The complete removal of cancerous tissue is a central aim of surgical oncology, but is difficult to achieve in certain cases, especially when the removal of surrounding normal tissues must be minimized. Therefore, when post-operative pathology identifies residual tumor at the surgical margins, re-excision surgeries are often necessary. An intraoperative approach for tumor-margin assessment, insensitive to nonspecific sources of molecular probe accumulation and contrast, is presented employing kinetic-modeling analysis of dual-probe staining using surface-enhanced Raman scattering nanoparticles (SERS NPs). Human glioma (U251) and epidermoid (A431) tumors were implanted subcutaneously in six athymic mice. Fresh resected tissues were stained with an equimolar mixture of epidermal growth factor receptor (EGFR)-targeted and untargeted SERS NPs. The binding potential (BP; proportional to receptor concentration) of EGFR – a cell-surface receptor associated with cancer – was estimated from kinetic modeling of targeted and untargeted NP concentrations in response to serial rinsing. EGFR BPs in healthy, U251, and A431 tissues were 0.06 ± 0.14, 1.13 ± 0.40, and 2.23 ± 0.86, respectively, which agree with flow-cytometry measurements and published reports. The ability of this approach to quantify the BP of cell-surface biomarkers in fresh tissues opens up an accurate new approach to analyze tumor margins intraoperatively.

Optical imaging offers exquisite sensitivity and resolution for assessing biological tissue in microscopy applications; however, for samples that are greater than a few hundred microns in thickness (such as whole tissue biopsies), spatial resolution is substantially limited by the effects of light scattering. To improve resolution, time-and angular-domain methods have been developed to reject detection of highly scattered light. This work utilizes a modified version of a commonly used Monte Carlo light propagation software package (MCML) to present the first comparison of time-and angular-domain improvements in spatial resolution with respect to varying sample thickness and optical properties (absorption and scattering). Specific comparisons were made at various tissue thicknesses (1-6 mm) assuming either typical (average) soft tissue scattering properties, μ s ' = 10 cm −1 , or low scattering properties, μ s ' = 3.4 cm −1 , as measured in lymph nodes.

Optical tomography can yield anatomical and molecular information about biological tissue. However, its spatial resolution is poor in thick samples owing to high scatter. Early photon approaches, where photon arrival times are measured with time-resolved detectors, provide one means of improving spatial resolution through selection of photons that travel a straighter path. Here, a novel approach to significantly enhance detection of early photons in time-correlated single photon counting with avalanche photodiodes has been discussed. Results suggest that the early photon detection rate can be increased by about 10 orders of magnitude by running the detector in a dead-time regime.

Scattering of visible and near-infrared light in biological tissue reduces spatial resolution for imaging of tissues thicker than 100 μm. In this study, an optical projection imaging system is presented and characterized that exploits the dead-time characteristics typical of photon counting modules based on single photon avalanche diodes (SPADs). With this system, it is possible to attenuate the detection of more scattered late-arriving photons, such that detection of less scattered early-arriving photons can be enhanced with increased light intensity, without being impeded by the maximum count rate of the SPADs. The system has the potential to provide transmittance-based anatomical information or fluorescence-based functional information (with slight modification in the instrumentation) of biological samples with improved resolution in the mesoscopic domain (0.1-2 cm). The system design, calibration, stability, and performance were evaluated using simulation and experimental phantom studies. The proposed system allows for the detection of very-rare early-photons at a higher frequency and with a better signal-to-noise ratio. The experimental results demonstrated over a 3.4-fold improvement in the spatial resolution using early photon detection vs. conventional detection, and a 1000-fold improvement in imaging time using enhanced early detection vs. conventional early photon detection in a 4-mm thick phantom with a tissue-equivalent absorption coefficient of μ = 0.05 mm and a reduced scattering coefficient of μ' = 5 mm.

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