications involving in vivo excitation in deep tissue, we demonstrated that an increase in the branch number of GNS can shift their peak absorption band into the near-infrared (NIR) region (700–1200 nm). This spectral range is very important for in vivo medical applications, and is often called the “tissue optical window,” where photons are less absorbed by tissue, and can travel further in tissue for sensing and therapy.

We can also control the size of GNS such that they can passively accumulate in tumors selectively by taking advantage of the enhanced permeability and retention (EPR) effect of tumor vasculature for cancer therapy. The EPR effect is a result of the inherent leakiness of the tumor vasculature, which allows nanoparticles having certain sizes to escape the circulation and accumulate passively in tumors. In addition, retention of nanoparticles in the tumor is enhanced by the lack of an efficient lymphatic system that would normally carry extravasated fluid back to the circulatory system.

For light-induced photothermal therapy applications, we showed that GNS are excellent nano-sources for heating tumor cells” from the inside.” In hyperthermia treatment of tumors, elevating the temperature to more than 42 °C, malignant cells are killed through apoptosis or necrosis. Traditional hyperthermia modalities, such as microwave, or radiofrequency, and focused ultrasound, have been used to control macroscopic heating around the tumor region, but cannot target or ablate cancer cells at the microprecision scale. GNS, which accumulate preferentially in and around cancer cells, can be triggered with light to rapidly achieve high ablative intratumorally temperatures, and can also induce milder fever-range hyperthermia in the tumor microenvironment. We demonstrated that GNS-mediated treatment with a near-infrared laser provides a controllable method to photothermally treat sarcoma in the murine tumor model. We also showed that, in a brain cancer mouse model, it was possible to achieve selective accumulation of GNS within target brain tumors but not in adjacent normal brain tissue, demonstrating spatial specificity in focal disruption of brain tumor vasculature and the blood-brain barrier for potential drug delivery applications. The combination of the EPR effect and the capacity for efficient photon to heat conversion make GNS an ideal photothermal transducer for selective cancer therapy at the cellular level.

Your research in 2000 described an antibody-based nanoprobe for in situ measurements of a single cell (6). The nanoprobe you selected used antibody-based receptors targeted to a fluorescent analyte, benzopyrene tetrol (BPT). You had hoped that the detection of this BPT nanoprobe could be used as a biomarker for monitoring DNA damage due to exposure to the carcinogen benzo[a]pyrene (BaP), and that this may result in a technique for possible precancer diagnosis. Would you tell our readers how these types of probes have been applied, and what clinical applications or additional research has resulted from this work?

Two decades ago, we developed the first antibody-based nanosensor capable of monitoring chemicals in a single living cell. The nanosensors had tiny probes fabricated with optical fibers pulled down to tips with distal ends of approximately 30 nm. The nano-scale size of this new class of sensors allows for measurements in the smallest of environments. One such environment that has evoked a great deal of interest is that of individual living cells. Using these nano-biosensors, it has become possible to probe individual chemical species in specific locations throughout a cell. The fiberoptics nanoprobes were covalently bound with antibodies designed to target benzopyrene tetrol (BPT), a biomarker of DNA-adducts of the carcinogen benzo[a]pyrene (BaP). DNA adduct formation involves the binding of a compound to DNA, a process that could damage DNA, resulting in abnormal replication and, without proper DNA repair, could lead to a cancerous cell. Monitoring DNA adducts is a strategy that could be used to monitor the extent of environmental exposure and health effects on people before occurrence of cancer. Such a precancer diagnosis approach could lead to appropriate pretreatment or early prevention actions. We focused on the detection of BPT because it is a biomarker of human exposure to BaP, a byproduct of combustion processes and a compound of great environmental and toxicological interest, due to its mutagenic and carcinogenic properties and its ubiquitous presence in the environment.

We also used nanobiosensors to monitor a molecular signaling process, apoptosis, in a single living cell that was treated with a photodynamic drug for cancer. Apoptosis or programmed cell death is a process by which cells degenerate during normal development, aging, or disease. When the process of apoptosis malfunctions, it can lead to clinical problems such as cancer and neurodegenerative disease. Drugs are often designed to provoke apoptosis selectively in cancer cells. Unlike traditional bioanalytical techniques, the nanobiosensor technology could probe the cell machinery, elucidating life-and-death processes such as apoptosis. This will help us better understand and cure disease occurring at the cellular and molecular level that were heretofore invisible to human inquiry.

In your research you have introduced the concept of synergistic immune photothermal nanotherapy (SYMPHONY) for potential cancer treatment of inoperable cancer sites (7). Would you explain this approach and any further results or insights that have been gained from this work? How does your approach differ from other forms of photodynamic therapy?

From disease diagnostics, my research interests also expanded to the treatment of illnesses such as cancer. We have recently seen an increasing interest in immunotherapy. Molecular processes, called immune checkpoints, are normally safeguards used by the body to prevent inappropriate processes or control overactivation of the immune response. However, many tumors have acquired the ability to manipulate these checkpoints and to block the action of the immune system. Drugs designed to counterblock immune checkpoints have recently shown efficacy in the treatment of certain cancers. In this interdisciplinary study, I collaborated with a group of bioengineers, medical researchers, and clinicians throughout the Duke campus and the Duke medical school. Our team demonstrated that the use of GNS in combination with a checkpoint blockade immunotherapy drug (antiPD-L1)—a treatment we referred to as synergistic immune-photothermal nanotherapy (SYMPHONY)—can dramatically enhance the efficacy of immunotherapy. Our studies indicated it is possible to achieve complete eradication of primary treated tumors as well as distant untreated tumors in mice implanted with a bladder cancer cell line. GNS were designed to efficiently absorb laser light for conversion into heat and to accumulate preferentially within a tumor due to the EPR effect. We now have the combined ability to use laser light in order to selectively and effectively treat tumor areas where GNS are located while keeping surrounding healthy tissues at significantly lower and safer temperatures. This GNS-mediated photothermal treatment strategy offers significant advantages over other traditional thermal therapies such as ultrasound, microwaves, and radiofrequency, which can control macroscopic heating around the tumor region, but cannot target or ablate cancer cells at microprecision scale and cannot differentiate tumor cells from healthy cells. Our studies showed that the effectiveness of the combination therapy was synergistic and not just additive. Remarkably, we found that SYMPHONY not only eradicated primary tumors treated by the laser, but also induced immune-mediated destruction of distant metastatic tumors that were untreated by the laser. The approach could provide an effective treatment when aggressive tumors that spread throughout the body cannot be surgically removed. It is our hope that the SYMPHONY strategy could lead to an entirely new treatment paradigm that challenges traditional surgical resection approaches for many cancers and metastases.

Of great interest was our observation that the SYMPHONY approach was capable of inducing long-term immunological memory that can provide protection against future tumor recurrence. In our studies involving two murine models of bladder cancer and brain tumor (glioblastoma), delayed rechallenge with repeated tumor injections did not lead to new tumor formation, indicating that the combined treatment induced effective long-lasting immunity that could provide protection against tumor recurrence long after treatment of the initial tumors, like an “anticancer vaccine” effect. We are in the initial stage of this SYMPHONY project and more studies involving larger cohorts of laboratory animals and different types of cancer cell lines are being performed. Further fundamental studies are also being conducted by our team to obtain better understanding of the mechanisms underlying these novel synergistic treatment modalities, in order to help us enhance and broaden the effect of immune-checkpoint inhibitors for successful eradication of metastatic cancer.

What were some of the key challenges you have encountered during your career of laboratory research and teaching?

Life presents plenty of challenges to us, and challenges often create new opportunities. There are important challenges facing scientists today. With our planet’s limited resources, we are now witnessing a paradigm shift from a development-driven society (20th century) to a sustainability-driven society (21st century). Scientists and engineers in photonics, like me, all have many opportunities to use our knowledge, learn new skills, and apply our innovativeness to address the global sustainability and environmental challenges, and ultimately contribute to a sustainable future as best as we can.

Another great challenge of our time is the exponential increase in the complexity of knowledge. In my teaching classes, I try to instill in my students a certain sense of scientific humility by telling them that no single discipline can solve big problems. I hope this statement will encourage my students to pursue teamwork and establish cross-disciplinary collaborations in their future careers. We are now entering a new phase where the knowledge of individual elements is no longer sufficient but should be combined and integrated in order to attain knowledge at the next level; that is at the multi-scale systems level, where the information on organization, activity and function requires a much higher level of complexity and sophistication. This transition from a knowledge base of individual elements to a systems level is one of the major paradigm shifts of the 21st century and can be achieved only by integrating multiple disciplines and different domains of knowledge. Education and research in this and coming decades will evolve into a framework to fit the new reality of our world, a world that will be faced with and embrace cross-disciplinary, systems level, and global challenges. It is important for us to educate our students, the next generation of innovators and leaders, not only to solve scientific and technical problems, but also to understand societal connections between various human activities and create bridges between elements spanning multiple disciplines in order to ultimately build a better interconnected world.

