Fig. 3. Enhanced SBR with increased Ca²⁺ flux during DOP recording.

(A) Imaging results (top) and the corresponding 2D Gaussian fittings (bottom) acquired from DOP recording. The CaCl₂ concentration in trans was increased when the KCl concentration in cis was adjusted so that the osmolarity concentrations were kept isotonic. The fluorescence spot, which corresponds to Ca²⁺ flux through a WT α-HL nanopore, becomes brighter with an increased Ca²⁺ flux. Scale bar, 4 μm. (B) The FWHM and SBR of the fluorescence imaging signals with different electrolyte osmolarity concentrations (n = 12). The DOP recordings in (A) and (B) were carried out with 0.75 to 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, 10 mM HEPES, pH 7.0 in cis and 0.5–1.5 M CaCl₂, 10 mM HEPES, pH 7.0 in trans. (C) A representative fluorescence trace shows PEG 1500 translocation signals through a WT α-HL nanopore, as acquired by DOP recording. PEG 1500 was added to the agarose substrate reaching a final concentration of 20 mM. The DOP recording was carried out with 2.25 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl₂ and 10 mM HEPES (pH 7.0) in trans. (D) Simulated total fluorescence intensity as a function of osmolarity concentration, shown for four different pore sizes with a diameter of 2, 4, 6, and 8 nm, respectively. The electrolyte concentrations were kept isotonic to avoid the interference of osmosis in this demonstration. (E) Simultaneously imaging of WT α-HL and ClyA-RR nanopores in the same DIB. Because of a larger channel conductance, ClyA-RR appears as a larger and brighter spot in comparison with WT α-HL in the same field of view (yellow dashed circles, WT α-HL; red dashed circles, ClyA-RR). Scale bar, 20 μm. (F) Left: Simultaneous imaging of an α-HL and a ClyA-RR. Right: The fluorescence intensity profile along vertical lines as marked by location 1 and 2, respectively. The fluorescence intensity profile is fitted with a Gaussian distribution. Scale bar, 5 μm. (G) The FWHM and SBR of the fluorescence imaging signals of WT α-HL and ClyA-RR (n = 5). DOP recordings as demonstrated in (E) and (F) were carried out with 1.5 M KCl, 400 μM EDTA, 40 μM Fluo-8, and 10 mM HEPES (pH 7.0) in cis and 1.5 M CaCl₂ and 10 mM HEPES (pH 7.0) in trans.