A Noninvasive Method for Deep Raman Spectroscopy of Living Tissue and Powders

Many analytical applications require fast, noninvasive characterization of deep regions of diffusely scattering (turbid) media, such as living tissue and powders, with high chemical specificity. Such information is important, for example, in disease diagnosis, noninvasive probing of pharmaceutical products for quality control, drug authentication, and security screening.

Laser spectroscopy holds particular promise in this area due to its ease and speed of implementation. Presently, fundamental optical spectroscopic techniques that are applicable to this class of problems include near- and midinfrared (NIR and MIR) absorption spectroscopy and Raman spectroscopy. Although NIR absorption is widely used, it often suffers from low chemical specificity.¹ In comparison, both MIR and Raman spectroscopy offer a substantially higher degree of chemical specificity but have, thus far, been confined to applications involving mainly surface layers of turbid media. In the case of MIR, the principal reason for this is the excessively high absorption of its signal by water or sample matrix. From this perspective, Raman spectroscopy holds great potential since it does not suffer from these fundamental limitations.

Conventional Raman spectroscopy

The Raman effect is the inelastic scattering of photons from molecules via interactions with the vibrational modes of the analyte molecule.¹ In this process, a photon typically transfers a fraction of its energy to a vibrational mode within the molecule. Consequently, the wavelength of the scattered photon is spectrally red-shifted, with the degree of the shift indicating the amount of energy uptake by the molecule. Since the vibrational modes are quantized and molecule specific, the molecular identity can be determined from the distribution of the observed shifts in wavelength. The shift pattern serves, in essence, as a unique fingerprint for the molecule, specific to its structure and conformation.

The general applicability of this technique is limited to samples that do not exhibit strong fluorescence emission in the spectral Raman region, since this can easily swamp the relatively weak Raman signals. However, this problem can often be avoided by using near-infrared excitation away from the electronic absorption bands of most fluorescing species, thus preventing their excitation and consequently the generation of fluorescence emission in the first place.

Figure 1 - Illustration of the conventional backscattering Raman and spatially offset Raman spectroscopy (SORS) geometries.

To date, the Raman method has been used predominantly in the backscattering collection mode (see Figure 1, left) capable of probing only shallow depths of turbid media where the medium still appears semitransparent.¹ For example, in living tissue, depths of up to several hundred micrometers are accessible. However, the deeper layers remain inaccessible due to the surface Raman or interfering fluorescence signals masking much weaker Raman spectra of the deeper components. The penetration depth is somewhat higher with pharmaceutical products such as tablets, typically 1 mm or so, but this is still insufficient for probing the overall bulk content of the tablet or its composition through thicker packaging.

Spatially offset Raman spectroscopy

A substantial extension of the Raman technique penetration depth was recently accomplished using the diffuse component of light in a manner similar to NIR absorption tomography. The developed methods fall into two categories: temporal and spatial. The temporal approach relies on the impulsive excitation of sample and fast, time-resolved detection of the Raman signal.^2–4 The spatial method (SORS,⁵ see Figure 1, right), which is instrumentally much simpler since it does not require a pulsed laser and time-gated detection, is based on the collection of Raman signal from spatially offset regions away from the point of illumination on the sample surface. Since its development, the technique has been used in numerous applications, including the noninvasive Raman spectroscopy of bones,^6,7 and in pharmaceutical⁸ and security applications.⁹

In such measurements, the laterally offset spectra contain different relative contributions from sample layers located at different depths. This difference is a result of the wider spread of the deep-layer Raman photons emerging from the surface in comparison with surface Raman contributions. This difference is brought about by the lateral diffusion of photons; photons penetrating to deeper layers are influenced to a greater degree than photons interacting with the surface layers alone. It is also beneficial that the SORS approach is not only capable of effectively suppressing Raman signals from the surface layers, but also the interfering fluorescence originating from surface layers. This is in contrast to conventional Raman spectroscopy, where a Raman spectrum is collected directly from the laser deposition area. Consequently, its signal is dominated by the intense surface layer components.

