Supporting Information - PNAS · 2016-04-22 · At higher potentials, the current recording can...
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Supporting Information Sengel and Wallace 10.1073/pnas.1517437113 SI Experimental Methods Device Preparation. Coverslips were subjected to oxygen plasma for 8 min. A volume of 140 μL of 0.75% (wt/vol) aqueous agarose solution was pipetted onto the center of a coverslip, which was subsequently spun at 4,000 rpm for 30 s (WS-650Mz-23NPP/LITE spin coater, Laurell Technologies). The coated coverslip was placed within a 0.2-mm recess on the underside of the device and affixed in place using adhesive tape. A volume of 165 μL of a hydrating agarose solution [2% (wt/vol), 750 mM CaCl 2 ; or 1.5%, 1.5 M KCl] was injected via a pipette into the device. An Ag/AgCl electrode (ground electrode) was inserted into the device and the entrance sealed with vacuum grease. See Fig. S1 for details of the device and a schematic of the device setup. Experimental Setup. Prepared devices were placed within a Faraday cage on the stage of an inverted microscope (Eclipse TiE; Nikon). The ground electrode was plugged into the headstage of an Axo- patch 200B patch-clamp amplifier, which sits inside the Faraday cage and is separate from the main unit. A volume of 60 μL of DPhPC solution (8.7 mg·mL −1 in hexadecane) was pipetted into the device; at the base of each well, lipids associate with the sub- strate agarose, forming a monolayer. Aqueous droplets ranging from 18 to 50 nL were pipetted into a poly(methyl methacrylate) (PMMA) incubating tank containing the lipid solution. Droplets were allowed to rest for 30 min to ensure a complete lipid mono- layer had formed; the device was similarly incubated for a period of 15 min. After this time, droplets were pipetted into the wells and the two monolayers contact under gravity. An agarose-coated [0.75% (wt/vol)] active electrode affixed to a micromanipulator was then inserted into the top of the droplet. All experiments were conducted at room temperature. Electrical Recording. The patch-clamp amplifier (voltage-clamp mode) was interfaced with a computer via a BNC2090A rack- mountable connector block and an NI PCIe-6251 M-series data acquisition card (both National Instruments); all unnecessary electrical equipment was switched off, including lights, to minimize noise. Electrical measurements were digitized during acquisition at 1 kHz. Traces were recorded using WinEDR software (version 3.2.6; John Dempster, University of Strathclyde, Glasgow, UK). Fluorescence Imaging. Droplets were formed from a solution con- taining Fluo-8 (46 μM, 1.5 M KCl, 370 μM EDTA). DIBs were observed using a 60× TIRF oil-immersion objective lens (Nikon), with the fluorophore excited by a 473-nm continuous-wave laser, coupled to the inverted microscope by an optical fiber (power at fiber output, ∼4 mW). The fluorescence and excitation signals were separated by dichroic (XF2037) and bandpass (XF3080 510/23; both Omega Optical) filters before being imaged onto an electron- multiplying CCD (iXon3 897; Andor). Image Analysis. Analyses of electrical and optical data were carried out in IGOR Pro-6.2 (Wavemetrics). Spots were detected by Trackmate using its Laplacian of Gaussian algorithm and tracked with the Linear Assignment Problem tracker, allowing for gap closing over a maximum distance of ∼5 pixels over 10 frames. Detected spots and their associated tracks were manually checked and filtered for quality. For Gaussian fitting in Igor Pro, a 2D Gaussian was fitted to the brightest pore within an image stack, and then a patch containing the fit subtracted from the original image. This process was repeated until no trajectories remained with intensities 3σ above the SD of the background. We have previously used this CLEAN algorithm (46) to perform super- resolution imaging of single molecules (47). Pore Radius Estimation. The nature of the field around an elec- tropore is complex owing to its geometry, but we approximate the conductance of a cylindrical defect of radius r to be as follows: G = πr 2 g=d, where d is the membrane thickness and g is the conductivity of the solution filling the pore. We assume a DPhPC bilayer to be 4.8 nm thick. Conductance G was obtained from the current recordings of pores exhibiting discrete conductance fluctuations (upper traces in Fig. S5 A and B). SI Movies The image correction process is as follows, carried out in ImageJ. A Fourier transform (FT) bandpass filter is applied with a feature diameter of between 50 and 100 pixels to remove large diffraction or interference features. Next, a region containing no pore throughout the movie is extracted (∼10,000 pixel 2 ), and the mean intensity value for each frame is calculated. This value is subtracted from its (complete) host image and repeated across the movie (mean-of- patch subtraction). This removes “flickering” of the movie due to laser variation. In some cases, a median stack is formed and a Gaussian blur (σ = 5–10) applied; this image is subtracted from all frames to remove small laser profile features (blurred median subtraction). In the legends of Movies S1–S5, we use the fol- lowing shorthand to indicate which correction processes have been used: FT, Fourier transform bandpass; MoPS, mean-of- patch subtraction; BMS, blurred median subtraction. Sengel and Wallace www.pnas.org/cgi/content/short/1517437113 1 of 7
Transcript of Supporting Information - PNAS · 2016-04-22 · At higher potentials, the current recording can...
