Integrated system for combined Raman spectroscopy-spectral domain optical coherence tomography

Abstract

Raman spectroscopy (RS) and optical coherence tomography (OCT) are powerful tools for optical analysis of tissues with mutually complementary strengths and limitations. OCT excels at visualizing tissue microstructure but lacks molecular specificity, while RS can relay tissue biochemical composition but typically cannot relate microstructure. Previous implementations of combined RS-OCT have utilized a common sample arm while maintaining independent RS and OCT detection arms. We present the design and application of an integrated RS-OCT instrument with a common detection arm for both RS and OCT. The detector is a spectrograph capable of sequential detection of the 855-nm OCT signal and the Raman scatter generated by a 785-nm source. The capabilities of the instrument are demonstrated ex vivo in the calvaria and retina of rodents, as well as in vivo in human skin.

Figure 1

Illustration of the CCD array illumination for RS and OCT, along with the spectral overlap of an example Raman spectrum (acetaminophen) and the OCT source.

Figure 2

Overall RS-SDOCT system schematic. Configurations of sampling optics used for collection from retina (config A), and from standard samples (config B). PC – polarization control paddles, ND – neutral density filter, WC – water-filled cuvette, TM – translatable mirror, LP – long pass filter, DM – dichroic mirror, BP – band pass filter, SF – spatial filter, xy – xy galvanometer pair, MOS – MEMS optical switch, and NI-DAQ – National Instruments multifunction DAQ.

Figure 3

RS-OCT of a dissected murine calvaria. (a) OCT image. Arrow indicates dark hyporeflective region likely associated with the suture of the left and right parietal skull plates. Red area indicates the region where the corresponding Raman spectrum was acquired. Axial scale assumes n_calvaria = 1.55. (b) Raman spectrum, normalized to mean spectral intensity. Arrows indicate positions of peaks typically utilized in Raman spectral analysis of mineralized tissues, including the proline peak at 857 cm⁻1, the phosphate peak at 960 cm⁻¹, the carbonate peak at 1072 cm⁻¹, the amide I peak at 1667 cm⁻¹.

Figure 4

RS-OCT of the rodent retina. (a) OCT image. The layers of the retina visible, from inner to outer layers, include the thin, bright nerve fiber layer (NFL), hyporeflective ganglion cell layer (GCL), thicker hyper-reflective inner plexiform layer (IPL), the hyporeflective inner nuclear layer (INL), the thin, bright outer plexiform layer (OPL), the hyporeflective outer nuclear layer (ONL), and the photoreceptor layer and choroid (PR and C), which are difficult to distinguish and are labeled as a single layer. (b) Corresponding histology. (c) Mean Raman spectrum acquired from five axes equally spread across the retina. The set of peaks most prominent in the retina are identified (arrows), and include amide III (1265 cm⁻¹), CH_x (1440 cm⁻¹), and amide I (1660 cm⁻¹), as well as those from DNA∕RNA at 723, 1003, and 1094 cm⁻¹.

Figure 5

RS-OCT of in-vivo human skin on the palm of the hand. (a) OCT image. A hyper-reflective feature that is likely a sweat gland (SG) is seen within the stratum corneum (SC). Red area indicates location of Raman spectrum. Axial scale assumes n_skin = 1.38. (b) Raman spectrum of skin. The prominent skin peaks are identified (arrows) at 936 cm⁻¹ (C-C backbone of collagen protein), 1003 cm⁻¹ (phenylalanine), 1280 cm⁻¹ (amide III), 1335 cm⁻¹ (C-H), 1440 cm⁻¹ (CH_x), and 1660 cm⁻¹ (amide I). (Color online only.)

Figure 6

RS-OCT of the region surrounding the proximal nail fold. (a) OCT image. Nail is labeled on the right side of the image, and inserts into the nail bed beneath the cuticle, which is located in the transverse dimension from 2.5 to 3.0 mm. To the left of the cuticle is the skin on the back of the finger. Raman spectra acquired from the regions are indicated from the red overlays. (b) Raman spectra of the skin and nail. Peaks of interest are indicated with arrows, and include the sharp 620-cm⁻¹ C-S peak, the 936-cm⁻¹ C-C protein backbone peak, the relative intensity and position of the amide III (1251 cm⁻¹), and C-H (1317 cm⁻¹) peaks, all of which are indicative of the β-sheet keratin proteins that make up the nail. In contrast, the 1770-cm⁻¹ lipid peak seen in soft tissue and skin is not seen in the nail.(Color online only.)