John A. Reffner

Although 69 years have passed since infrared spectroscopy and microscopy were first united, these technologies continue to advance. Since 1983, the catalysts for rapid commercial growth and broad applications were Fourier transform infrared (FT-IR) spectrometers, resulting in advances in all reflecting microscope optics and high sensitivity detectors. Today, quantum cascade lasers and scanning microscopy are emerging technologies that are demonstrating the ability that drastically improves resolving power and quality of spectral data collected from submicrometer-sized samples. This installment of "Molecular Spectroscopy Workbench" is a brief history and reports on the status of infrared microspectroscopy and its future promise.

	Art conservationists, biologists, chemists, environmentalists, forensic scientists, geologists and materials scientists need the combination of chemical composition and morphological structure to analyze complex matter, solve problems, or identify small particulates. Scanning electron microscopy (SEM) provides imaging of morphological structure, and can be combined with X-ray spectroscopy for elemental analysis. However, molecular composition may only be inferred from X-ray spectra by quantitative elemental analysis. Infrared microspectroscopy provides both molecular chemical information and imaging of morphological structure, but is limited for elemental analysis. The success of both of these techniques is dependent on their ability to provide high quality spectral data at the highest possible spatial resolution. Recent discoveries in infrared microspectroscopy are advancing this approach to new levels of spectral quality and resolving power.

	First, a brief history of infrared microspectroscopy. In 1949, two publications (1,2) heralded the practicality of infrared spectroscopy. Perkin-Elmer Corp. introduced the first commercial infrared microspectrometer in 1953, the Model 85 (3). A single-beam, sodium chloride prism, dispersive infrared spectrometer was coupled to an all-reflective light microscope. A four-bladed variable aperture located in the primary image plane defined the area of the sample that produced the transmission spectrum. The detector was a bolometer and the intensity of transmitted infrared radiation at each wavelength was plotted by a strip chart recorder. A string connected to the variable slit in the spectrometer was attached to a cardioid cam, increasing the slit width to maintain a level of incident intensity as the wavelength was increased. However, the spectral resolution varied across the spectrum. Obtaining a transmittance spectrum required recording an 

 single beam spectra, tabulating the intensity of each at periodic wavelengths, calculating the 

 ratio, and replotting the data. Although this system was a novel research tool, it was not a commercial success.

	Infrared microspectroscopy was essentially dormant until 1978, when V. Coats introduced the Nanospec IR20 microspectrometer. By combining a microprocessor computer, a liquid nitrogen–cooled solid-state detector, a variable-wavelength interference filter monochromator, and an all-reflecting optical microscope, Coats resurrected interest in IR microspectroscopy. His revolutionary contribution was overshadowed when Fourier transform infrared (FT-IR) spectrometers became commercially available. Attaching all reflecting microscopes to FT-IR spectrometers let the genie out of the bottle. Three decades of improvement to FT-IR microspectrometers, and sustained growth applications, established infrared microspectroscopy as a vital analytical technique.

	The motto "good enough is never good enough" is a driving force for advancement. In microspectroscopy, improving spectral quality, detection limits, and resolving power are challenges. The quality of an infrared spectrum is limited by the intensity of incident radiation, wavelength resolution, sample size, and detector sensitivity. Detection limits are dependent on the sample's area, thickness, and composition. Resolving power-the minimal distance of separation of two objects-using visible or infrared radiation is limited by wavelength, numerical aperture, optics and contrast. The resolving power of spectral maps created using an FT-IR microspectrometer is limited by the wavelength and spectral contrast. The spectral quality and contrast of maps are reduced as the dimensions of the sample are reduced. When spectra are collected from a diffraction-limited point using an FT-IR spectrometer with a 1200 K ceramic source, spectral quality and contrast are reduced. Higher intensity sources, such as the synchrotron or quantum cascade (QC) lasers, have demonstrated improved spectral quality and mapping speed of performance at the wavelength diffraction limit.

