Methods that can control and vary the solution environment around single cells are abundant. In contrast, methods that offer direct access to the intracellular proteome and genome in single cells with the control, flexibility, and convenience given by microfluidic methods are both scarce and in great demand. Here, we present such a method based on using a microfluidic device mounted on a programmable scanning stage and cells on-chip permeabilized by the pore-forming glycoside digitonin. We characterized the on-chip digitonin poration, as well as the solution exchange within cells. Intracellular solution exchange times vary with the dose of exposure to digitonin from less than a second to tens of seconds. Also, the degree of permeabilization obtained for cells treated with the same dose varies considerably, especially for low doses of digitonin exposure and low permeabilities. With the use of the presented setup, the degree of permeabilization can be measured during the permeabilization process, which allows for "on-line" optimization of the digitonin exposure time. Using this calibrated permeabilization method, we demonstrate the generation of intracellular oscillations, intracellular gradients, and the delivery of substrate to initiate enzymatic reactions in situ. This method holds the potential to screen and titrate intracellular receptors or enzymes or to generate intracellular oscillations, useful in the study of signaling pathways and oscillation decoding among other applications.