Comparison of diffuse versus inverse spatially-offset Raman spectroscopy modalities for analyte detection through barriers

Abstract

Spatially-offset Raman spectroscopy (SORS) is gaining an increasing popularity in detection of analytes through barriers. SORS can be applied in a number of different excitation modes and head-to-head comparison of different SORS modes are limited in the literature. In this study, a custom system that is capable of switching between two spatially offset Raman excitation modes, namely inverse (iSORS, ring) and diffuse (dSORS, enlarged flat-top circle), were compared at the same power for depth-probing Raman spectroscopy applications. Specifically, depth of detection, signal-to-noise ratio, and intensity ratio as a measure of elimination of barrier contribution to the Raman spectra were compared between the two methods. Transparent and opaque polymeric materials as well as skin tissue were used as barriers to characteristic Raman peaks from other polymers, pharmaceutical tablets or joint-borne gout crystals. Results indicated that diffuse SORS mode provides signal collection from depth layers at significantly greater intensities and at higher signal-to-noise ratio when compared to iSORS. On the other hand, iSORS generally provided a higher depth layer to barrier layer intensity ratio, indicating that iSORS is more effective than diffuse SORS in terms of the elimination of barrier layer contribution. We also demonstrate that barrier signal can be eliminated in diffuse-SORS mode as well by reducing collection zone area using a variable pinhole. Overall, the results of this study indicate that diffuse SORS mode to be useful in detecting the analyte particularly when the barrier layer is thick, or, when presence of barrier layer peaks in a spectrum does not confound the analysis.

Introduction

A number of applications in the fields of healthcare, pharmaceuticals, defense, security, agriculture and counterfeit detection necessitate the detection of analytes through barriers such as containers [[1], [2], [3], [4], [5], [6], [7]]. In medical diagnostics, overlying fat or skin may hide the feature of interest. Presence of barriers has prevented Raman spectroscopy from being a viable tool for such applications until the discovery of spatially offset Raman spectroscopy (SORS) in 2005 [8]. The basic principle of SORS involves spatial uncoupling of excitation and emission, resulting in detection of photons originating from deeper regions which probabilistically deviate from the point of excitation [8,9]. Conventional confocal Raman spectroscopy systems can be used for subsurface analysis by employing off-confocal detection [10] or defocusing micro-scale spatially offset modes [[11], [12], [13]]. Since the debut of SORS as a concept, a number of studies have shown the enhancement of signal collection from sample depth with SORS when compared to conventional Raman spectroscopy [8,14]. The concept has been demonstrated for transparent polymers, opaque polymers, paper envelope and various types of glass [15,16]. In the biomedical research, SORS was used to detect bone signal beyond the skin [17], and to detect micro-calcifications in the context of breast cancer [18].

Spatial uncoupling of excitation from emission during SORS has been realized by arranging these domains, respectively, as point vs. point, point vs. ring or point vs. line [[19], [20], [21]]. Application of excitation as an outer ring with a central collection point is coined as the inverse SORS (iSORS) which eliminates of the excitation simply by using a pinhole on the reflected light path [22]. So far iSORS has been an efficient modality for analyte detection through-containers. The method is reported to collect data beyond 25 mm thick transparent polymer and 9 mm thick opaque polymer [7]. The method is also reported to eliminate barrier/container contribution to Raman spectra depending on the ring dimensions [23].

Diffuse (large spot) excitation approach, also known as global illumination, is not well-investigated. Thus far there has been only one peer-reviewed research study in the literature on diffuse SORS [24]. In diffuse SORS, the excitation beam illuminates the entire field of view, resulting in interrogation of a greater fraction of sample volume, potentially increasing the throughput of the analysis. Therefore, diffuse SORS may provide greater signal intensity at higher signal to noise ratios than iSORS. However, there are no reported studies that compared the performances of diffuse versus inverse SORS modes to support or refute these claims. In this article we are reporting an experimental study comparing diffuse versus iSORS excitation modes head-to-head using a single platform to determine the merits of each method.