Methods and systems for alignment of a subject for medical imaging are disclosed, and involve providing a reference image of an anatomical region of the subject, the anatomical region comprising a target tissue, processing the reference image to generate an alignment reference image, displaying the alignment reference image concurrently with real-time video of the anatomical region, and aligning the real-time video with the alignment reference image to overlay the real-time video with the alignment reference image. Following such alignment, the subject may be imaged using, for example, fluorescence imaging, wherein the fluorescence imaging may be performed by an image acquisition assembly aligned in accordance with the alignment.