What would you consider to be the most meaningful contributions of your work, including patents?

An invention or new technique is, in an intellectual sense, like a child that you have created and nurtured throughout your work and scientific career. Each has its own unique features and values. Therefore, I have no favorite invention or contribution; I cherish all of them. They all have a favorite place in my heart. I feel very fortunate to have the opportunity to contribute in the important field of photonics. I believe that photonics is one of the ultimate enabling technologies that has played a crucial role by contributing key revolutionary and disruptive advances and will open new avenues and new possibilities that will define the next century. Light influences our lives today in new ways that we could never have imagined just a decade ago. As we enter a new decade, light will play an even more significant role, further empowering the information revolution in global communications, creating new nanotools to unveil the inner world of matter, uncover medical cures to save us from emerging global health threats, inventing new renewable energy sources, and galvanizing human exploration to the frontiers of the universe.

Would you share with our readers to describe your work ethic, philosophy, and how you plan your daily or weekly work schedule?

The career of a scientist requires a passion to learn and discover new things with the hope that the fruit of his or her efforts can contribute, even a very small part, to society. I would also say that it involves tremendous efforts in innovative thinking and intellectual pursuits. It also requires large amounts of energy for the long working hours required for inventing new ideas, designing new devices, planning new experiments, writing reports, publications, and research grants, as well as many other professional duties. However, if you have a great passion for what you are doing, this is a small price to pay for a very intellectually rewarding life experience. Stimulating collaborative work, learning new knowledge, and exchanges of new ideas with colleagues from other disciplines are added benefits that I truly cherish. I have had the opportunity to work with many talented coworkers, postdocs, and students; they all share with me, as a team, all the recognitions and honors that I have received throughout my career.

What words of wisdom do you have for any young people interested in a scientific research career?

This is a most important and exciting time in science. Dream out of the box. Think long-term. Be patient.

1. T. Vo-Dinh, B.J. Tromberg, G.D. Griffin, K.R. Ambrose, M.J. Sepaniak, and E.M. Gardenhire, 

2. T. Vo-Dinh, M. Panjehpour and B.F. Overholt, “Laser-Induced Differential Normalized Fluorescence Method for Cancer Diagnosis,” US Patent 5,579,773 (1996).

3. T. Vo-Dinh, M.Y.K. Hiromoto, G.M. Begun, and R.L. Moody, 

6. T. Vo-Dinh, J.P. Alarie, B.M. Cullum, and G.D. Griffin, 

7. Y. Liu, P. Maccarini, G.M. Palmer, W. Etienne, Y. Zhao, C.T. Lee, X. Ma, B.A. Inman, and T. VoDinh, 

Tuan Vo-Dinh is R. Eugene and Susie E. Goodson Distinguished Professor of Biomedical Engineering, Professor of Chemistry, and Director of the Fitzpatrick Institute for Photonics at Duke University. After high school in Vietnam, he pursued studies in Europe, receiving a BS in physics at EPFL-Lausanne (1970) and a PhD in physical chemistry at ETH-Zurich (1975). Before joining Duke University in 2006, he was Director of the Center for Advanced Biomedical Photonics and a Corporate Fellow, one of the highest honors for distinguished scientists at Oak Ridge National Laboratory (ORNL). His main research goal is focused on developing advanced technologies to protect the environment and human health. His research has centered on the development, integration and application of biophotonics, molecular spectroscopy, molecular biology and nanotechnology for biomedical diagnostics, photoimmunotherapy, precision medicine, and global health. Dr. Vo-Dinh has received seven R&D 100 Awards for Most Significant Advance in Research and Development; the Gold Medal Award, Society for Applied Spectroscopy (1988); the LanguedocRoussillon Award (France) (1989); the Scientist of the Year Award, ORNL (1992); the Thomas Jefferson Award, Martin Marietta Corporation (1992); two Awards for Excellence in Technology Transfer, Federal Laboratory Consortium (1995, 1986); the Lockheed Martin Technology Commercialization Award (1998); the Distinguished Inventors Award, UTBattelle (2003); the Distinguished Scientist of the Year Award, ORNL (2003); the Exceptional Services Award, U.S. Department of Energy (1997); the Award for Spectrochemical Analysis, American Chemical Society (ACS) Division of Analytical Chemistry (2011); and the Sir George Stokes Award, Royal Society of Chemistry (United Kingdom (2019). He has authored over 500 publications, is a Fellow of the U.S. National Academy of Inventors and holds over 50 patents.

Spectroscopy interviewed Tuan Vo-Dinh of Duke University, discussing his work in spectroscopy and photonics.

RSC Award: the Sir George Stokes Prize Spectroscopy for Medicine: From SERS Laser-Induced Fluorescence and Biosensors to Analytical Nanotools 

Infrared (IR) spectroscopy had its beginning in the early 1900s, when William Weber Coblentz demonstrated that chemical functional groups exhibited specific and characteristic IR absorptions. In this early work, Coblentz collected the IR spectra of ~135 compounds with an accuracy that still stands the test of time some 60 years later. Interest in the method grew during World War II, when a method for characterizing synthetic rubber formulations was needed for the war effort. The early to mid 1940s saw the first commercial instruments come on the market from both Beckman and Perkin Elmer. In 1957, Perkin Elmer introduced the first low-cost IR spectrophotometer, the Model 137, priced at just $3800. Several years later, the Coblentz Society was formed to educate early practitioners in the art. The IR method was used widely, but experienced a significant resurgence in the sciences with the advent of Fourier transform IR (FT-IR) instruments in the late 1960s and early 1970s. These instruments could collect spectra in a matter of seconds, and, by signal averaging, spectra of very high quality could be measured. Although instruments of that era were very large and bulky, current instruments are approaching the size of a cell phone. Today, the method is widely used in fields as diverse as planetary modeling, chemical characterization, climate monitoring, chemical threat detection, forensics, and disease detection, to name a few.

The mid-IR spectrum ranges in wavelengths from 2.5 to 50 micrometers. To correlate these wavelengths with bond energies, the wavenumber (cm

) is used, which is simply the number of wavelengths contained in one centimeter. The corresponding wavenumbers relating to the above wavelength range is 4000 to 200 cm

, respectively. For qualitative work, a transmission spectrum is collected by passing these wavelengths through a very thin sample (~10 micrometers thick), and recording how much light is absorbed by the sample. Figure 1 illustrates the IR and Raman spectra of acetyl salicylic acid (aspirin). 

Both spectra are rich in information, but to contrast the two methods, the IR method is sensitive to molecules possessing heteronuclear bonds and molecular vibrations that are asymmetric. For example, the features near 1751 and 1683 cm

 arise as a result of the C=O stretch of the ester and acid carbonyl groups, respectively. In contrast, the Raman method is sensitive to homonuclear bonds and molecular vibrations that are symmetric. Thus, the C=C stretch located near 1600 cm

 is the most intense transition in the Raman spectrum. Both IR and Raman spectra are unique for a given compound and are considered fingerprints for that compound. Indeed, in 1962, Ellis R. Lippincott testified before the United States Congress that an IR spectrum is a unique fingerprint that could be used in patent litigation.

In the late 1980s, two more significant developments took place: the development of novel sampling accessories, most notably attenuated total internal reflection (ATR) sampling accessories, and the development of IR microscopes. ATR accessories allowed any sample to be measured on a routine basis. Previously, the samples had to be in a specific form (a thin film less than 10 micrometers thick) to obtain a suitable transmission spectrum. This requirement involved considerable sample preparation or specialized accessories. Using the ATR method, the sample is brought into intimate contact with an internal reflection element (IRE). Figure 2 illustrates a typical ATR accessory. 

Contact between the sample and the IRE is achieved using a pressure applicator. Although several IREs are available, the most common ones are made of synthetic diamond. Diamond is both chemically inert and IR-transparent, but is also hard, and, as a result, can withstand high pressures. At the diamond–sample interface, the IR light penetrates into the sample only a few micrometers. As a result, highly absorbing samples like aqueous solutions can be easily studied without significant sample preparation. Today, the vast majority of IR spectra are collected using ATR accessories.

