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. 2013 Jul 23;110(30):12408-13.
doi: 10.1073/pnas.1301379110. Epub 2013 Jul 2.

A Small Animal Raman Instrument for Rapid, Wide-Area, Spectroscopic Imaging

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Free PMC article

A Small Animal Raman Instrument for Rapid, Wide-Area, Spectroscopic Imaging

Sarah E Bohndiek et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Raman spectroscopy, amplified by surface enhanced Raman scattering (SERS) nanoparticles, is a molecular imaging modality with ultra-high sensitivity and the unique ability to multiplex readouts from different molecular targets using a single wavelength of excitation. This approach holds exciting prospects for a range of applications in medicine, including identification and characterization of malignancy during endoscopy and intraoperative image guidance of surgical resection. The development of Raman molecular imaging with SERS nanoparticles is presently limited by long acquisition times, poor spatial resolution, small field of view, and difficulty in animal handling with existing Raman spectroscopy instruments. Our goal is to overcome these limitations by designing a bespoke instrument for Raman molecular imaging in small animals. Here, we present a unique and dedicated small-animal Raman imaging instrument that enables rapid, high-spatial resolution, spectroscopic imaging over a wide field of view (> 6 cm(2)), with simplified animal handling. Imaging of SERS nanoparticles in small animals demonstrated that this small animal Raman imaging system can detect multiplexed SERS signals in both superficial and deep tissue locations at least an order of magnitude faster than existing systems without compromising sensitivity.

Conflict of interest statement

Conflict of interest statement: A.W and S.Y. are employees of General Electric.

Figures

Fig. 1.
Fig. 1.
Schematic representation of SERS nanoparticles and their recorded Raman spectra. (A) Gold nanoparticles with a 60-nm–diameter gold core are covered with a layer of Raman active material, then a silica coating yielding a particle of 120 nm diameter. The Raman active materials were 4,4′-dipyridyl (S420), d8-4,4′-dipyridyl (S421), trans-1,2-bis(4-pyridyl)-ethylene (S440), and 1,2-di(4-pyridyl) acetylene (S470) (4). (B) The molecular vibration of the different chemical bonds in the Raman active material after laser excitation at 785 nm yields a unique spectral fingerprint. The background spectrum (free space background) is acquired in the same experimental arrangement without nanoparticles present.
Fig. 2.
Fig. 2.
Optical design of SARI system for wide-area line mapping and characterization of performance. (A) Photograph of a system with the illumination path overlaid in red. (B) Layout of the optical system. (C) Line-spread function. Perpendicular to the laser line, the full widths at half-maximum and at tenth-maximum are 250 μm and 675 μm, respectively. (D) Raman scatter intensity from S440 SERS nanoparticles as a function of vertical displacement from the focal plane and (E) depth in tissue equivalent material. (F) System response in the focal plane measured from a full-area (31 × 25-mm) scan of a uniform phantom containing S440 nanoparticles. These data illustrate the Gaussian profile along the laser beam, resulting in lower signal intensity at the edges of the laser line (y), and the fall-off in signal intensity at the extreme (x,y) positions; in both these circumstances, there is an increased signal contribution from the mean free space background (G). (H) The four principal signal components composing the Raman scatter intensity recorded from the phantom, offset for clarity.
Fig. 3.
Fig. 3.
System sensitivity to detection of S440 nanoparticles compared with the Renishaw inVia microscopy system. (A) The limit of detection for superficial SERS nanoparticles in our system is 3.1 pM. As expected, the Renishaw microscope, with its 400-fold higher irradiance, has a higher sensitivity, detecting subpicomolar concentrations (B). (Inset) Close-up of the lowest concentrations, showing that the signal falls into the noise below 3.1 pM on our system but is still linear with concentration at 0.78 pM on the inVia system. Important for quantification purposes, both systems show a linear dependence of recorded Raman scattered intensity with increasing nanoparticle concentration.
Fig. 4.
Fig. 4.
Rapid wide-area imaging of SERS nanoparticle distribution in living mice. (A) Subcutaneous injections of SERS nanoparticle flavors, both individual (Upper) and multiplexed (Lower), were successfully detected and unmixed. The vertical scale is the same in all images. Irregularities in the shapes of these injection sites arise from the curvature of the mouse flank and the stitching of data recorded at different heights. (B) Our line-scanning system provides a 3.5-fold improvement in setup time and a 10-fold improvement in scan time compared with the Renishaw inVia microscope operating in the high-speed Streamline mode with matched spectral and spatial resolution. Differences in signal-to-noise ratio in the observed spectra are a result of the higher irradiance provided by the inVia system. (C) Mapping of the full mouse torso on our system following i.v. injection of two SERS flavors (S421 + S440, 1 h post injection; Left) and a narrower region after injection of four flavors (2 h post injection; Right) shows the accumulation of nanoparticles in the liver as expected for clearance through the reticuloendothelial system. The distribution of particles within the different lobes of the liver can be seen to change between these two time points, with greater accumulation in the median and left lobes by 2 h post injection. The vertical scale is the same in all liver images.

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