A schematic of the MSI platform is presented in Fig. 2. A predetermined narrowband high-power light-emitting diode (LED) light source (SR-02, Quadica Developments Inc., Ontario, Canada) was used with a fiber-optic ring light guide and a condensing lens to generate three uniform illumination lights (center wavelengths 470, 600, and 770 nm) in series. These wavelengths in the visible spectrum were selected to demonstrate hemoglobin absorption and to examine the effects of wavelengths on penetration depth.24 Since the LED light was unpolarized and the use of nonpolarization-maintaining fibers randomized the polarization state of the light,25 we applied a polarizing sheet onto the distal end of the ring light guide to create linearly polarized illumination. Reflected light from the tissue passes back through the empty space of a ring light guide, a rotating linear polarizer filter (46 mm, Prinz Optics GmbH, Stromberg, Germany), a macrolens (Fujinon HF 12.5 SA-1, Phoenix Imaging Ltd., Michigan), and finally reaches a near-infrared camera (acA2000-50gmNIR, Basler, Pennsylvania). By adjusting the angle of the linear polarizer attached to the camera, we can effectively control the amount of polarization effects. To reduce specular reflections from the tissue surfaces, two linear polarizers were set orthogonal to each other. Three different spectral images were acquired at 6 fps, with the image size of . LED-based MSI has an advantage over hyperspectral imaging in that it enables high-speed image acquisition and data processing, which could be potentially useful for real-time guidance. Both LED control and image acquisition were programmed using a custom C# script (Visual Studio 2010, Microsoft). The acquired images were cropped to obtain the tissue region of interest () for image processing. The 470-nm band images were selected for blood vessel segmentation.26 In addition, all three spectral band images were combined to form composite images that were used for the multispectral analysis.