Advancements in Molecular Spectroscopy: Instrumentation and Applications



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Highlights of recent advances in three major areas of molecular spectroscopy: infrared (IR), Raman, and fluorescence.

Recently, the field of molecular spectroscopy has expanded into new and exciting biological, medical, and sensing applications. This expansion resulted from both improvements in existing instrumentation and the development of new techniques in the fields of Raman, fluorescence, and infrared spectroscopy. Of particular interest, is how these techniques have moved beyond previous limitations, such as the diffraction limit of light or direct imaging through difficult media like plastic or bone. Although much of this work was carried out in an academic setting, the results provide important directions for future industrial endeavors.

A broad field encompassing several techniques, molecular spectroscopy has revealed fundamental chemical and biological information in a variety of applications. Although there are many different types of molecular spectroscopy, these processes involve how analytes interact with light. The following sections highlight recent advancements in three major areas of molecular spectroscopy: infrared (IR), Raman, and fluorescence. Improvements in instrumentation and the development of new techniques have greatly enhanced each field in imaging, sensing, and understanding fundamental chemical principles. Each one has certain advantages and problems that confine them to certain disciplines, and in some cases, these limitations have been overcome for future applications.

Infrared Spectroscopy

IR spectroscopy uses infrared light sources to irradiate molecules, which absorb at specific frequencies. These frequencies depend on the molecular size, structure, and composition, with the measured frequencies corresponding to certain motions. Furthermore, the vibrational and rotational motions of the molecule also depend on the functional groups, and these can be used to differentiate similar compounds with small differences. The broad vibrational bands of water are one of the primary limitations in IR spectroscopy, but this limitation has been addressed by advancements in spectral processing. Other alternatives include working with solid samples and collecting reflectance measurements using techniques such as attenuated total reflectance IR spectroscopy. Although the details of IR spectroscopy have been reviewed and highlighted elsewhere, the following section describes some recent advances in sensing, protein folding, and new applications through coupling to another instrument.

The most common IR technique, Fourier transform infrared (FT-IR) spectroscopy, is a widely used approach in fields ranging from forensics to art conservation. Recently, it has become a popular analytical tool in detecting adulterated food. This field encompasses industry as well as government in testing the authenticity and safety of different foods, plants, and herbs. Some of the advantages of FT-IR for food detection are ease of use, quick read out, small waste, and retention of the original sample (1). The primary concerns in food safety include watered-down products, cheap unnatural fillers, and, most importantly, toxic contaminants. Melamine is a common industrial plastic that can react with cyanuric acid or other additives and become toxic and lead to kidney damage. The Food and Drug Administration (FDA), European Union (EU), and World Health Organization (WHO) have set standards on the amount of melamine that can be consumed, which became an international health concern when it was discovered in infant formula. Although mass spectrometry (MS) can identify melamine, it requires longer processing times with a relatively large sample volume. In the case of trying to test several shipments of a product entering a country, such as formula, this would be difficult to implement. Researchers used FT-IR and multivariate analysis to detect melamine in tainted formula based on stretches from the amino groups (1).

To explore complex biological processes such as protein folding, two-dimensional (2D) IR spectroscopy uses ultrafast spectroscopy to probe mechanical motions on picosecond timescales (2). Two-dimensional IR is still an emerging technique, but it has successfully determined the structure of small-unknown peptides and has similar capabilities to 2D nuclear magnetic resonance (NMR) spectroscopy. With the addition of isotopic labeling, Buchanan and coworkers (3) directly explored the misfolding and kinetics of an amyloid peptide, amylin, associated with type II diabetes. By monitoring a carbonyl backbone isotope, they discovered important information related to the secondary structure of this peptide in forming fibrils, which has further implications for other diseases such as Alzheimer's disease and Parkinson's disease. Although NMR provides structural information with good resolution, it cannot determine intermediates on this timescale.

Even though IR spectroscopy has biological limitations, new progress in ionic sources and techniques are making IR more competitive and universal (4). One such example is IR ion spectroscopy coupled with MS, which has the capability of discerning small molecules. An area of high interest for identifying such compounds is metabolomics, which involves the study of residual small analytes to gain new insight about intracellular processes. The addition of IR to MS aided in identifying a group of peptides that all have a mass at ~669 Da, but contain unique functional groups such as a phosphate or amide stretch in the molecule. Such subtle details hidden within the mass spectrum were actually quantified using IR spectroscopy.

