## 2 Materials and Methods
### Characterization Methods
#### 2.4.4 Atomic Force Microscopy (AFM)-Based Methods  
The morphology and mechanical properties of the as-prepared and annealed electrospun PLLA nanofibers were inspected by a Dimension Icon AFM instrument (Bruker Nano Inc., Santa Barbara, CA, USA). AFM Tapping mode was employed for studying the morphology of the nanofibers using silicon cantilevers (RTESPA from Bruker Nano Inc.) with a spring constant of \(\sim\)42 N/m and an apex radius of curvature \(\sim\)10 nm. For the investigation of the local mechanical properties, pre-calibrated RTESPA-300-30 tips from Bruker were used in PFQNM mode. These tips had a well-controlled tip end radius of 30 nm (\(\pm\) 15%) and the spring constant of each tip was individually calibrated by the laser doppler vibrometer method. The information related to tip calibration was stored and can be easily retrieved by scanning over the QR code on the tip container. In this study, the tip used had a tip radius of 29 nm and a spring constant of 49.487 N/m. For PFQNM experiments, the deflection sensitivity was estimated before the measurements from the linear part of a force-distance curve obtained by ramping the tip onto a clean Sapphire surface. The peak force amplitude was 30 nm, the scan rate was 0.5 Hz and all the captured images were recorded with a resolution of \(256\times 256\) data points. A force curve was also recorded for each pixel of the image. After the calibration step was done, the same tip and parameters (peak force amplitude, scan rate and resolution) were applied to measure the contact modulus of the PLLA electrospun nanofibers. Only the maximum force (peak force) was kept at F = 30 nN to ensure enough indentation (a few nm) into the samples while still keeping the deformation in the elastic region.  
ImAFM experiments were conducted with the same AFM apparatus equipped with an intermodulation lockin analyzer and ImAFM software supplied by Intermodulation Products Company (Segersta, Sweden). We used the NCHV-A cantilever (from Bruker Nano Inc.) which is similar to NCHV used in tapping mode except that the back side of this cantilever is coated with a reflective layer of aluminum. The dynamic stiffness in air of the cantilever was calculated as \(\text{k}_{\text{air}}\) = 37.52 N/m by the Sader method using the expression for the hydrodynamic damping and the calibrated inverse optical lever responsibility invOLR = 56.04 nm/V. The nanoscale electroactive responses were probed by using the dual AC resonance tracking (DART) method [36] of the piezoresponse force microscopy (PFM) technique in air at room temperature with a commercial AFM microscope (Asylum Research/Oxford Instruments, MFP-3D, Santa Barbara, CA, USA). For these experiments, nanofibers were directly deposited on conductive substrates (i.e., indium-tin-oxide-coated glass) with carbon paste in-between and the nanometric tip probed individual nanofiber. Pt/Ir-coated silicon tips and cantilevers with stiffness of about 3.5 N/m (PPP-EFM, Nanosensors) were used as the conductive probe. More specifically, PFM in spectroscopic mode was used for recording remnant PFM loops (at zero DC bias) after a continuous AC signal superimposed on an intermittent DC bias voltage was applied to the fibers to promote the electromechanical response at the expense of the electrostatic contribution.