Although the first IR microscope (the PerkinElmer 85) was introduced in the 1950s, its use was limited because it was interfaced to a prism or grating spectrometer. The limited light throughput associated with micro-metersized samples and the serial data collection of the spectrophotometer translated into long scan times by today’s standards. In the mid 1980s, these microscopes were interfaced to FT-IR instruments. The reduced collection times, along with computer averaging, made it possible to analyze samples whose size approached the wavelengths of IR light.

In the late 1990s and early 2000s, the IR microscopes began to be outfitted with both linear and two-dimensional array detectors, along with motorized stages. These detectors allowed one to obtain IR maps and images from micrometer-sized spatial domains over large areas. In addition, the IR microscope could be used in a variety of different modes which included transmission, reflection, and ATR microspectroscopy. This latter method had the advantage of increasing the spatial resolution of the measurement by changing the refractive index of the IRE employed.

Recently, IR spectroscopy has been coupled with atomic force microscopy (AFM), and a new generation of IR microscopes has been outfitted with tunable IR lasers. AFM-coupled IR microscopes allow one to collect spectra on spatial domains much smaller than the wavelength of light. However, these microscopes have a high price tag, and are not considered routine analytical instruments. Similar to the AFM–IR microscopes, IR microscopes outfitted with solid-state lasers are expensive, and not considered routine. In addition, the spectral range of these systems is limited to ~1800–900 cm

It remains to be seen where the technology will take IR analysis in the future, but the method is one of the most important tools in the arsenal of many scientists throughout the world.

Infrared Spectroscopy

For more than 20 years, Heidi Goenaga-Infante, a science fellow and the leader of the inorganic analysis team at LGC Ltd., has been working on elemental and speciation analysis. Two recent areas of investigation include the analysis of trace metals in biological samples and the study of nanomaterials. In these studies, Goenaga-Infante puts particular emphasis on metrology—advancing this work by developing validated reference methodologies. Goenaga-Infante is the 2020 recipient of the Lester W. Strock Award from Society of Applied Spectroscopy (SAS) and the SAS New England Regional Section in recognition of her contributions to the field of analytical atomic spectrometry, and she recently spoke to us about her work. This interview is part of an ongoing series of interviews with the winners of awards that are presented at the SciX conference.

What led you to study trace elemental and speciation analysis?

My PhD supervisor, Professor Alfredo Sanz-Medel at the University of Oviedo, inspired me with a fascinating lecture on the speciation work by his group in the 1990s. This was at the very early stages of my university journey, and my heart and head fell in love with the topic. Professor Sanz-Medel supervised my PhD on the “Speciation Analysis of Cadmium in Biological Samples,” and this was followed by postdoctoral research on trace elemental and speciation analysis at the University of Antwerp under the supervision of Professor C. Freddy Adams. I was then fortunate to be offered a permanent position at LGC Ltd. in Teddington, UK, and asked to lead a small group developing metrological applications of speciation analysis.

Your research covers a range of areas-from metallomics research to the characterization of nanoparticles. How do you choose your projects, and what are your biggest challenges in these areas?

The UK National Measurement Laboratory (NML) at LGC is one of the top three institutes worldwide for chemical and bioanalytical measurements. Our world-leading science and method development helps solve measurement challenges across areas such as healthcare and food safety, both in the United Kingdom and globally. As such, our project areas are underpinned by a range of stakeholder and customer needs, prioritized and reviewed by an expert group involving academia, industry, clinical experts, and government representatives. LGC’s presence in the atomic spectrometry community is invaluable in identifying emerging areas of high impact in which metrological principles can play an important role to improve the quality of measurements. Moreover, within the organization as a whole there are many opportunities to work with different disciplines that benefit the output of our work. One important thing to highlight is the importance of recruiting high-caliber scientists and having access to state-of-the art instrumentation. Without that, we would not be able to deliver high-quality science in such a broad portfolio of measurement areas. My biggest overall challenge has been coaching emerging scientists to deliver first-class measurement solutions to key problems as well as helping develop them to develop their careers within LGC.

You coauthored a study on quantitated iron spatial distribution in biological tissues using online double isotope dilution analysis with laser ablation– inductively coupled plasma–mass spectrometry (LA-ICP-MS) (1). Can you explain the significance of this work and how it can advance disease model development and detection of neurodegenerative diseases?

I wish I could say that our work directly impacts the treatment and cure of these chronic diseases affecting many of our friends and families, but our contribution is rather modest, although important. The role of metals, including iron (Fe) in the diagnosis or treatment of neurodegenerative diseases, has been studied by many groups. Having reliable methods to image key disease biomarkers and monitor targeted treatments or drugs is essential to the development of improved therapies. LA-ICP-MS is a powerful tool for determining the spatial distribution of elements, both endogenous and externally tagged to biological samples, such as preclinical or post-mortem brain tissue. However, the relationship between the intensity of the element and the concentration at which it is present is often biased by matrix effects. Therefore, absolute concentrations obtained by reliable calibration strategies in the absence of reference materials are urgently needed to help validate measurements by nondestructive techniques. With this in mind, our work has described a reference methodology based on the use of LAICP-MS with isotope dilution analysis for the accurate quantification of the spatial distribution of iron in a model-tissue sample. A comprehensive estimation of the associated measurement uncertainty for the analyte spatial distribution was also achieved. Our high-level approach is invaluable to validate existing higher throughput methods used for medical research as carried out by our collaborators and stakeholders across the United Kingdom.

You developed a systematic approach to accurately quantitate plasma selenoprotein P (SEPP1)—a biomarker for human nutrition and disease—at clinical levels using species-specific double isotope dilution MS (2). What is the impact of this work?

The output of this work was a validated reference methodology for SEPP1. This is the major selenoprotein in plasma responsible for transport and distribution of selenium (Se), which is a key essential element to human health. SEPP1 has been associated with neurodegenerative diseases, such as Alzheimer’s disease and type 2 diabetes. Considering that SEPP1 is a biological active form of selenium and most of the plasma selenium is associated with is this protein, it represents one of the most accurate selenium-status biomarkers for human nutrition. For clinical purposes, SEPP1 has usually been characterized and quantified using antibody-based enzyme immunoassays such as ELISA. However, those assays often suffer from a lack in selectivity, and results are usually associated with high standard deviation values. Hence, there are some inconsistencies and lack of data comparability in the results obtained for SEPP1 in humans. Since developed, our methodology has been actively used to estimate the bias of ELISA results for SEPP1 in clinical research led by collaborators from hospitals in the United Kingdom and from overseas. It is planned to use such a valuable measurement tool for the production of new speciated reference materials that the community can use to validate their measurements and also to provide reference values to clinical trials involving selenium and its species as mediating agents.

In a recent study, you developed and validated a methodology for the accurate determination of number concentration of inorganic nanoparticles using single-particle ICP-MS (spICP-MS) without a nanoparticle reference material (3). Why did you undertake this project? What challenges did you face in this work?

Nanomaterials are increasingly being used in innovative products manufactured by advanced industries and provide enhanced, unique properties of great commercial and societal value. Measuring the number concentration of nanoparticles in colloidal suspension is a major interest for a large range of industries, including the pharmaceutical, personal care, cosmetics, and food packaging industries. Such methods are invaluable to underpin industry measurements. They also help industry to ensure the quality and efficacy of products and compliance with legislation. A number of methods capable of measuring particle number concentration in colloidal suspensions have been proposed, but neither SI-traceable approaches nor reference materials certified for nanoparticle number concentration were available when we did the work. Therefore, in our role as a National Measurement Laboratory, we undertook the challenge of developing and validating a methodology traceable to the SI, enabling the determination of analyte transport efficiency (TE) on the basis of weight measurements, only without the need for a reference material for calibration. Because the approach is based on direct and continuous measurements of the weight of sample uptake and the weight of sample reaching the plasma online over time (sample mass flow) while the ICP-MS system is in equilibrium, we named it the “dynamic mass flow” method. We faced great challenge; in particular, elucidating whether there was agreement between the mass-based TE of the sample solution and the nanoparticle-based TE was the most critical. It is important to note that in this work, a conventional nebulizer with a cooled spray chamber (2 °C) was used. This helped reduce the amount of water vapor (produced from evaporation of water from the aerosol in the spray chamber) entering the plasma, thus minimizing the contribution of this source of error to the uncertainty of the mass-based TE. Therefore, investigation of the feasibility of the dynamic mass flow (DMF) approach to obtain accurate nanoparticle number concentration data when using interfaces operated at ambient temperature or other types of nebulizers (such as high transport efficiency nebulizers) is still ongoing. Having said that, the DMF method shows promise for the validation of other laboratory techniques for particle number concentration. It has also been demonstrated to be useful for the characterization of quality control nanoobjects (such as LGCQC5050) that can be used with the particle frequency or size methods in a more targeted manner for a range of applications.