Although the examples outlined here describe the broad applications of IR, several challenges remain. In the case of FT-IR, the overtones associated with water — especially in the near-infrared region — completely overwhelm the spectra, and this can prevent moisture-rich foods from being detected. Moreover, in detecting melamine, while MS was more time consuming, the detection limit was ~250 ppb, but FT-IR only reached ~75 ppm. While efficiency and sample preparation have some importance, the primary aim remains sensitivity. As the 2D IR technique develops and researchers continue to explore problems like protein misfolding, it still requires careful labeling and only a few bands or stretches can be monitored at a time. Furthermore, 2D NMR still provides better structural resolution and ease in assignment of spectral peaks even at slower timescales. The coupling of IR with MS bridges the advantages of both techniques, but the difficulty lies in the ionization of biomolecular analytes. In addition, the complex nature of such a high concentration of metabolites requires an initial screening with a high-resolution combination MS technique before IR. Some of the challenges discussed here can be addressed or avoided using other techniques in Raman or fluorescence spectroscopy.

Raman Spectroscopy

Raman spectroscopy reveals chemical information about a compound based on the inelastic scattering of light. However, most of the light undergoes Rayleigh scattering, so only a few Raman-active molecules are available. To increase the inelastic scattering signal, fields have emerged such as surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), and resonance Raman spectroscopy, to name a few. Most importantly, Raman measurements can be performed in an aqueous environment, giving it a huge advantage over IR, and with light sources ranging from the ultraviolet to the near infrared regions of the spectrum.

SERS is one of the most popular methods and has been used in a variety of applications related to sensing proteins, cells, and chemical warfare agents. A gold or silver substrate can support surface plasmons, and when irradiated with light, the enhanced electromagnetic fields increase the Raman signal of analytes close to the substrate. A unique application of SERS has been in identifying dye components to verify the authenticity and degradation of art works. A major advantage of SERS over techniques such as high performance liquid chromatography (HPLC) is the small sample volume required. In particular, a tiny sample removed from a piece of art with a needle can be used with SERS. In an effort to analyze works in a nondestructive manner, Leona and colleagues (5)developed a residue-free gel containing nanoparticles, which directly attaches to art works and actually captures a small amount of dye. This is incredibly important because now virtually no sample removal is required and even areas that have small amounts of paint or dye can be analyzed without hurting the integrity of the art.

Similar to SERS, which uses a substrate, TERS uses a plasmonic metal tip such as a scanning tunneling microscope (STM) or atomic force microscopy (AFM) tip. TERS combines the spatial resolution of scanning probe microscopy with the chemical information of SERS. This typically involves the tip interacting with a metallic substrate to get the greatest enhancement, but there has been a lot of work done with tips on nonplasmonic surfaces. Paulite and colleagues (6) were the first to image an amyloid β peptide nanotape, and one of only a few to do so without a plasmonic hot spot. Typically, these peptides have weak Raman cross sections, but the enhancement at the tip field allowed them to map and actually collect images of the peptide, which matched images gathered using scanning tunneling microscopy. This greatly expands the capability of TERS beyond Raman active dyes and molecules and moves it toward exciting biological applications on different substrates.

Spatially offset Raman spectroscopy (SORS), which is a relatively new technique, has been used to identify samples hidden below the surface of different media such as layers of plastic and deep into tissue. In this system, the collected Raman signal is offset from the excitation region. Recently, Sharma and colleagues (7) combined the penetrating depth of SORS with the enhancing signal of SERS to perform surface-enhanced spatially offset Raman spectroscopy (SESORS). Using SERS-active nanotags, they detected the SESORS signal through thick layers of bone for the first time, which has many potential imaging and biomedical applications in the future.

A new imaging modality has emerged comprised of a magnetic photoacoustoic Raman (MPR) active nanoparticle that combines the advances of magnetic resonance imaging (MRI), photoacoustic imaging, and Raman microscopy for detecting tumors in the brain (8). These MPR named probes target only the cancerous cells as confirmed by both Raman imaging and histological samples collected from mice. In addition to tumor detection, Raman imaging confirmed in vivo that all of the cancerous cells had been removed from an anesthetized mouse. The future of these particles and Raman imaging could aid surgeons in confirming that all of the cancerous cells have actually been removed. Bridging all of these techniques with a single particle harnesses the advantages of each one to a promising goal.