	United States Patent 457,709, filed April 4, 1994, lists the inventors F. Capasso, A.Y. Cho, J. Faist, A.I. Hutchinson, S. Luryi, C. Sirtori, and D.L. Sivco (4). The abstract of this patent states: "This application discloses, to the best of our knowledge, the first unipolar laser. An exemplary embodiment of the laser was implemented in the GaInAs/AlInAs system and emits radiation of about 4.2 µm wavelength. Embodiments in other material systems are possible, and the lasers can be readily designed to emit at a predetermined wavelength in a wide spectral region. We have designated the laser the 'Quantum Cascade' (QC) laser."

	The foundation for the QC laser is the use of intersubband transitions for amplification of radiation (5). Over the past two decades, an abundance of QC laser research and development has been taking place (6). Today, QC lasers are commercially available from several sources. The power output has increased, and the operating temperature can be above room temperature. They can operate in either a continuous or pulsed mode. They are capable of emitting mid-infrared radiation in the 2857–400 cm

 range. The successful application of QC lasers in medical, biological, environmental, communication, and chemical research has driven their expanding growth.

	QC laser technology provides a new direction for advancing infrared microspectroscopy. The major avantages of QC lasers for infrared microspectroscopy are broad tunablity over the mid-infrared wavelength range, intensity, low divergence, and high frequency rate. Three approaches have been advanced for improving spatial resolution and quality of spectral data using QC lasers. The first approach was to replace the infrared radiation source with a broadly tunable QC laser for an all-reflecting microscope. The second approach was to couple the broadly tunable QC laser with scanning probe microscope to detect the photothermal expansion created by a modulated QC laser beam. The third approach was to use a visible light probe to detect the photothermal expansion created by a modulated QC laser beam. These three approaches are addressed chronologically.

	Replacing the thermal source with QC lasers was first demonstrated by the R. Bhargava research group at University of Illinois in Urbana, Illinois, in 2012. An in-house infrared microspectrometer was developed that used an array of tunable QC lasers supplied by DRS Daylight Solutions, tunable over the range of 1738 –1570 cm

 as a transmitted radiation source (7). This microspectrometer system demonstrated that major advantage of using the QC laser over FT-IR was the speed of collection spectral maps using a single wavelength. In 2014, DSR Daylight Solutions introduced the first commercially available QC laser–powered chemical imaging microscope for IR spectroscopy. The Spero system (DSR Daylight Solutions) proclaimed real time, high-throughput spectral imaging enabled by ultrahigh-brightness QC laser technology. The spectral range of the Spero imaging system was expanded to 1800–900 cm

. This expanded range was a significant development. The initial applications these advances were in label-free histochemical imaging, and classification in tissues (8–11).

	Combining QC laser technology with scanning probe microscopy (SPM) is the invention of C. Prater and K. Kjoller in 2011 (12–14). This invention makes possible a novel method for obtaining nanometer resolving power and high-resolution infrared spectra. The method includes using infrared radiation from a quantum cascade laser directed onto a small area of a sample. The QC laser array is tuned over a broad range of infrared wavelengths. When the sample adsorbs radiation of a certain wavelength, the energy of the adsorbed photon is transformed into heat, causing a thermal expansion of the sample. This photothermal event is monitored using a scanning probe microscope. The probe of the microscope is mounted on a cantilever. The QC laser is operated in a pulsed mode, creating a periodic motion of the cantilever. The amplitude of this osculation is a measure of the magnitude of the photothermal event. The magnitude of the thermal event plotted against the wavelength infrared radiation to produce a photothermal IR (PTIR) spectrum. This unique combination of QC laser and scanning probe technologies produces infrared spectra that are consistent with spectra collected by FT-IR. Photothermal IR is also known as atomic force infrared spectroscopy (AFM-IR).

