Phase transitions in purple membrane have been a topic of debate for the past two decades. In this work we present studies of a reversible transition of purple membrane in the 50-60 °C range in zeptoliter volumes under different heating regimes (global heating and local heating). The temperature of the reversible phase transition is 52 ± 5 °C for both local and global heating, supporting the hypothesis that this transition is mainly due to a structural rearrangement of bR molecules and trimers. To achieve high resolution measurements of temperature-dependent phase transitions, a new scanning probe microscopy-based method was developed. We believe that our new technique can be extended to other biological systems and can contribute to the understanding of inhomogeneous phase transitions in complex systems.