What does the Lester W. Strock award mean to you?

Receiving such a prestigious award made me feel very honored, and to be honest, it took me by surprise. It increases my motivation to achieve my best, and to keep seeking new opportunities. I wish to express my thanks those that have helped me and mentored me during my career, to my wonderful team at LGC, and last but not least, to those who nominated me, and to the Society for Applied Spectroscopy (SAS) for the award.

Our mission is to maintain our capability on core areas of metrology (high accuracy elemental, isotope ratio, and speciation and metallomic analysis), as well as continue bringing our metrology impact to emerging areas of research such as the characterization of nanomaterials. This work will focus on application areas driven by legislation and with a keen interest in nanomedicine. Also, more effort will be put into establishing a multidisciplinary platform for biomarker quantification and imaging of tissue down to single cells. This work will support medical research into the diagnosis and treatment of chronic diseases like cancer, neurogenerative diseases, and Wilson’s disease. A significant amount of our time and effort will continue to be dedicated to providing calibration services for reference material certification and for proficiency testing and clinical trials.

1. D.N. Douglas, J. O’Reilly, C. O’Connor, B.L. Sharp, and H. Goenaga-Infante, 

, 270–279 (2016). DOI: 10.1039/c5ja00351b.

2. C.L. Deitrich, S. Cuello-Nuñez, D. Kmiotek, F.A. Torma, M.-E. Del Castillo Busto, P. Fisicaro, and H. Goenaga-Infante, 

3. S. Cuello-Nuñez, I. Abad-Alvaro, D. Bartczak, M.E. del Castillo Busto, D.A. Ramsay, F. Pellegrino, and H. Goenaga-Infante, 

 Advance Article (2020). DOI: 10.1039/c9ja00415g.

Heidi Goenaga-Infante has more than 20 years of experience in elemental and speciation analysis, starting with her PhD at Oviedo University, Spain. She joined LGC in 2003 as a senior researcher in speciation analysis and is currently a science fellow. She is also the principal scientist and team leader of the Inorganic Analysis team, leading 14 PhD and postgraduate scientists. Her group currently focuses on trace element speciation analysis, metallomics research, the characterization of nanomaterials, high-accuracy isotope ratio analysis, quantitative elemental bio-imaging, and the characterization of “speciated” reference materials and standards.

Goenaga-Infante is the UK representative at the Inorganic Analysis Working Group of the international Consultative Committee for Metrology in Chemistry (CCQM). She is also a member of the international advisory boards of Analytical and Bioanalytical Chemistry and the RSC journal Metallomics and of the Editorial Board of the Journal of Analytical Atomic Spectrometry, and a member of IUPAC. She is the Government Chemist representative on the Nanomaterials Environment and Health Government Group chaired by the UK Department of Environment, Food, and Rural Affairs (DEFRA) and the LGC representative to ISO TC 24 (Particle characterization). She is the EURAMET representative for inorganic analysis at the CCQM Key Comparison Working Group. She has acted as the coordinator of the EU EUROPEAN Metrology Research Proposal (EMRP) NanoChop “Chemical, Optical and Biological characterization of Nanomaterials in Biological Samples.” She is the lead author of more than 106 scientific research papers and five book chapters.

Spectroscopy Magazine spoke with Heidi Goenaga-Infante about her work on elemental and speciation analysis.

SAS Strock Award: Advancing Trace Elemental and Speciation Analysis with a Focus on Metrology 

Laser-induced breakdown spectroscopy (LIBS) is principally an elemental analytical approach. It relies on the sampling and excitation of the material by a laser pulse. The excitation is sustained by the laser-induced plasma, and the emission of each element provides the spectral fingerprint we rely on to identify and quantify it.

LIBS benefits from the unique features of laser ablation (LA) and the accessibility of optical emission spectroscopy (OES). Given that sampling is controlled by the laser– matter interaction, sampling can be designed to attain micrometer resolution, and still obtain an analytical signal from the plasma. Given that the excitation medium is a plasma, it can be controlled (although not necessarily) by the background gas and the ablation chamber. Finally, the OES from such a short-lived plasma does not suffer from as much interference from atmospheric dimers as a signal from a torch plasma would. OES also allows much more freedom in the choice of the analyte signal than a mass spectrum would, circumventing interferences with ease.

LIBS has increasingly been used in many fields for imaging or for field applications when elemental information is required. Its main advantages result from the fact that the sampling is done by laser ablation, allowing both elemental analysis and chemical mapping, as well as emission spectroscopy, making it possible to exploit the technological miniaturization of spectrometers.

LIBS provides important chemical information in quasi-real time, and is very versatile, capable of detecting both major and minor elements in the sample. New approaches using LIBS provide protocols for the analysis of halogens via molecular emission in the plasma that are easier to accomplish than mass spectrometry methods for such analyses.

I have explored and contributed to the development of LIBS for 15 years, ever since I used LIBS for microbiological applications during my MS internship. After having explored many applications throughout these years, I have settled on two main areas of focus for my forensic research portfolio: the forensic analysis of rubber (tire forensics), and the development of matrix-matched standards for the analysis of hard biological materials (forensic anthropology). This focus is motivated both by the needs in forensic analysis but also by the interesting challenges these two applications pose.

Rubber is a very tricky material for analytical laser ablation. Because rubber is highly susceptible to melting, it is usually hard to ablate it efficiently and get a good elemental signature from it. But LIBS offers a unique approach to perform elemental analysis of the overall organic matrix of rubber, as well as the vulcanization agents it may contain, such as sulfur and various metals. This capability enables the analysis of few micrograms of tire skid mark material and a search for its provenance. We have shown that, by conducting elemental analysis with LIBS, it is possible to classify tire chemistry and origin; we now are expanding our research to describe a full analytical approach from crime scene collection to statistical decision making.

Forensic anthropology has typically relied on X-ray fluorescence (XRF) for elemental analysis in the field as well as in the laboratory. LIBS, however, can provide so much more, thanks to its efficiency. For example, it can provide information about low-Z elements beyond C, N, and O; hydrogen, lithium, and boron can now be easily detected. But elemental trace analysis requires reliable standards. And, just as XRF is matrix-sensitive, LIBS analysis of bones, hair, and teeth cannot rely on compressed standard powders. This is why my research group is working to develop such standards for hard biological materials. Our research has succeeded in the creation of elemental standards for the analysis of hair, teeth, and fingernails. We are also now exploring new approaches for more reliable standards and a wider breadth of applications such as medical analysis and bio-archaeology. 

The LIBS community has known me with multiple hats: from an impulsive graduate student working on bacteria analysis and polymer laser ablation in a physics department, to a research scientist then faculty member focusing on defense applications of LIBS within the UCF College of Optics, to a cheerful member of the chemistry faculty establishing his own research in the National Center for Forensic Science at UCF. Every time, I have found challenges and motivation to develop new approaches for the main goal of what LIBS is for: quantitative analytical science.

Dedicating my research to analytical spectroscopy and its applications, I have always mixed all the evolving knowledge I could find from physics, chemistry, optics, engineering, data science, material development, and more. It is this sense of connection and the “multilingual” blend of all this knowledge that keeps me excited and passionate about LIBS. Laser technology keeps providing better tools for us as LIBS spectrometers become more popular, and better optics get integrated into instruments. Data analysis becomes more and more powerful, and can take into account the highly non-linear aspects of LIBS. Now, discovering the potential of material science for quantitative analysis, I feel like a DJ in a record store: Every single track, whatever the genre, can have a jewel sample, and we have the potential to put these pieces together to form a masterpiece. This is what makes me passionate about LIBS— the opportunity to put many pieces together to solve analytical problems.

Laser-Induced Breakdown Spectroscopy

Kay Sowoidnich, PhD, is a research associate with Laser Sensors Lab at the Ferdinand-BraunInstitut, Leibniz-Institut für Höchstfrequenztechnik, and one of the 2020 winners of the Society for Applied Spectroscopy William F. Meggers Award. His group have been able to demonstrate the potential of shifted-excitation Raman difference spectroscopy (SERDS) as an efficient tool for soil nutrient analysis. Additionally, his previous research work applied SERDS for the analysis of meat could enable rapid screening of large numbers of meat samples for authentication and quality monitoring purposes at different points along the distribution chain from the slaughterhouse to the supermarket. We spoke with Sowoidnich about his work applying SERDS, its impact and limitations, and his work using spatially offset Raman spectroscopy (SORS) to diagnosis skeletal disorders.