	AFM-IR advanced infrared spectroscopy from a resolving power limited by the wavelength of the infrared radiation to a limit defined by the tip size of the scanning probe, a change of scale from ~10 µm to ~10 nm. QC lasers provide the intensity to produce photothermal events that the scanning probe microscope detects to produce infrared spectra at subnanometer spatial resolution. The rapid scanning of the sample, while the it is exposed to a single wavelength of infrared radiation, produces molecular composition maps with unprecedented resolution.

	AFM-IR spectroscopy was the basis for the development by Anasys Instruments of the first commercial instrument, the nanoIR system (Anasys Instruments). The nanoIR instrument became available in 2012. In the past six years, AFM-IR technology has become a prominent tool in nano research in the biological, medical, chemical and materials sciences (14-16). The best evidence for the success of AFM–IR is the recent acquisition of Anasys Instruments by Bruker.


	Figure 1: A schematic diagram illustrating the optical components of the mIRage photothermal infrared microspectrometer. 

	Replacing the mechanical probe of a SPM with a focused monochromatic visible light probe to detect the photothermal event created by the incident infrared radiation provides a new instrumental approach to infrared microspectroscopy. The Anasys group developed this approach and created a new company, the Photothermal Spectroscopy Corp. The company's new mIRage instrument fills the resolving power gap between FT-IR and AFM-IR. At the heart of this approach, the photon probe is focused on the sample's surface, and is coaxial and confocal with the mid-infrared radiation. This relationship is illustrated in Figure 1. The infrared source is a broadly tunable pulsed QC laser. The photothermal event, produced when the sample absorbs the infrared radiation, creates a change in the reflected or scattered light's intensity. This change in the visible light intensity is proportional to the absorption of infrared radiation used to create a photothermal infrared (O-PTIR) spectrum. The O-PTIR spectra, collected using the mIRage micro, are comparable to spectra in the KnowItAll Spectra FT-IR database (Bio-Rad). Illustrated in Figure 3, the O-PTIR spectra are well matched to the database spectra, all with a correlation factor above 98%.


	Figure 2: Infrared absorption spectra of poly(styrene) (PS), poly(ethyleneterephthalate) (PET), and poly(methylmethacrylate) (PMMA), collected using the mIRage photothermal infrared microspectrometer compared against the KnowItAll FT-IR spectral database (Bio-Rad) (in red).

	With the optical probe system of the mIRage system, the resolving power in spectral imaging is improved to ~0.45 nm. Importantly, the resolving power in spectral images is limited by the wavelength of the visible-light laser and is constant across the infrared spectral range. In spectral images collected using FT-IR or QC laser micro spectrometers, the resolving power is a function of the infrared radiation, and therefore varies across the spectrum.


	Figure 3: (Left) Photothermal infrared spectra collected using the mIRage system. (Right) Micrograph showing a digital image record of the cross-section of multilayered laminated sample. Two points, 500 nm apart, are marked with red and blue X's. The infrared spectra were collected at the indicated locations.

	Operating in the noncontact reflection mode simplifies sample preparation. For example, Figure 4 shows the spectrum an individual fiber in a fabric acquired directly with no special sample preparation. Thick samples can be analyzed in the single point mode. Mapping of large areas require flat surfaces normal to incident radiation. The variety of samples, improved resolving power, quality of O-PTIR spectra and ease of sample preparation, dramatically advance microspectroscopy.


	Figure 4: Optical image and O-PTIR spectrum of test fabric consisting of 13 different textile materials all woven together into a single sample. Data collected using the mIRage instrumentThe blue cross indicates the location where the O-PTIR spectrum was collected.

	There are numerous examples of applications of infrared microspectroscopy in all academia, national laboratories, government agencies, hospitals, industries; all of them benefiting from the advances in the field (17–19). It is stimulating to see the advances of infrared microspectroscopy. The history of progress of this powerful and diverse technology is very impressive. It is great to be part of the evolution from its humble beginnings to major factor in our growing technology. Biologists, chemists, geologists, materials scientists, microscopists, and spectroscopists around the world are awakening to the values of nanotechnology. The small world is getting larger.

