Subcellular biopsy (SCB) using atomic force microscopy (AFM) with a microfluidic cantilever enables the extraction of biological samples from living cells. Traditional cryo-electron microscopy (cryo-EM) sample preparation methods are unsuitable for the small volumes from SCB. This study presents a novel method to prepare cryo-EM samples directly from SCB samples, integrating dispensing, thickness measurement, and local temperature control to minimize evaporation. The method uses a microfluidic AFM cantilever for dispensing, a Mach-Zehnder interferometer for real-time thickness measurement, and plunge freezing for vitrification. The system successfully demonstrated dispensing picoliter droplets, with potential for femtoliter volumes. The temperature-controlled grid holder reduced evaporation rates, allowing sufficient time for real-time thickness measurement. The system has demonstrated the required transition speed and plunge velocity. Initial cryo-EM thickness measurements demonstrate the method's potential. In conclusion, a novel system for subcellular biopsy sample preparation for cryo-EM was designed, fabricated, and tested. It maintains picoliter sample volumes on a temperature-controlled EM grid, enabling real-time thickness measurement and immediate plunge freezing.