You’ve been applying SERDS for the analysis of soil, food, and other constituents for several years (1, 2). How has this analytical technique proven to be advantageous for soil and food analysis? What type of impact could it have on precision agriculture and for food supplies?

Conventional Raman spectroscopy allows obtaining chemically specific information from numerous samples, and is a powerful tool for qualitative and quantitative analysis. As Raman scattering is usually a weak effect, interfering contributions can easily mask the characteristic Raman signals,and thus complicate or prevent meaningful sample investigation. Prominent examples of such interferences are laser-induced fluorescence and, driven by improvements in the sensitivity of portable Raman instrumentation, various types of ambient light. Among other techniques, SERDS is a powerful instrumental approach to address this issue by separating Raman signals from unwanted light contributions using a dualwavelength excitation light source. If two slightly shifted excitation wavelengths are used to record two Raman spectra, then the Raman signal positions will follow that shift in wavelength, while fluorescence and other quasistatic contributions will remain virtually unchanged. Subtracting the two recorded Raman spectra can thus effectively retain the Raman spectroscopic information while removing the unwanted interferences.

Diode lasers are well-suited as excitation light source for SERDS, as they offer compact size, low-power consumption, and high efficiency. Such innovative and customized lasers are developed and realized at FerdinandBraun-Institut, Leibniz-Institut für Höchstfrequenztechnik (FBH). HeinzDetlef Kronfeldt’s laser spectroscopy group at Technical University Berlin had established a fruitful collaboration with Dr. Bernd Sumpf and Dr. Martin Maiwald from FBH, and we were able to use unique diode lasers for our Raman and SERDS experiments. Within a collaborative research project, SERDS has successfully been applied for the noninvasive optical discrimination between meat of selected animal species, exemplarily demonstrated for beef, pork, chicken, and turkey.

Furthermore, Raman investigations could demonstrate the noninvasive quantitative detection of meat spoilage with respect to legal limits. In the future, this could enable rapid screening of large numbers of meat samples for authentication, and quality monitoring purposes at different points along the distribution chain from the slaughterhouse to the supermarket. The nondestructive Raman technique could become a valuable asset for food inspectors, enabling analysis of large production batches rather than relying on random sampling and cumbersome laboratory analysis. The topic of Raman spectroscopy for food analysis has further been developed by my former colleague Dr. Heinar Schmidt at University of Bayreuth. His group conducted successful Raman trials for meat inspection in a slaughterhouse as well for on-site inspection of Australian lamb meat using a portable device at 671 nm excitation.

During my current role within the Laser Sensors Lab at FBH, our group was able to demonstrate the potential of SERDS as an efficient tool for soil nutrient analysis. Currently, soil investigations require sample collection followed by complex and time-consuming laboratory analysis. Optical methods could be very beneficial in this instance, as they enable rapid analysis without prior sample collection. Conventional Raman spectroscopy can provide information on the present molecular species, but suffers from fluorescence from soil organic matter. Here, SERDS could enable in situ soil investigations at a large number of spots across the whole field, thus allowing mapping the distribution of relevant soil components, including nutrients. This could be a major advantage, as the needs-based precision fertilization is already possible with state-of-the-art farming equipment. But to fully exploit this benefit, it would require knowledge of spatially-resolved information about the actual soil status that is not yet available from current analytical methods.

What types of limitations does SERDS present when, for example, using it to investigate fluorescent heterogenous samples (3, 4)? How do you overcome these limitations or challenges?

SERDS is based on the alternate and subsequent recording of Raman spectra excited at two slightly shifted wavelengths. Using a conventional charge-coupled device (CCD) detector, one SERDS cycle would then comprise the following steps: 1) exposure at first wavelength, 2) readout of Raman spectrum recorded at first wavelength, 3) exposure at second wavelength, and 4) read-out of Raman spectrum recorded at second wavelength. If, during that period of time ,unwanted background interferences are not constant, residual artifacts could remain after subtraction of the two spectra, and this would compromise qualitative and quantitative analysis. Potential examples are fluorescence quenching, sample or instrument movement in case of heterogeneous specimens, and varying ambient light intensities. Faster, ideally quasi-simultaneous, acquisition of SERDS spectra would therefore be highly beneficial to address this issue. On the excitation side, this will require a dualwavelength light source with reproducible and fast-shifting capabilities between the two emission lines. Such lasers have readily been demonstrated, for example, by FBH with rapid modulation up to the kilohertz range. The limitation rather lies on the detection side, where one could reduce the individual exposure times. However, there is a fundamental physical limit to the time required to read out the charges from the CCD in conventional operation mode.

During my time at the United Kingdom’s Science and Technology Facilities Council (STFC), I was working in the Central Laser Facility (CLF) under the supervision of Professor Pavel Matousek, who is a brilliant scientist full of exciting new ideas. He came up with a neat solution by combining the advantages of SERDS with the benefits of a charge-shifting approach. The basic concept of charge-shifting CCD operation was proposed in 1990, while a pilot study in 2008 by others has shown its suitability for conventional Raman spectroscopy. The major advantage here is that the read-out steps after each single exposure are omitted. Instead, the charges generated by Raman scattered light excited at the first excitation wavelength are electronically shifted towards a non-illuminated area on the CCD before charges are generated by Raman scattered light excited at the second excitation wavelength. These charges, in turn, are then shifted to another unilluminated area on the CCD when additional charges are generated by Raman scattered light excited at the first excitation wavelength. This alternate shifting procedure is repeated until sufficient charge in the two distinct areas on the CCD, corresponding to charges generated by Raman scattered light at the two excitation wavelengths, is accumulated. Finally, only one read-out step at the very end is performed.

Through a prolific cooperation between STFC and FBH, which developed a custom 830 nm diode laser with tailored electro-optical properties, putting special emphasis on fast wavelength switching, the charge-shifting SERDS approach has been realized experimentally. Using a stock CCD that has been modified according to our requirements, we could successfully demonstrate alternate recording of Raman spectra at frequencies of 1000 Hz, and that is about two orders of magnitude faster than speeds achievable in conventional CCD read-out mode. The reproducibility of SERDS spectra recorded on exemplarily chosen heterogeneous rock samples in charge-shifting mode was superior to spectra recorded in conventional CCD operation. This led to improved classification accuracy for the discrimination between rock species. We also applied the charge-shifting concept to suppress rapidly varying ambient light interference (5) and successfully translated it toward sub-surface analysis using spatially offset Raman spectroscopy (SORS).

Through your work using SORS to diagnosis skeletal disorders as well as other medical conditions, what did you learn about the technique (6)? How is it advantageous as a medical diagnostic technique? Is it currently used clinically or are there plans for clinical deployment?

The current gold standard for the diagnosis of bone conditions are X-ray-based methods. Despite being able to assess the mineral phase of the bone very well, this kind of technique is unfortunately insensitive to collagen, the second important component of the composite material bone. Raman spectroscopy, in contrast, is able to retrieve chemically specific information from both phases, for example, mineral and collagen, thus giving a more complete picture of the overall bone status. As conventional Raman spectroscopy is restricted to the investigation of surface-near layers, we applied SORS for efficient subsurface analysis. This enables us to measure the bone composition, noninvasively, through the skin at selected anatomical locations, such as at lower legs and fingers. SORS works in such a way that the point of laser illumination on the surface is set at a certain spatial distance from the point where the Raman-scattered photons are collected, thus probing subsurface layers within the sample. For the development of SORS as medical diagnostic technique it is essential to know from what depth the detected Raman spectroscopic information is actually emerging for a chosen spatial offset. Our photon migration study was addressing this important point, using typical long bone tissue, as well as antler and bulla, to include two types of bone exhibiting among the lowest and highest mineralization levels found in nature. Results indicated that photons can more easily migrate inside less mineralized bone tissue and that porosity can play an important role as well. We were also able to estimate the approximate depth from which the major Raman signal contribution is collected for a given spatial offset within exposed bone during SORS investigations. Under safe laser illumination conditions compatible with in vivo applications even in highly mineralized bulla depths of up to 3.8 mm are still accessible using large spatial offsets in the range of 9 to10 mm. The findings of our study significantly increased our understanding of SORS analysis through bones of different composition, thus providing vital information to assess the potential of SORS for medical diagnostics.

