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There is emerging evidence that microvascular alterations may occur early, even at early stages of carcinogenesis, as indispensable participants in tumor growth. However, the exact spatial extents of such alterations remain unclear, in part, because detailed microvascular alterations in relatively deep tissue over a relative large area are not easily visualized. Due to the heterogeneous nature of tissue microvasculature, microscopic evaluations with a small field of view often fail to provide a representative assessment. On the other hand, conventional whole-body small-animal optical imaging techniques suffer from unwanted diffuse light, which would otherwise deteriorate image contrast and resolution. To fill such a gap, we take advantage of the high anisotropic property of biological tissue by implementing back-directional gating into an imaging platform to suppress unwanted diffuse light. We further combine a spectral analysis of microvascular hemoglobin (Hb) absorption with back-directional gated imaging to improve image resolution, contrast, and penetration depth that are required for subcutaneous mouse xenograft models. In tissue phantom and pilot animal studies, we demonstrate that our diffuse-light-suppressed spectroscopic imaging platform can be a simple, yet effective, imaging setup to visualize subcutaneous microvascular Hb content over a relatively large area.