Tip-enhanced Raman scattering is used to investigate the surface structure of the cell wall of Staphylococcus epidermidis with high lateral resolution and chemical specificity. For most biological samples, the transmission tip-enhanced Raman scattering set-up is ideally suited because it allows the specimen to be kept under specific environmental conditions, whereas it most efficiently collects the signal created at the field-enhancing probe. Special emphasis is given here to the parameters required to reproducibly set up the instrument, such that field-enhancement factors can be estimated properly. Also the importance of control experiments to avoid misinterpretation of signals will be emphasized by an example.