	(3) V.J. Coats, A. Offner, and E.H. Siegler, 

	(4) F. Capasso et al., US Patent, 5,457,709, Unipolar Semiconductor Laser, 1994.

	(5) F. Capasso et al., US Patent, 5,936,989, Quantum, 1997.

	(7) K. Yeh, M. Schulmerich, and R. Bhargava, "Mid-infrared Microspectorscopic Imaging with a Quantum Cascade Laser," in 

Next Generation Spectroscopic Technologies VI

, edited by M.A. Druy and R.A. Crocombe, Proc. of SPIE vol. 8726 8720E-1(2013).

	(8) K. Yeh, S. Kenkel, J-N. Liu, and R. Bhargava, 

	(9) H. Sreedhar, V.K. Varma, F.V. Gambacorta, G. Guzman, and M.J. Walsh, 

	(10) J.B. Bird and J. Rowlett, "A Protocol for Rapid, Label-free Histochemical Imaging of Fibrotic Liver," 

	(11) C. Kuepper et al., "Quantum Cascade Laser-Based Infrared Microscopy for Label-Free and Automated Cancer Classification in Tissue Sections," 

	(12) C. Prater and K. Kjoller, US Patent 8,680,467 (2013).

	(13) L. Brown, M. Davanco, Z. Sun, A. Kretinin, Y. Chen, J.R. Matson, I. Vurgaftman, N. Sharac, A.J. Giles, M.M. Fogler, T. Taniguchi, K. Watanabe, K.S. Novoselov, S.A. Maier, A. Centrone, and J.D. Caldwell, 

	(15) M. Belkin, F. Lu, V. Yakolev, C. Prater, K. Kjoller, and M. Raschke, US Patent US8869602B2, High frequency deflection measurement of IR absorption (2014).

	(17) F. Huth, Al. Govyadinov, S. Amarie, W. Nuansing, F. Keilmann, and R. Hillenbrand, 

	(19) J. Chae, Qi. Dong , J. Huang, and A. Centrone, 

is a professor at John Jay College of Criminal Justice in New York, New York. Direct correspondence to: 

Articles

Infrared microspectroscopy has led to important advances in a wide range of fields, as biologists, chemists, geologists, materials scientists, microscopists, and spectroscopists around the world have awakened to the values of nanotechnology. The small world is getting larger.

Advances in Infrared Microspectroscopy and Mapping Molecular Chemical Composition at Submicrometer Spatial Resolution

The authors discuss the use of vibrational spectroscopy to differentiate an authentic article from a counterfeit one throughout a product's lifecycle, from component receipt at the site of manufacture, to product receipt by the end user.

While the challenge of facing this type of illicit activity might seem overwhelming for a variety of reasons, the need to do so has never been more critical. Aside from the loss of revenue, there are other considerations that are just as significant. Many of these counterfeit products pose a significant threat to health and safety due to substandard quality control, and some have even been shown to be harmful to the user (2). Counterfeiting and piracy also have a long-term negative impact on innovation and brand recognition, and channel a considerable portion of revenue to criminal networks, organized crime, and other groups that disrupt and corrupt society (3). In addition, intellectual property in the form of copyrights, patents, and trademarks promotes cultural development, fosters innovation and growth, and protects public health and safety (4). Theft of these goods affects all industry sectors and results in substantial economic, social, and developmental costs that have increased exponentially in recent years. Piracy and counterfeiting have been shown to decrease employment opportunities, diminish tax revenues for governments, and reduce incentives to invest (5). 