SORS technique application areas encompass pharmaceutical, chemical, and food analysis applications; security screening for harmful or illegal substances; and disease recognition of subsurface tissue components such as bones and breast cancer tissue. Below are two examples from the pharmaceutical and security screening areas.

Noninvasive authentication of pharmaceutical drugs

The first example is the authentication of drugs in the supply chain. The need for such authentication is driven by the increasing infiltration of the market by counterfeit drugs.^8,10,11 For example, the occurrence of fake antimalarial drugs presents a serious threat to health in eastern Asia. This problem is exacerbated by the rapid spread of Internet vendors, which significantly increases the complexity of the supply chain.

The existing monitoring tools include NIR absorption spectroscopy, which has limited chemical specificity restricting its effectiveness, and Raman spectroscopy, which is used in its conventional backscattering collection mode, limiting its use to surface layers of turbid samples. Although the Raman technique is effective with many pharmaceutical blister packs, darkly colored tablet coatings or capsules or thick packaging yield excessively intense Raman and/or fluorescence signals. This reduces the technique’s sensitivity and, in some cases, prevents its deployment entirely.

Recently, it was demonstrated⁸ that SORS can substantially enhance the detection sensitivity and even permit the probing of pharmaceutical products through opaque plastic bottles, which often present a major insurmountable challenge to the existing Raman approach. In this study, SORS outperformed, in all investigated cases, the conventional Raman technique by effectively suppressing the contributions from the blister packs and capsule shells, and permitted unobstructed probing of the content of white plastic bottles.

Figure 2 - Noninvasive Raman spectra of paracetamol tablets measured through a white, diffusely scattering 1.7-mm-thick plastic container in drug authentication. Conventional Raman and SORS raw data are shown together with the tablet’s reference Raman spectrum. The conventional Raman spectrum is overwhelmed with the Raman signal of the container, masking the Raman signal of paracetamol held within.

Figure 2 shows the performance of SORS and conventional backscattering Raman geometry in probing noninvasively white plastic bottles containing pharmaceutical tablets. In this application, the conventional Raman approach was ineffective due to the presence of very intense interfering Raman signals from the container. Even under such challenging conditions, the SORS approach, after the scaled subtraction of two SORS spectra measured at different spatial offsets (0 and 3 mm), yielded a clean Raman spectrum of the pharmaceutical tablets within the bottle. The experiments were performed at 830 nm, with an acquisition time of 10 sec and a laser power of 50 mW.

Security screening

The recent heightened terrorist threat underlines the importance of the availability of robust security screening techniques with high chemical specificity. Examples include the screening of passengers and their luggage at airports or the screening of envelopes at mail sorting centers.

Figure 3 - Noninvasive measurement of powders in envelopes in security screening applications. The measurement was performed using an envelope containing sugar representing a hidden illegal or harmful substance. a) Raw conventional and SORS spectra, b) background-subtracted SORS spectrum (eliminating fluorescence contribution) and sugar reference spectrum. The conventional Raman spectrum is dominated with interfering fluorescence background originating from the envelope material masking the Raman signal of the substance within.

The use of SORS was reported⁹ for probing powders held in envelopes, and the results were compared with those attained using conventional backscattering Raman spectroscopy. In these measurements, a standard brown envelope containing white sugar, representing a harmful substance, was used. Comparative conventional backscattering Raman measurement yielded a massive fluorescence background originating from the envelope material, entirely masking the Raman signal of the substance contained within (see Figure 3). In contrast, the SORS spectrum clearly shows the Raman components of sugar held within the envelope. The experiments were performed using 830 nm, and the acquisition time was 10 sec with a laser power of 55 mW.

Conclusion

The SORS method has stimulated the development of numerous new analytical methods for probing tissue and powders at previously inaccessible depths. Potential application areas include disease diagnosis, such as osteoporosis, brittle bone disease, and breast cancer; quality control and authentication of pharmaceutical products; and security screening applications.

References

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The authors are with the Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire OX11 0QX, U.K.; tel.: +44 1235 445377; fax: +44 1235 445693; e-mail: [email protected].