We report on the development of a novel approach for constant-distance mode imaging using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry. The nano-DESI probe is comprised of two glass capillaries: the promary capillary delivers solvent to the sample while the secondary capillary removes the solvent containing analyte molecules extracted from the sample and delivers the resulting solution to a mass spectrometer inlet. The new approach for precise control of the distance between the sample and the nano-DESI probe relies on real-time shear force distance measurement. This enables high-resolution imaging of biological samples with complex morphology including living microbial communities, yeast colonies, and tissue sections.