Optical imaging and spectroscopic modalities are of broad interest for in-vivo molecular imaging, fluorescence guided cancer surgery, minimally invasive diagnostic procedures, and wearable devices. However, considerable debate still exists as to how deeply visible and near-infrared (NIR) light could penetrate normal and diseased tissues under clinically relevant conditions. Here we report the use of surface-enhanced Raman scattering (SERS) nanotags embedded in ex-vivo animal tissues for direct and quantitative measurements of light attenuation and spectroscopic detection depth at both the NIR-I and NIR-II spectral windows. SERS nanotags are well suited for this purpose because of their sharp spectral features that can be accurately differentiated from fluorescence and background emission. For the first time, the spectroscopic detection depth is quantitatively defined and measured as the maximal thickness of tissues through which the embedded SERS nanotags are still detected at a signal-to-noise ratio (SNR) of three (99.7% confidence level). Based on data from six types of fresh ex-vivo tissues (brain, kidney, liver, muscle, fat, and skin), we find that the maximum detection depth values range from 1—3 mm in the NIR-I window, to 3—6 mm in the NIR-II window. The depth values are largely determined by two factors – the intrinsic optical properties of the tissue, and the overall SNRs of the system without the tissue (system SNR, a result of nanotag brightness, instrument efficiency, and data acquisition parameters). In particular, there is an approximately linear-logarithmic relationship between the system SNR and maximum detection depth. Thus, the detection of hidden or occult lesions can be improved by three strategies – reducing tissue attenuation, minimizing background noise, and maximizing the system’s performance as judged by SNR.Graphical
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