Fig. 3. Microscopic imaging of M2 MspA. (A) The schematic diagram of the measurement device. A DIB was formed between the droplet and the hydrogel substrate. M2 MspA spontaneously inserts itself into the DIB, forming channels connecting the electrolyte in the droplet and the hydrogel substrate. Transport of calcium ions from the substrate to the droplet results in the appearance of fluorescence spots.²⁷ A positively applied bias enhances the transport of calcium ions which results in an enhanced fluorescence intensity. (B) A frame containing multiple fluorescence spots representing inserted M2 MspA nanopores (red circles) in the DIB. Scale bar: 20 μm. (C) A representative fluorescence-time trace. A square wave voltage protocol was applied. The fluorescence intensity (FI) change was modulated by the voltage protocol indicating that the fluorescent spot results from inserted channel proteins. Cropped images containing a single pore at different frames of the measurements were placed on top of the fluorescence-time trace. The measurements were performed as described in ESI Methods.† A buffer combination of 1.5 M KCl buffer (cis, droplet), and 0.75 M CaCl₂ buffer (trans, hydrogel substrate) was applied during the measurements. The M2 MspA applied for these measurements was from the corresponding sample as demonstrated in the gel results of Fig. 1.