We are aiming to develop SORS as a valuable tool for the noninvasive in vivo diagnosis of bone disorders and diseases. In close collaboration with the Royal National Orthopaedic Hospital in London, we were running a preclinical SORS trial with a custom instrument to apply the technique in a clinical environment. More than 150 patients were recruited to date, either having one of the diagnosed bone conditions—osteoporosis, osteoarthritis, or osteogenesis imperfecta (also known as brittle bone disease)—or belonging to a matched healthy control group without any bone condition. Within the frame of this SORS trial, for the first time, the in vivo bone disease detection in case of osteogenesis imperfecta was demonstrated. For the other bone conditions, particularly for less severe cases, our results indicate a difference between healthy and diseased bone, but the differentiation is not yet statistically significant. We are, however, confident that improvements in instrument hardware and data analysis will pave the way for SORS to become a routine analytical technique for bone quality assessment, complementing or even partially replacing existing methods.

What would you consider to be the most meaningful contributions of your work?

In my opinion, the charge-shifting SERDS approach has made a significant impact, as it has overcome existing fundamental limitations of conventional CCD operation. The potential applications are numerous, and the examples described in our corresponding publications only cover a fraction of what will actually be possible. The underlying technology has already been implemented into a novel CCD camera of one of the world’s leading companies for spectroscopic equipment. The future will show what exciting new research fields can be explored by the enabling capabilities of the charge-shifting technique. This is actually a hot topic, as in parallel to our study, similar research has been undertaken independently by LeibnizInstitut für Astrophysik Potsdam (Germany) and University of Tokyo (Japan).

Besides doing cutting-edge research, it is equally important to communicate the findings not only to scientific experts but also addressing the layman. Another important contribution is therefore related to public outreach activities. During my time at STFC’s Rutherford Appleton Laboratory, our group has developed a SORS demonstration setup within the frame of the International Year of Light 2015. The instrument was basically a simplified version of the commercial SORS devices used at airports to scan liquids inside containers. During numerous exhibitions, the SORS demo setup was one of the most popular and most reliable exhibits highlighting an important part of STFC’s science to the public. The instrument has been also presented to key stakeholders and decision-makers, including the UK science minister. Due to its tremendous success, a second device was realized that is now permanently installed in the CLF visitor’s centers at STFC’s Rutherford Appleton Laboratory.

What are your plans for the future? Your next steps?

My immediate next steps are further research efforts to establish SERDS as a valuable tool for the analysis of soil, paving the way for efficient nutrient management in the frame of precision agriculture (the consortium Intelligence for Soil (I4S) in funding measure Soil as a Sustainable Resource for the Bioeconomy (BonaRes) by Federal Ministry of Education and Research). On a long-term scale, at FBH the Laser Sensors Lab provides excellent working conditions for me to make further significant contributions to application-oriented research aiming to address current as well as upcoming societal needs and global challenges. FBH’s big advantage is that the whole value chain is located in-house, with expertise reaching from the design and manufacture of novel diode lasers with unique properties to the application of these laser sources as part of customized portable SERDS instruments for in situ field experiments. As the area of photonic technologies in general and optical spectroscopy in particular is a very dynamic field, I am keen to see what opportunities and scientific challenges I will come across in the future.

1. FBH research, April 16, 2020. https://www.fbhberlin.de/forschung/forschungsnews/detail/ramanspectroscopic-investigations-on-soil-using-a-785- nm-dual-wavelength-diode-laser.

https://doi.org/10.1002/9780470027318.a9510

3. K. Sowoidnich, M. Towrie, M. Maiwald, B. Sumpf, and P. Matousek, 

4. K. Sowoidnich, M. Maiwald, B. Sumpf, M. Towrie, and P. Matousek, Proc. SPIE11236, 112360K (2020).

5. K. Sowoidnich, M. Towrie, and P. Matousek, 

6. K. Sowoidnich, J.H. Churchwell, K. Buckley, A.E. Goodship, A.W. Parker, and P. Matousek, 

Spectroscopy Magazine sat down with Kay Sowoidnich to talk about how his group has demonstrated the potential of shifted-excitation Raman difference spectroscopy (SERDS) as an efficient tool for soil nutrient analysis.

SAS Meggers Award: Analyzing Soil and Meat with Shifted Excitation Raman Difference Spectroscopy 

Overall, the application of Raman spectroscopy has yielded a greater understanding of multiple biomedical problems. We interviewed Yukihiro Ozaki, professor emeritus and a university fellow at Kwansei Gakuin University in Japan, about his work in this field. Ozaki is the winner of the 2020 Charles Mann Award for Applied Raman Spectroscopy, to be given at the 2020 SciX conference, for his Raman work in biomedical applications. This interview is part of a series of interviews with winners of awards presented at SciX.

Raman spectroscopy has become an important analytical technique for exploration of organic and inorganic compounds for applications from astrobiology to zoology. Since the 1980s, there has been an interest in exploring the use of Raman spectroscopy for biomedical applications requiring in situ measurements where no sample preparation or manipulated pathlength of samples is permitted. Raman spectroscopy is being used for tissue examination, for discovering disease mechanisms and etiology, and for determining the detailed spatial chemical composition of tissue structures for diseased and normal tissues. In biology, Raman spectroscopy has been used to nondestructively measure component changes in proteins and other metabolites affected by variations in environmental conditions, genetics, and metabolic activity. The enhancement of Raman signals by the use of surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy has led to an improved understanding of their mechanisms and also to the development of bioassays, such as enzyme immunoassays and quantitative analysis of double-stranded DNA, amplified by polymerase chain reaction (PCR). Overall, the application of Raman spectroscopy has yielded greater understanding of multiple biomedical problems. We interviewed Professor Yukihiro Ozaki, professor emeritus and a university fellow at Kwansei Gakuin University in Japan, about his work in this field. Ozaki is the winner of the 2020 Charles Mann Award for Applied Raman Spectroscopy to be given at the 2020 SciX conference for his Raman work in biomedical applications. This interview is part of a series of interviews with winners of awards presented at SciX.

One of your most cited papers to date on the subject of Raman spectroscopy (1) reviews the development of SERS-based methods for proteins. The emphasis of this paper is to describe protein mediated SERS-active substrates for application as both label-free and Raman dye-labeled protein detection methods These methods provide tools for the study of protein-controlled processes within living cells. How did you become interested in this subject?

When I was a PhD student at Osaka University, I investigated resonance Raman spectroscopy of hemeproteins at the Institute for Protein Research of Osaka University. After I got my PhD, I became a research associate at the National Research Council of Canada, where I was involved in enzyme research using a resonance Raman label method. Therefore, I have strong background for both Raman spectroscopy and protein research since the 1970s. When I encountered SERS, I just thought of highly sensitive detection of proteins by SERS. Then, I started studies on protein-mediated SERS-active substrates for application as both label-free and Raman dye-labeled protein detection methods with Dr. Xiaoxia Han, who was a postdoc in our group.

In your work, you have described both resonance Raman and SERS spectroscopy. Why are these techniques important and what are the differences between resonance Raman spectroscopy and SERS for biomedical applications, in terms of sensitivity and specificity?

For biomedical applications, normal Raman, resonance Raman, and SERS are all very important. The sensitivity and specificity are low for normal Raman, but normal Raman will detect most biomolecules nonspecifically, such as proteins, lipids, nucleic acids, and so forth. It can detect everything in tissues and cells. Resonance Raman spectroscopy has much higher sensitivity and specificity than normal Raman, but resonance Raman cannot detect lipids and proteins, except for those such as hemeproteins and metalloproteins, unless one uses UV-resonance Raman spectroscopy. However, resonance Raman is useful for highly sensitive and selective detection of biological pigments such as carotenoids. SERS has the highest sensitivity and selectivity, but SERS always requires silver or gold nanoparticles, which are not always biocompatible. Therefore, it is important to use these three methods properly, depending on the research purpose.

How is the Raman spectroscopy work you have done different from what others have done previously?

In my opinion, among various optical molecular spectroscopy techniques, Raman, near-infrared (NIR), and fluorescence spectroscopy are the most important for biomedical applications. Fluorescence spectroscopy has very high sensitivity, and, thus, it is very suitable to detect a tiny number of biological components. Fluorescent spectroscopic imaging is also a powerful technique for biomedical applications. A disadvantage of fluorescence spectroscopy is that one cannot explore structural changes of biological molecules such as proteins. The advantage of NIR spectroscopy is that it has very high permeability. NIR enables us to probe whole biomedical materials or whole living bodies in situ; one can examine even their internal structures nondestructively. There is now a big research filed named fNIR (functional NIR) spectroscopy. The disadvantages of NIR spectroscopy are that sensitivity and specificity are relatively low. Spectral analysis is also not straightforward for NIR. Compared with fluorescence spectroscopy and NIR spectroscopy, Raman spectroscopy can detect proteins, lipids, and nucleic acids separately and one can disclose their structural changes. However, sensitivity for normal Raman spectroscopy is rather low.

