SQUID (superconducting quantum interference device) microscopes are versatile instruments for biosensing applications, in particular for magnetic nanoparticle detection in immunoassay experiments. We are developing a SQUID microscope based on an HTS rf SQUID magnetometer sensor with a substrate resonator. For the cryogenic set-up, a configuration was realized in which the cryostat is continuously refilled and kept at a constant liquid nitrogen level by an isolated tube connection to a large liquid nitrogen reservoir. The SQUID is mounted on top of a sapphire finger, connected to the inner vessel of the stainless steel cryostat. The vacuum gap between the cold SQUID and room temperature sample is adjusted by the precise approach of a 50 νm thin sapphire window using a single fine thread wheel. We investigated possible sensing tip configurations and different sensor integration techniques in order to achieve an optimized design. A new scheme of coupling the rf SQUID from its back to a SrTiO3 substrate resonator was adopted for the purpose of minimization of the sensor-to-sample spacing. By SQUID substrate thinning and washer size reduction, the optimum coupling conditions for back coupling were determined for different rf SQUID magnetometers prepared on LaAlO3 and SrTiO3 substrates. The SQUID microscope system is characterized with respect to its spatial resolution and its magnetic field noise. The SQUID microscope instrument will be used for magnetic nanoparticle marker detection.