Product Authentication Using Analytical Chemistry

Defense of intellectual property assumes that it is possible to differentiate an authentic article from a counterfeit one. Frequently this is a relatively simple task. As counterfeiters become more experienced in their trade, though, differentiation becomes more and more challenging. In many instances, it is necessary to perform chemical analysis before a conclusion about authenticity can be made. In addition, due to the complex nature of the lifecycle of a counterfeit article, it has become necessary for brand owners across many different industries to employ methods for detection and authentication at various levels within their legitimate supply chains. For instance, in the pharmaceutical industry, many companies employ in-house forensic scientists to address product authentication concerns. Within the textile industry, safeguarding the integrity of natural organic fibers is an absolute requirement, as contamination with pesticide-tainted counterfeits undermines quality and, ultimately, revenue. In the electronics industry, counterfeiting has been a problem for a long time, but the current economic challenges appear to be making things even worse. The industry has even begun to adopt practices like qualifying reliable vendors to help prevent counterfeits from entering their supply chain and marketplace. Although this is a common practice in the pharmaceutical industry, it has been adopted only recently within the electronics arena. 

The level of testing performed to determine authenticity is variable, but there are analytical methods known to be effective at providing proven results that enable effective chemical differentiation. For instance, vibrational spectroscopy in the form of both infrared (IR) and Raman spectroscopy has been used with success to perform chemical identifications on both the counterfeit article itself and on the packaging used for transport of the article (6). Figure 1 shows the IR spectra collected from packaging from genuine and replicate medications. These spectra show that the chemical information contained within an IR spectrum frequently can be quickly and easily (within seconds) used to differentiate the coating layer of packaging used. Differences between these two samples are evident across the range of the mid-IR spectrum. Analysis was performed using the IdentifyIR portable Fourier-transform infrared (FT-IR) spectrometer (Smiths Detection, Alcoa, Tennessee).  

Figure 1: Infrared spectra collected from the outside packaging of genuine and replicate samples. 

Vibrational microspectroscopy also can be used to differentiate components of the counterfeit product itself. Figure 2 shows the IR spectrum and photomicrograph collected from a cross-section of a genuine tablet, as well as the IR spectrum of a known reference standard of pseudoephedrine hydrochloride. These data show that the active pharmaceutical ingredient can be selected for microspectroscopic analysis using polarized light microscopy or other contrast-enhancement techniques. The coupling of the microscope with microspectroscopy makes it possible to collect spectra from discrete locations within a relatively complex matrix. In this instance, IR microspectroscopy was used to perform the chemical identification using an IlluminatIR IR microprobe (Smiths Detection). This IR microprobe allows the user to employ contrast-enhancement methods such as polarized light or fluorescence microscopy to visualize the sample and selectively isolate particles for microspectroscopic identification. It is also possible to use Raman microspectroscopy to perform a similar type of chemical analysis. Frequently, although not always, the chemical composition of counterfeit samples will not only have different excipients (nonactive components) from the replicate sample, but it might not even contain the active pharmaceutical ingredient at all (7). 

Figure 2: Infrared spectrum showing the active pharmaceutical ingredient contained within tablet cross-section. 

Product Authentication and Brand-Management Protection

Advances in technology make it possible to perform these and other types of chemical analysis, but protection of brand owner rights is not simply based upon detection of a counterfeit article when one presents itself. It relies upon maintaining control throughout a product's lifecycle. This can be a very difficult challenge, especially considering the global economy within which many commercial products are manufactured. Products frequently contain components from around the world, regardless of where final assembly or manufacture of the product might occur. The ability to establish control can be challenging in the best of circumstances and is almost always difficult to manage. The IntelliMark Solution product identification system (Smiths Detection in partnership with ARmark Authentication Technologies, LLC, Glen Rock, Pennsylvania) enables control and authentication of both the components used during manufacture or assembly and of the finished product. Product authentication can be verified anywhere along its lifecycle. 

The product identification system is a complex, customized product authentication solution that makes it possible for the brand owner to establish and maintain control by building it in at the beginning of the product's lifecycle. Information-laden, custom-designed covert markers are either incorporated into the raw materials and components, or they can be placed within the final product or its packaging and shipping containers. Product authentication can occur in transit or when the product is delivered to its final destination. The industries within which this technology can be applied effectively are virtually unlimited, ranging from the food industry, where the covert markers are manufactured using ingredients that are generally recognized as safe (GRAS) (8) for human consumption, to the electronics industry, where even fluorescent or other types of chemically elegant markers can be designed and used. 