You have edited a text on the analytical methods of Raman, infrared (IR), and NIR chemical imaging (2). You are one of the few researchers who has made major contributions using all three spectral measurement regions. From your perspective, what are the applications of Raman imaging that highlight its advantages over IR and NIR imaging?

One of my strong points is I have been involved in all three spectral measurement regions: Raman, IR, and NIR spectroscopy. So, I can evaluate fairly these spectroscopies. The advantage of Raman imaging over IR and NIR imaging is its high special resolution. Even without using tipenhanced Raman spectroscopy (TERS), one can obtain a spatial resolution of 300 nm by using Raman. If one uses SERS imaging, one can expect higher sensitivity and selectivity over IR and NIR imaging. In Raman imaging, it is possible to properly use normal Raman imaging, resonance Raman imaging, SERS imaging, and TERS imaging. So, Raman imaging offers a very wide set of imaging capabilities. The existing issues of spectroscopic imaging are the speed of the imaging measurement, the ability to image materials with roughness, and the analysis of depth direction. Raman spectroscopy imaging has strong potential for solving these issues.

In an earlier article, you wrote about the use of Raman spectroscopic imaging with an emphasis on the chemometrics used for data processing (3). What did you learn regarding the unique properties of Raman spectra and how chemometrics can best be applied to Raman data?

Chemometrics was used first for NIR spectroscopy, because NIR spectra often have high multicollinearity. Nowadays, chemometrics is powerful for all kinds of spectroscopy. For Raman spectroscopy, from time to time chemometrics is highly useful (3), but sometimes Raman does not require chemometrics because Raman bands are sharp compared with NIR bands. Generally speaking, chemometrics is useful for the discrimination of Raman spectra and qualitative analysis of Raman spectra, but not always suitable for quantitative analysis of Raman spectroscopy, particularly when Raman spectral intensity is fluctuating.

You have made significant contributions to the biomedical applications of SERS. Your contributions include the elucidation of the SERS electromagnetic mechanism (4), as well as discovery research related to the chemical mechanism of SERS (5,6). Would you summarize for our readers what your main discoveries were in this work?

The SERS mechanism had been investigated by an ensemble nanoparticle system, where the cause of the enhancement (plasmon resonance) was averaged, and, thus, it was difficult to elucidate directly the relation between the cause and the result (SERS). Dr. Tamitake Itoh, who was a postdoc in my group, thought of using a single nanoparticle system to solve this problem (4). We developed a new experimental setup for studying the SERS mechanism consisting of an electron microscope (EM), a plasmon resonance measurement system, and a SERS measurement system for the same single nanoparticle. We measured SERS and plasmon resonance spectra, and EM images, and based on this information we calculated the SERS spectra. By comparing the experimental SERS spectra with the calculated ones, we succeeded in quantitative verification of the electromagnetic enhancement effect. A group at Jilin University in China and our group also contributed to the studies of chemical effects and semiconductor-enhanced Raman scattering (5). Our recent study on the Mie resonance scattered nearfield effect provided a coherence framework for modeling the electromagnetic mechanism of SERS on semiconductors (6).

What were some of the key challenges you encountered during your career of teaching and research?

The key challenges I encountered during my career of teaching and research were finding my academic position; recruiting students, postdocs, and collaborators; and getting funding. Here, I would like to mention something about the second point. For recruiting, I placed an emphasis on collecting people who have different backgrounds. I belonged to a chemistry department, so I had enough chemists. But in my group, I always included a few people who had a strong background in physics. This has been quite successful. I also accepted people from bioscience, pharmaceutical science, agricultural science, and so on. As a result, our group was always full of new ideas. Moreover, our group was always quite international and big, and, thus again, I have been surrounded by new ideas and high energy. From our group, about 50 people have obtained their PhD. Also, about 50 people have joined our group as postdoctoral fellows and assistant professors. About half of them were foreigners; we were able to establish quite an international group. Currently, many of them are very active in academia and industries.

I have three most meaningful contributions. The first is the first trial of the studies of mechanism of disease by Raman spectroscopy (7). The second is the establishment of application of NIR spectroscopy to physical chemistry (8). The third is the proposal of ATR-far-ultraviolet (FUV) spectroscopy (9).

Would you share with our readers your philosophy for work and success in an academic research career?

The group motto of our research group is “top among the top” and our mission is “promoting science and application of molecular spectroscopy throughout the world.” The most important things in my philosophy for work and success are to have ambition and consider working on significant contributions that benefit the world.

Raman, Infrared, and Near-Infrared Chemical Imaging

 (John Wiley & Sons, Hoboken, New Jersey, 2011).

3. H. Shinzawa, K. Awa, W. Kanematsu, and Y. Ozaki, 

4. T. Itoh, Y.S. Yamamoto, and Y. Ozaki, 

5. X.X. Han, W. Ji, B. Zhao, and Y. Ozaki, 

6. W. Ji, L. Li, W. Song, X. Wang, B. Zhao, and Y. Ozaki, 

8. M. A. Czarnecki, Y. Morisawa, Y. Futami, and Y. Ozaki, 

9. Y. Ozaki, Y. Morisawa, A. Ikehata, and N. Higashi, 

Yukihiro Ozaki graduated from Osaka University in 1973 with a BS degree in chemistry and MS (1975) and PhD (1978) degrees in physical chemistry from the same university. After he had spent for two and a half years as a research associate at the National Research Council, Canada, he joined the Jikei University School of Medicine in Tokyo as an assistant professor in 1981. In 1989, he moved to Kwansei Gakuin University, Hyogo, Japan as an associate professor in the Chemistry Department. Since 1993, he has been a professor in the Department of Chemistry, School of Science and Technology, Kwansei Gakuin University. He was a Vice Rector of Kwansei Gakuin University from 2013 to 2018. 

Currently, Ozaki is a professor emeritus and a university fellow at the Kwansei Gakuin University, a guest fellow of Toyota RIKEN, a guest professor of Peking University, Kobe University, and Fukui University, and a guest principal researcher of RIKEN. Ozaki has been internationally known in the research and education of physical chemistry, analytical chemistry, and spectroscopy for the past four decades. He was a senior research fellow of the Chemistry Department of Princeton University in 1993. He is a fellow of the Society for Applied Spectroscopy (since 2010), the Royal Society of Chemistry (since 2015), The Chemical Society of Japan (since 2016), and the International Council of Near-infrared Spectroscopy (since 2017). Ozaki was also given a Doctorate Honoris Causa from Jagiellonian University, Poland (2016) and from the University of Wroclaw, Poland (2019). He has received multiple international awards, such as the Bomem Michaelson Award and Pittsburgh Spectroscopy Award. Ozaki has published six textbooks and approximately 1000 papers with an H-index of 72. The number of citations of his papers is more than 24500.

Spectroscopy interviewed Yukihiro Ozaki about his work in the field of Raman spectroscopy.

Charles Mann Award Biomedical Applications of Raman Spectroscopy Over Several Decades 

The “Atomic Spectroscopy” section of the SciX program focuses on many areas of research that include low pressure glow discharge, atmospheric pressure glow discharge, inductively coupled plasma, and laser ablation. This article provides an educational summary on inductively coupled plasma–based instrument platforms. There is an imminent need for routine trace element and isotopic analysis, expanding across many fields including, but not limited to, geological, industrial, nuclear, health, and environmental analysis. Arguably, the gold-standard technique for the majority of these measurements is through an inductively coupled plasma (ICP) excitation–ionization source coupled to an optical emission spectroscopy (OES) or mass spectrometry (MS) detection platform (1). The ICP, initially employed as an excitation source for OES detection, was first implemented by Fassel and colleagues (2,3), later by Greenfield and coworkers (4), and subsequentially realized as an ionization source for MS detection (5). The ICP is formed when the energy of a radio frequency (RF) generator interacts with a tangential flow of argon gas (~15 L/min) in a quartz tube often referred to as the plasma torch. This process subsequentially ionizes the gas forming a high-temperature plasma, in which photons and ions can be generated.

Sample introduction schemes in ICP-OES and ICP-MS are dominated by solution-based analysis. Ultimately, an aerosol must be generated before the sample enters the ICP, because of the plasma’s inefficiency at dissociating bulk solution (large droplets). Traditionally, a solution is carried into a concentric nebulizer, where a Venturi effect is achieved via argon gas and low pressure to generate a fine aerosol. Next, the aerosolized sample is further separated such that the larger droplets dissipate (becoming solution waste), and the fine droplets exit the spray chamber and are directed into the torch. Many advances over the years have been focused on improving nebulizers, spray chambers, and membrane desolvation to improve sensitivity and precision for elemental and isotopic analysis.

