Imaging cells and tissues through opaque and turbid media is challenging and presents a major barrier for monitoring maturation and remodeling of bioengineered tissues. The fiber optics based imaging system described here offers a new approach for fluorescent cell imaging. A micro imaging channel is embedded in a Polycaprolactone (PCL) electrospun scaffold designed for cell seeding, which allows us to use an optical fiber to locally deliver excitation laser close to the fluorescent cells. The emission is detected by an Electron Multiplying Charge Coupled Device (EMCCD) detector and image reconstruction of multiple excitation points is achieved with a working distance of several centimeters. The objective of this study is to assess the effects of system parameters on image reconstruction outcomes. Initial studies using fluorescent beads indicated that scaffold thickness had a small effect on image quality, whereas scaffold composition (collagen content), fluorophore spectra, and the reconstruction window size had a large effect. The results also suggest that a far-red fluorescent emission is preferential when using collagenous scaffolds with a thickness of up to 500 μm. Using these optimized parameters, we were able to image fluorescently labeled cells on a scaffold with a resolution of 15-20 μm, and have also measured muscle progenitor cell differentiation and scaffold surface coverage with endothelial cells. In the future, this imaging platform can be applied to other bioengineered tissues for non-invasive monitoring both in vitro and in vivo.