Probe and imaging fluid contributions to substratum root-mean-square (rms) roughness and feature heights, as measured with atomic force microscopy (AFM), were explored. A unique method to measure the roughness of a singular location (+/-70 nm) was developed. Probe type [glass (hydrophilic) and octadecyltrichlorosilane (OTS)-coated glass (hydrophobic) colloids and Si3N4 (hydrophilic)] were observed to influence feature heights but not surface rms averaged roughness of the glass substratum. Probe type significantly impacted both rms roughness and feature heights on the OTS-coated substratum. Use of smaller Si3N4 probes (80 nm diameter versus 1 mum diameter colloid probes) in air resulted in the largest surface roughness measurements for both substrata. Changes in perceived OTS-coated substratum roughness as a function of probe type were postulated to derive from disordered organosilane surface layers. Interactions of OTS-coated glass colloid probes with the OTS-coated glass substratum decreased observed feature heights. Roughness of the OTS-coated substratum increased when imaged in organic fluids while individual feature heights decreased with increasing fluid polarity. Finally, surface-averaged rms roughness correlated positively with measured adhesive forces. In sum, what were thought to be intrinsic surface properties (topography and chemistry) can be modified by imaging conditions (probe type and imaging fluid).