Another means of sample introduction for ICP-based analysis is laser ablation sampling. With this approach, a pulsed laser ablates a sample surface within an ablation cell, and the subsequently formed nanoparticles are carried into the ICP via a carrier gas (such as helium) (6,7). Most commonly, laser ablation is combined with an ICP-MS instrument; however, it is not out of the realm to utilize laser ablation with an ICP-OES instrument. Techniques such as laser ablation have become popular because of the reduction in sample preparation (little to no sample preparation is required), and the ability to obtain spatial (lateral or depth) elemental information, rather than the bulk analysis that is more typical of solution-based ICP analysis.

The combination of laser ablation with ICP-MS is not the only hyphenated configuration that is employed; the coupling of liquid chromatography or ion chromatography are extremely useful sample introduction techniques for ICP-MS. These techniques offer the ability to separate compounds, isotopes, proteins, or elemental species based on column chemistry prior to introducing into the ICP-MS instrument, which ultimately acts as an element-specific detector.

The detection platforms for ICP instrumentation consist of OES and MS instrumentation. For OES detection, the photons emitted from the ICP are commonly collected via radial or axial viewing, each with their respective advantages and disadvantages. Axial viewing, collecting photons down the longitudinal axis of the plasma, is often preferred for enhanced sensitivity, because more photons are collected; however, more photons could be detrimental if there are large amounts of total dissolved solids (TDS). Radial viewing, collecting photons perpendicular of the plasma, is less sensitive and less hindered by interferences from background and complex matrices. Spectrometers employed with OES detection can primarily be found in two configurations: scanning-based Czerny-Turner spectrometers, or simultaneous-based echelle spectrometers. The Czerny-Turner design allows for higher resolution, which can be favorable for complex samples; however, it is accompanied by longer analysis time, particularly if many elements are of interest. Echelle spectrometers allow for the simultaneous measurement of many wavelengths in a timely manner, but often lack spectral resolution compared to Czerny-Turner spectrometers.

For ICP-MS detection platforms, commercially available options primarily consist of quadrupole, time-of-flight, and magnetic sector-based mass analyzers. The quadrupole platform is ultimately the workhorse ICP-MS system for routine trace elemental analysis. This approach has been enhanced with the introduction of collision–reaction cells that facilitate the reduction or elimination of polyatomic interferences caused by sample matrix or plasma gas (8). More recently, the focus has been on the introduction of triple-quadrupole ICP-MS systems, utilizing the first quadrupole as a mass filter, followed by collision or reaction gas in the second; the third quadrupole acts as a pass-through or mass filter, depending on application needs (9). Sector-based platforms are often sought out for enhanced capabilities when performing isotope ratio analysis, particularly if multiple detectors are employed such as in multi-collector (MC) ICP-MS. Sector-based platforms can also offer higher mass resolution for separating polyatomic or isobaric interferences. Finally, time-of-flight–based ICP-MS instruments offer the ability to collect every mass simultaneously rather than sequentially, as is done in the aforementioned quadrupole-based platforms. Time-of-flight instruments have become increasingly popular for single particle and laser ablation imaging applications.

Practical Guide to ICP-MS–A Tutorial for Beginners

 (CRC Press, Boca Raton, Florida, 3rd Ed., 2013).

4. S. Greenfield, I.L. Jones, and C. Berry, 

5. R.S. Houk, V.A. Fassel, G.D. Flesch, H.J. Svec, A.L. Gray, and C.E. Taylor, 

6. R.E. Russo, X. Mao, H. Liu, J. Gonzalez, and S.S. Mao, 

8. D.W. Koppenaal, G.C. Eiden, and C.J. Barinaga, 

9. L. Balcaen, E. Bolea-Fernandez, M. Resano, and F. Vanhaecke, 

Inductively Coupled Plasma–Based Techniques (ICP-OES and ICP-MS) 

Surface enhanced Raman scattering (SERS), and the high-resolution imaging analog tip-enhanced Raman scattering (TERS), have emerged as ultrasensitive methods of chemical analysis (1). SERS derives from Raman scattering, and is a nondestructive, label-free, chemicalspecific method. In Raman spectroscopy, laser radiation incident on a molecule is inelastically scattered at frequencies that can be correlated to the vibrational modes of the molecule (2). These vibrational modes are similar to the energy levels probed in infrared spectroscopy and arise from how elements are connected within a molecule. The energy of each vibrational mode is sensitive to interactions with other molecules, temperature, and other conditions, providing insight into physical properties. Additionally, the unique organization and combination of elements that compose a molecule give rise to unique combinations of bands in the Raman spectrum, which is often considered a molecular fingerprint for identification. The challenge with Raman spectroscopy is that the probability of a photon inelastically scattering is very low (about a billion times less than that of a photon fluorescing). Fortunately, in the presence of silver and gold nanostructures, SERS can enhance Raman scattering signals by as much as 1011, such that single molecules can be investigated (3).

The general phenomenon responsible for SERS is shown in Figure 1 and arises from an intense localized electromagnetic field on the nanostructures when the laser excites a localized surface plasmon resonance (LSPR) (4). The LSPR is the collective oscillation of electrons in the conduction band of the metal nanoparticle. Silver and gold nanoparticles are most commonly used because their LSPR is found in the visible region of the electromagnetic spectrum, where most Raman spectroscopy experiments are performed. Other materials can also generate SERS; for example, aluminum nanoparticles exhibit a LSPR at ultraviolet wavelengths and can generate SERS (5). When excited, the oscillation of the electrons concentrates the laser radiation into intense local electromagenetic fields at the nanostructure surface. Molecules in close proximity (generally <5nm) to the nanostructure surface experience this field and generate increased Raman scattering. The Raman scattering occurs at a wavelength similar to the LSPR, causing the nanoparticle to further re-radiate the increased Raman scattering from nearby molecules. In essence, the nanoparticle acts as an antenna to amplify the Raman signal from molecules (6–8).

The electric field in the junction between two plasmonic nanoparticles is significantly larger than on the surface of a single particle and gives rise to very large Raman enhancements. Similarly, asymmetric nanoparticles, such as nanorods and nanostars (9,10), have higher enhancements at points and vertices. Highly enhancing locations are often called “hotspots” and have been shown to dominate the SERS signal (11). Plasmon-enhanced scattering has enabled Raman detection of single molecules and, in some applications, is more sensitive than fluorescence (12,13). At this point, it is well understood the physical interactions that generate a large SERS enhancement; however, controlling the interactions for quantitative analysis has been more challenging. The observed Raman intensity arises from the organization of the nanoparticles and the location of the analyte molecule interacting with the nanoparticles. Although nanoparticles are often modeled as spheres, small features on the surface can modulate the enhanced fields. Additionally, the spacing between particles affects the resulting fields, and forming reproducible aggregates can be challenging and affect the observed signals. Because only molecules near the surface can be detected, the signal saturates at higher concentrations.

The plasmonic effects that give rise to SERS can also be localized at the apex of a scanning probe microscope tip to perform TERS (14,15). This approach has been demonstrated using both atomic force microscopy (AFM) and scanning tunneling microscopy (STM) to control the position of the tip. The advantage of AFM-based TERS is it can be done on almost any sample, while STM–TERS requires a conductive sample. In either approach, a plasmonic metal nanoparticle (or sharp metal point) is located at the very apex of the tip to generate the Raman signal enhancement from molecules beneath the tip. By controlling the location and configuration of the enhancing nanostructure, high sensitivity spectroscopy can be obtained from precise locations on a sample (16). Figure 2 illustrates how, by scanning the sample under the TERS tip in the laser focus, high spatial-resolution images of samples can be obtained. In ultrahigh-vacuum (UHV) experiments, where molecular motion can be minimized, TERS imaging within a molecule has been demonstrated. Under ambient conditions, TERS experiments are capable of imaging with a spatial resolution of 1 to 2 nm (17–19).

A number of applications of SERS and TERS have been reported in the literature. Many SERS applications use the enhanced signal for trace detection. A large number of papers have been published describing many ways to generate a SERS substrate, and commercial solutions are available.

One common use is to create “SERS tags,” where a reporter molecule with a strong Raman response is attached to the surface of the nanoparticle along with some kind of recognition agent (such as an antibody or nucleic acid sequence) (20,21). The unique SERS signal from the reporter molecule is used to quantify, and image, the detected analyte. Although this is similar to fluorescence, SERS tags do not photobleach, and the narrow Raman bands allow multiple different tags to be detected and unmixed simultaneously (22). 

Special Issues-09-02-2020