Supporting InformationSengel and Wallace 10.1073/pnas.1517437113SI Experimental MethodsDevice Preparation.Coverslips were subjected to oxygen plasma for8 min. A volume of 140 μL of 0.75% (wt/vol) aqueous agarosesolution was pipetted onto the center of a coverslip, which wassubsequently spun at 4,000 rpm for 30 s (WS-650Mz-23NPP/LITEspin coater, Laurell Technologies). The coated coverslip was placedwithin a 0.2-mm recess on the underside of the device and affixedin place using adhesive tape. A volume of 165 μL of a hydratingagarose solution [2% (wt/vol), 750 mM CaCl2; or 1.5%, 1.5 M KCl]was injected via a pipette into the device. An Ag/AgCl electrode(ground electrode) was inserted into the device and the entrancesealed with vacuum grease. See Fig. S1 for details of the device anda schematic of the device setup.
Experimental Setup. Prepared devices were placed within a Faradaycage on the stage of an inverted microscope (Eclipse TiE; Nikon).The ground electrode was plugged into the headstage of an Axo-patch 200B patch-clamp amplifier, which sits inside the Faradaycage and is separate from the main unit. A volume of 60 μL ofDPhPC solution (8.7 mg·mL−1 in hexadecane) was pipetted intothe device; at the base of each well, lipids associate with the sub-strate agarose, forming a monolayer. Aqueous droplets rangingfrom 18 to 50 nL were pipetted into a poly(methyl methacrylate)(PMMA) incubating tank containing the lipid solution. Dropletswere allowed to rest for 30 min to ensure a complete lipid mono-layer had formed; the device was similarly incubated for a period of15 min. After this time, droplets were pipetted into the wells andthe two monolayers contact under gravity. An agarose-coated[0.75% (wt/vol)] active electrode affixed to a micromanipulator wasthen inserted into the top of the droplet. All experiments wereconducted at room temperature.
Electrical Recording. The patch-clamp amplifier (voltage-clampmode) was interfaced with a computer via a BNC2090A rack-mountable connector block and an NI PCIe-6251 M-series dataacquisition card (both National Instruments); all unnecessaryelectrical equipment was switched off, including lights, to minimizenoise. Electrical measurements were digitized during acquisition at1 kHz. Traces were recorded using WinEDR software (version3.2.6; John Dempster, University of Strathclyde, Glasgow, UK).
Fluorescence Imaging. Droplets were formed from a solution con-taining Fluo-8 (46 μM, 1.5 M KCl, 370 μM EDTA). DIBs wereobserved using a 60× TIRF oil-immersion objective lens (Nikon),with the fluorophore excited by a 473-nm continuous-wave laser,coupled to the inverted microscope by an optical fiber (power atfiber output, ∼4 mW). The fluorescence and excitation signals were
separated by dichroic (XF2037) and bandpass (XF3080 510/23;both Omega Optical) filters before being imaged onto an electron-multiplying CCD (iXon3 897; Andor).
Image Analysis.Analyses of electrical and optical data were carriedout in IGOR Pro-6.2 (Wavemetrics). Spots were detected byTrackmate using its Laplacian of Gaussian algorithm and trackedwith the Linear Assignment Problem tracker, allowing for gapclosing over a maximum distance of ∼5 pixels over 10 frames.Detected spots and their associated tracks were manually checkedand filtered for quality. For Gaussian fitting in Igor Pro, a 2DGaussian was fitted to the brightest pore within an image stack,and then a patch containing the fit subtracted from the originalimage. This process was repeated until no trajectories remainedwith intensities 3σ above the SD of the background. We havepreviously used this CLEAN algorithm (46) to perform super-resolution imaging of single molecules (47).
Pore Radius Estimation. The nature of the field around an elec-tropore is complex owing to its geometry, but we approximate theconductance of a cylindrical defect of radius r to be as follows:
G= πr2g=d,
where d is the membrane thickness and g is the conductivity of thesolution filling the pore. We assume a DPhPC bilayer to be 4.8 nmthick. ConductanceG was obtained from the current recordings ofpores exhibiting discrete conductance fluctuations (upper traces inFig. S5 A and B).