One of the strongest arguments for using Raman active or plasmonic materials is that, unlike a fluorophore, they do not photobleach and have larger cross sections. This presents strong challenges to techniques that use fluorescent probes, but the nanoparticles that are easily imaged tend to be much larger. Some of these nanoparticles can be hundreds of nanometers in diameter, unlike a fluorescent protein, which is much smaller and can easily be incorporated into a cell or other protein, such as green fluorescent protein. The relatively larger size of these particles can perturb the system and affect the results in measuring protein folding for example. In the case of TERS, outside of a simple biological system, multiple chemical components can overwhelm the signal and require further processing using a technique such as principal component analysis (9). Additionally, when dealing with small volumes of analyte, the TERS spectra can actually shift from changes in the sample or even to the tip itself. Raman spectroscopy has been rising to the challenge of overtaking fluorescence, but as you will read in the following section, fluorescence has already evolved beyond its own imaging limits.

Fluorescence Spectroscopy

Fluorescence spectroscopy has long been the biological gold standard, and it has been widely used to study everything from DNA folding to cellular imaging (10). This tunability arises from the many dyes, proteins, and tags that can easily be integrated to study a biophysical process. Some of these include total internal fluorescence microscopy, confocal fluorescence microscopy, and single molecule fluorescence spectroscopy.

A widely used fluorescence-based spectroscopic technique, Förster resonant energy transfer (FRET), involves the exchange of nonoverlapping energy between a donor and acceptor fluorescent probe. This reaction is distance dependent and has been used extensively to study biological processes, even down to the single-molecule level (11). Multiplexing can be difficult using FRET because of the number of chromophores required that also cannot overlap. This requirement greatly limits the detection capability and execution of the technique. To overcome this limitation, each antibody was tethered to a different dye and reacted with a single terbium complex by Geibler and colleagues (12). They easily differentiated and processed the resulting signal for each chromophore with minimal spectral overlap. Additionally, they simultaneously detected multiple tumor markers associated with different forms of lung cancer in serum with excellent detection limits. This approach has overcome one of the disadvantages in using FRET both in high-throughput assays and for multiplexing capabilities and has far-reaching potential in other spectroscopy applications.

As microscopes have continued to improve, the maximum spatial resolution was met at the diffraction limit of light, but recently super resolution microscopy has emerged as a way to explore processes beyond this. Before the emergence of this technique, fluorescent images collected below the diffraction limit could not be resolved, which limited the study of intracellular components. Techniques such as stochastic optical reconstruction microscopy (STORM) (13) and photoactivation localization microscopy (PALM) (14) have allowed us to explore new parts of the cell, such as tubules and chromatin, which are too small to image with conventional means. Both of these techniques, developed independently, involve pinpointing the location of different individual fluorescent dyes (two or more) that are activated at different nonoverlapping wavelengths. This process involves collecting a series of images with a few fluorophores per image and isolating the position of each in all the images to reconstruct the original (15).

The use of such dyes is only one type of approach: Researchers at the National Institutes of Health made improvements in linear structured illumination microscopy (SIM) and successfully demonstrated its super resolution capabilities (16). Instead of using multiple dyes, SIM involves advancements in optics through modulating light and postcollection algorithm processing to resolve images. These advancements allowed them to collect images of moving blood cells in embryonic zebrafish and the growth and development of tubules within the endoplasmic reticulum in lung cells.


Many of the techniques described here are still emerging in their field of application or instrumentation and have enabled the exploration of diverse problems in biology, chemistry, food, and art. Each technique has found different ways to maximize their advantages and compensate for their weaknesses. Although IR spectroscopy is a standard laboratory technique, it appears to have the most challenges in tackling complex biological or medical problems. The work done so far in 2D IR has opened an interesting direction for IR to move into fields such as understanding protein dynamics. In some cases, the coupling of a technique to another such as IR with MS allowed for identifying individual analytes in a largely homogenous looking sample based on MS alone. As researchers continue to improve on enhancing Raman signals, the applications in imaging and sensing continue to evolve. Additionally, the coupling of Raman with other techniques has found an exciting new avenue for both pre- and post-tumor removal in cancer patients. Super resolution imaging has overcome traditional limitations in fluorescence imaging to allow exploration in unknown parts of the cell. As this field develops even further, less understood cellular processes can be explored and imaged, potentially in real time. The continued effort of researchers to overcome some of the challenges across many of these fields of spectroscopy will open new avenues of instrumentation, improve disease diagnostics, and determine unknown biological processes.


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Laura Ruvuna is a Postdoctoral Fellow in the Van Duyne Research Group in the Department of Chemistry at Northwestern University in Evanston, Illinois. Direct correspondence to

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