Figure 3: Photomicrograph of covert marker (approximately 75 Î¼m) in the finished product for detection in transit or at final destination. 

Regardless of the industry served, the markers are detected easily using instruments designed to be used in the environments encountered along the supply chain. Figure 3 shows a covert marker (ARmark Authentication Technologies, LLC) detected using the IdentifyIR (Smiths Detection) spectrometer. This instrument is used in the laboratory or on the loading dock and is capable of both creating a visual image of the covert marker and collecting an IR spectrum for chemical identification of either the marker, the product, or its packaging or shipping container. Both the image and the IR spectrum can be used to set pass–fail criteria for authentication, which can be established as part of an interactive service between the brand owner and Smiths Detection. This interactive service is a critical component of the product identification system. The spectrum of the covert marker shown in Figure 3 as well as of the matrix within which the marker is incorporated is shown in Figure 4. 

Figure 4: Infrared spectrum of covert marker (red) and of the matrix (blue). 

Alternate wavelength light-source accessories can be used to help locate the covert markers when the markers are fluorescent, and also can serve as an additional level of security for authentication of the covert marker. Figure 5 shows the polymer matrix of an electronics board impregnated with covert markers. A handheld ultraviolet light source was used to induce fluorescence at the time this photomicrograph was collected. The type of covert marker offered is customized to meet the needs of the customer and can be simple or complex, depending upon the user's needs. 

Figure 5: Covert markers dispersed within polymer matrix visualized using an ultraviolet light source accessory with a digital microscope. 

In recent years, counterfeiting, piracy, and other intellectual property rights violations have grown in magnitude and complexity, costing businesses billions of dollars in lost revenue and often posing health and safety risks to consumers. This growth was fueled in part by the spread of enabling technology, allowing for simple and low-cost duplication of copyrighted products, as well as by the rise in organized crime groups that smuggle and distribute counterfeit merchandise for profit (9). Global access to the Internet also plays a critical role, especially with regard to accessibility to counterfeit pharmaceuticals. The Internet has become the primary tool for criminal organizations to advertise, communicate, and conduct sales of counterfeit pharmaceuticals, as well as being the primary mechanism for consumers to find, order, and make payments for counterfeit pharmaceuticals (10). Considering these and other facts, brand owners need to be vigilant about how they protect their intellectual property. Building control into a product's lifecycle provides the greatest level of security. These combined capabilities enable detection and authentication throughout a product's lifecycle from component receipt at the site of manufacture, to product receipt by the end user. 

is with John Jay College of Criminal Justice, CUNY, New York, New York. 

is with ARmark Authentication Technologies, LLC, Glen Rock, Pennsylvania. 

is with Smiths Detection, Alcoa, Tennessee. 

(1) The Economic Impact of Counterfeiting and Piracy, Executive Summary, 2008, 

http://www.ul.com/newsroom/anticounterfeiting/risk.html

(3) The Economic Impact of Counterfeiting and Piracy, Executive Summary, 2007, 

http://usinfo.state.gov/products/pubs/intelprp/protecting.htm

http://www.iccwbo.org/policy/ip/id2948/index.html

(6) Smiths Detection Application Brief AB-83, Trace Evidence Analysis Using the IdentifyIR Diamond ATR Spectrometer. 

http://www.usdoj.gov/dea/programs/forensicsci/microgram/mg0106/mg0106.html

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPackaging/ucm061846.htm

http://www.ice.gov/pi/cornerstone/ipr/index.htm

(10) ICE Efforts to Combat Counterfeit Pharmaceuticals, Fact Sheet, July 11, 2006. 

John A. Reffner

Articles

Advances in Infrared Microspectroscopy and Mapping Molecular Chemical Composition at Submicrometer Spatial Resolution

Detection and Sourcing of Counterfeit Pharmaceutical Products and Consumer Goods