In vivo imaging using surface enhanced spatially offset raman spectroscopy (SESORS): balancing sampling frequency to improve overall image acquisition

Abstract

In the field of optical imaging, the ability to image tumors at depth with high selectivity and specificity remains a challenge. Surface enhanced resonance Raman scattering (SERRS) nanoparticles (NPs) can be employed as image contrast agents to specifically target cells in vivo; however, this technique typically requires time-intensive point-by-point acquisition of Raman spectra. Here, we combine the use of "spatially offset Raman spectroscopy" (SORS) with that of SERRS in a technique known as "surface enhanced spatially offset resonance Raman spectroscopy" (SESORRS) to image deep-seated tumors in vivo. Additionally, by accounting for the laser spot size, we report an experimental approach for detecting both the bulk tumor, subsequent delineation of tumor margins at high speed, and the identification of a deeper secondary region of interest with fewer measurements than are typically applied. To enhance light collection efficiency, four modifications were made to a previously described custom-built SORS system. Specifically, the following parameters were increased: (i) the numerical aperture (NA) of the lens, from 0.2 to 0.34; (ii) the working distance of the probe, from 9 mm to 40 mm; (iii) the NA of the fiber, from 0.2 to 0.34; and (iv) the fiber diameter, from 100 μm to 400 μm. To calculate the sampling frequency, which refers to the number of data point spectra obtained for each image, we considered the laser spot size of the elliptical beam (6 × 4 mm). Using SERRS contrast agents, we performed in vivo SESORRS imaging on a GL261-Luc mouse model of glioblastoma at four distinct sampling frequencies: par-sampling frequency (12 data points collected), and over-frequency sampling by factors of 2 (35 data points collected), 5 (176 data points collected), and 10 (651 data points collected). In comparison to the previously reported SORS system, the modified SORS instrument showed a 300% improvement in signal-to-noise ratios (SNR). The results demonstrate the ability to acquire distinct Raman spectra from deep-seated glioblastomas in mice through the skull using a low power density (6.5 mW/mm²) and 30-times shorter integration times than a previous report (0.5 s versus 15 s). The ability to map the whole head of the mouse and determine a specific region of interest using as few as 12 spectra (6 s total acquisition time) is achieved. Subsequent use of a higher sampling frequency demonstrates it is possible to delineate the tumor margins in the region of interest with greater certainty. In addition, SESORRS images indicate the emergence of a secondary tumor region deeper within the brain in agreement with MRI and H&E staining. In comparison to traditional Raman imaging approaches, this approach enables improvements in the detection of deep-seated tumors in vivo through depths of several millimeters due to improvements in SNR, spectral resolution, and depth acquisition. This approach offers an opportunity to navigate larger areas of tissues in shorter time frames than previously reported, identify regions of interest, and then image the same area with greater resolution using a higher sampling frequency. Moreover, using a SESORRS approach, we demonstrate that it is possible to detect secondary, deeper-seated lesions through the intact skull.

Fig. 1. SORS Set-up using the custom-built probe.

A Diagram describing the internal optics of the custom-built SORS collection probe. B In-house SORS set-up using the custom-built probe with an optical mouse phantom placed on the xy stage. Incident light is delivered at 45° to the sample surface and light is collected through the custom-built collection probe.

Fig. 2. Comparison of the collection efficiencies of the commercially available collection probe and the custom-built collection probe.

A–C Peak intensity of PTFE at 739 cm⁻¹ through 8, 14, and 20 mm of polypropylene (PP) at 0–10 mm spatial offsets (1 mm increments). D–F Comparison of signal-to-noise ratios from each probe through 8, 14, and 20 mm of PP at 0–10 mm spatial offsets (1 mm increments). Spectra were acquired using a 4 s integration, 5 acquisitions, 785 nm laser, 500 mW laser power.

Fig. 3. Diagram demonstrating par-sampling and over-sampling by 2, 5 and 10 over a 12 × 12 mm area.

