STM images of single biomolecules adsorbed on conductive substrates do not reproduce the expected physical height, which generally appears underestimated. This may cause the tip to interfere with the soft biological sample during the imaging scans. Therefore, a key requirement to avoid invasive STM imaging is the knowledge, and the control, of the initial tip to substrate distance. This is connected to the setting of the tunnelling current and applied voltage, which define a tunnelling resistance. The height of the STM tip was measured by calibrating the tunnelling resistance, as a function of its vertical displacement until establishing a mechanical contact. At a tunnelling resistance of 4 x 10(9)Omega, distances of about 3 and 6 nm are estimated when flat Au substrates are imaged in water and in air, respectively. On such a ground, the relevance of the starting tip-substrate distance in determining a non-invasive imaging has been investigated for a plastocyanin mutant chemisorbed on Au(111) electrodes. At tunnelling distances sufficient to overcome the physical height of the imaged biomolecules, their lateral dimensions are found to be consistent with the crystallography, whereas they are significantly broadened for smaller distances.