In recent years numerous studies have shown the potential advantages of molecular imaging in vitro and in vivo using contrast agents based on surface enhanced Raman scattering (SERS), however the low throughput of traditional point-scanned imaging methodologies have limited their use in biological imaging. In this work we demonstrate that direct widefield Raman imaging based on a tunable filter is capable of quantitative multiplex SERS imaging in vivo, and that this imaging is possible with acquisition times which are orders of magnitude lower than achievable with comparable point-scanned methodologies. The system, designed for small animal imaging, has a linear response from (0.01 to 100 pM), acquires typical in vivo images in <10 s , and with suitable SERS reporter molecules is capable of multiplex imaging without compensation for spectral overlap. To demonstrate the utility of widefield Raman imaging in biological applications, we show quantitative imaging of four simultaneous SERS reporter molecules in vivo with resulting probe quantification that is in excellent agreement with known quantities (R 2 >0.98 ).
As molecular imaging moves towards lower detection limits, the elimination of endogenous background signals becomes imperative. We present a facile background-suppression technique that specifically segregates the signal from surface-enhanced Raman scattering (SERS)-active nanoparticles (NPs) from the tissue autofluorescence background in vivo. SERS NPs have extremely narrow spectral peaks that do not overlap significantly with endogenous Raman signals. This can be exploited, using specific narrow-band filters, to image picomolar (pM) concentrations of NPs against a broad tissue autofluorescence background in wide-field mode, with short integration times that compare favorably with point-by-point mapping typically used in SERS imaging. This advance will facilitate the potential applications of SERS NPs as contrast agents in wide-field multiplexed biomarker-targeted imaging in vivo.
We report on the design and testing of a prototype widefield surface enhanced Raman scattering (SERS) imaging system
based on a fiber optic bronchoscope using bandpass filters for Raman signal selection. The SERS contrast agents
employed consist of gold nanoparticles encoded with a Raman-active dye and made specific for lung adenocarcinoma
tissue through the use of an anti-epidermal growth factor receptor (EGFR) antibody. By exploiting the extremely narrow
SERS spectral peaks we demonstrate a facile method of background fluorescence rejection that can be implemented at
sub-video rates. The system has been tested on in-vivo tissues and performance metrics, including the maximum tissue
penetration and minimum detectable nanoparticle quantity have been determined in a standardized fashion.
Diffuse reflectance (DR) spectroscopy is a simple, low-cost, and noninvasive modality with potential for distinguishing oral precancer. Recently, in an ex vivo study, the DR spectral ratio (R545/R575) of oxygenated hemoglobin bands at 545 and 575 nm was used for grading malignancy. This work presents the results of clinical trials conducted in 29 patients to detect oral precancer using this ratio. We use site-specific normal spectra from a group of 36 healthy volunteers for comparison with those of patients. Toward this, in vivo DR spectra from 14 anatomical sites of the oral cavity of healthy volunteers are recorded on a miniature fiber optic spectrometer with white light excitation. The R545/R575 ratio is lowest for healthy tissues and appears to increase with the grade of malignancy. As compared to scatter plots that use the mean DR ratio from all anatomical sites, those using site-specific data show improved sensitivity and specificity for early diagnosis and grading of oral cancer. In the case of buccal mucosa, using scatter plots of R545/R575 ratio, we obtain a sensitivity of 100% and specificity of 86% for discriminating precancer (dysplasia) from hyperplasia, and a sensitivity of 97% and specificity of 86% for discriminating hyperplasia from normal.