A Laser spot-size. The beam is elliptical and thus has a diameter of 6 mm×4 mm. (B-E) diagram demonstrating the number of measurements per sampling frequency of a 12 × 12 mm area. Par sampling represents a step size of 6 mm in × and 4 mm in y, whereas a ×10 sampling frequency represents a 0.6 mm step size in x and a 0.4 mm step size in y. Stepwise measurements are performed in x and then in y to image the whole 12 × 12 mm area. B Par-sampling approach resulting in a total of 12 data points collected over the ROI. C Over-sampling by 2 resulting in a total of 35 data points collected over the ROI. D Over-sampling by 5 resulting in a total of 176 data points collected over the ROI. E Over-sampling by 10 resulting in a total of 651 data points collected over the ROI.

Fig. 4. Application of varying sampling frequency approaches (par, ×2, ×5 and ×10) in brain tumor phantoms at 1–3 mm spatial offsets (0.5 mm increments).

A PTFE placed under the mouse’s skull. B Mouse head placed in paraffin wax mold. C–F Mapping of the brain tumor phantom at using a par-sampling frequency and oversampling by 2, 5, and 10 at a 1 mm spatial offset. G–J Mapping of the brain tumor phantom using a par-sampling frequency and oversampling by 2, 5, and 10 at a 1.5 mm spatial offset. K–N Mapping of the brain tumor phantom using a par-sampling frequency and oversampling by 2, 5, and 10 at a 2 mm spatial offset. O–R Mapping of the brain tumor phantom using a par-sampling frequency and oversampling by 2, 5, and 10 at a 2.5 mm spatial offset. S–V Mapping of the brain tumor phantom using a par-sampling frequency and oversampling by 2, 5, and 10 at a 3 mm spatial offset. All measurements were acquired using the custom-built collection probe, 785 nm laser, 0.5 s integration time, 1 accumulation.

Fig. 5. Characterization of SERRS nanostars for in vivo administration.

A Conceptual figure showing gold nanostars administered via the tail vein. Gold nanostars were functionalized with a resonant Raman reporter molecule (IR780p) and then encased in a silica shell which was then functionalized with PEG₅₀₀₀. B The unique fingerprint spectrum of the SERRS nanostars functionalized with IR780p. The spectrum corresponds to that of IR780p. SERRS spectra were obtained using a 785 nm wavelength, 100 ms integration time. C Transmission electron microscope image of the PEGylated SERRS nanostars from two different areas on the TEM grid. The scale bar represents 100 nm. Together the nanostars and silica shell had a total average diameter of 133 ± 12 nm. Images were acquired using a JEOL 1200EX Transmission Electron Microscope at 80 kV.

Fig. 6. Representative imaging of GBM in mice (n = 3) using SESORS and optimized sampling approaches.

A–C Magnetic resonance imaging at 2 weeks post-injection of GL261-Luc cells confirms the presence of a tumor as indicated by the red arrows. Magnetic resonance images acquired at depths of 2.5 mm, 3.5 mm, and 4.25 mm respectively. MR images demonstrate the emergence of a secondary tumor region at greater depths within the brain. Following confirmation of tumor growth, non-targeted SERRS NPs were administered to tumor-bearing mice (2 nM, 100 µL) via the tail vein. In vivo SESORRS imaging was performed using a spatial offset of 1.5 mm, using (D) par sampling frequency (12 data points) (E) over-sampling frequency of: 2 (35 data points), (F) 5 (176 data points), and (G) 10 (651 data points). The respective times taken to acquire each image are also shown. Raman spectra were truncated, normalized, and principal component (PC) analysis was applied to generate false-color 2D heat maps. PC scores 2 and 3 were used to create SESORS images and represent the accumulation of SERRS NPs in the tumor. SESORRS measurements were acquired using a power density of 6.5 mW/mm², 1.5 mm spatial offset, 0.5 s integration time, 1 acquisition, 785 nm excitation wavelength. H–K Background removal of areas of non-intensity from the SESORRS images. Images were then rotated to match the orientation of the MR images. L–O SESORRS spectra taken at the point of minimum and maximum intensity from the respective SESORRS image using a par sampling frequency and an oversampling frequency of 2, 5 and 10 respectively. P–R H&E stained 5 µM section of the brain confirming the presence of a tumor and the emergence of a secondary tumor region as deeper regions are sliced.