Double sided flow cell and other substrate imaging systems, such as imaging systems used in nucleic acid sequencing and similar processes. In one example, the imaging system includes a flipper to facilitate imaging different surfaces of the flow cell or other substrate. In another example, the imaging system includes two optical systems for imaging different surfaces of the flow cell or other substrate. In another example, the imaging system is an immersion system. In these and other examples, the system may include an auto-focus sub-system configured to accurately focus the optics on one surface of the double sided flow cell without interference from the other surface of the flow cell.