We present a method - Spatialyse - to selectively lyse adherent cells for nucleic acid analyses (Fig. 1a) and deliver this lysate in minimum volume for downstream molecular analysis. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. This method can be used for spatio-temporal studies of heterogeneous cells while in culture in contrast to current approaches, providing insight into cellular signaling.