SI MoviesThe image correction process is as follows, carried out in ImageJ. AFourier transform (FT) bandpass filter is applied with a featurediameter of between 50 and 100 pixels to remove large diffraction orinterference features. Next, a region containing no pore throughoutthe movie is extracted (∼10,000 pixel2), and the mean intensity valuefor each frame is calculated. This value is subtracted from its(complete) host image and repeated across the movie (mean-of-patch subtraction). This removes “flickering” of the movie due tolaser variation. In some cases, a median stack is formed and aGaussian blur (σ = 5–10) applied; this image is subtracted fromall frames to remove small laser profile features (blurred mediansubtraction). In the legends of Movies S1–S5, we use the fol-lowing shorthand to indicate which correction processes havebeen used: FT, Fourier transform bandpass; MoPS, mean-of-patch subtraction; BMS, blurred median subtraction.
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Fig. S1. Detail of experimental setup, highlighting the main features of the PMMA device used in these experiments. (A) Cut-through view of the device.(B) Side view of A. Once the substrate agarose (dark blue) has been spun onto a coverslip, this is then placed within the recess seen in A. Hydrating agarose(light blue) is then flowed through the device, where it flows around the wells. An Ag/AgCl electrode is inserted into the hydrating agarose via one of thefilling holes. Roman numerals represent placing a droplet (yellow) within a well (i and ii), its sinking toward the substrate under gravity (ii and iii), andformation of a bilayer (iv). (Inset) Cartoon of a well containing a DIB. The hydrating agarose does not cover the substrate within the wells. Dotted arrowsindicate movement of water and electrolyte into the substrate from the hydrating agarose.
Fig. S2. Correlating fluorescence and electrical recording. A triplet of pores at 200 mV generates an electrical signal that comprises the three individual porecurrents, EA+B+C (red). These can be revealed using fluorescence data (FA, FB, and FC, black). Fluorescence traces are overlaid atop duplicates of the totalelectrical signal to ease visualization of its components. The image is the maximum intensity over 200 frames recorded at 32.9 Hz. (Scale bar: 25 μm.)
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Fig. S3. Electrical characterization of DIBs. (A) Voltage protocol applied to DIBs to obtain breakdown voltages and I–V curves such as shown in C. The droplet issubjected to incrementally increasing positive and negative potentials. Between each 180-s application of voltage, there is a 30-s rest period at 0 mV, indicated bythe dashed line. (B) Current response of a bilayer under a voltage protocol, shown from the period in which electroporation has started. Electrolytes were identicalto those used in oSCR experiments. Bilayer capacitance charging and the rest periods at 0 mV have been removed for clarity. (C) I–V response characteristic of aDPhPC DIB. A voltage protocol exposed the membrane to ±5-mV stepwise increases in potential up to ±300 mV for 180 s, with each increment separated by 30 s at0 mV. Points are the mean current value at each applied potential. Error bars are the SD. (Inset) Expanded region of the I–V curve.
Fig. S4. Typical fluorescence–voltage behavior during electroporation. Red squares: mean intensity of all pores within the field of view (512 × 512 pixels; visiblebilayer area, 0.0188 mm2) during an oSCR recording at increasing potential. Error bars represent ±1σ. Black circles: maximum fluorescence intensity across thesame run.
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Fig. S5. Electropore behavior. (A) Electrical traces. At low potentials, we observe electrical behavior consistent with that of a lone pore, although we can onlyassume a single defect is present. At higher potentials, the current recording can only report on the total bilayer conductance, and so the component defects areunresolved. (B) oSCR reveals individual pore behavior from an ensemble of pores. We observe a range of opening and closing processes, with discrete bursts,rapid fluctuations, sudden pore closure, and stable behaviors. At high potentials, all of these behaviors are observed and no one process dominates.
Fig. S6. Electropore diffusion in phase-separated membranes. Median images of between 500 and 7,000 frames recorded at 99.4 Hz (A) or 49.9 Hz (B). Coloredtrajectories show motion of electropores; their color indicates the time at which the trajectory begins with respect to the start of the recording. (A) DPhPC/DPPG/cholesterol, 1:1:1. Electropores in the Ld phase diffuse around, but not within, areas of Lo lipid. The lower panel shows an electropore passing between a gap inthe ordered region. (B) In a DIB formed of DPhPC/bSM/cholesterol, 1:1:1, electropores form and are trapped within the small regions of the Ld phase, marked byarrows. (Scale bars: 5 μm.)
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Fig. S7. Lifetimes for single pores. Analysis of isolated pores indicates that the double-exponential fits are appropriate for the ensemble histograms (Fig. 6 inmain manuscript), and that the two lifetimes are not a result of two distinct populations of pore. Pores 1 and 2 (A and B) were recorded on the same bilayerand 3 (C) from a different bilayer. All three exhibited different mobilities, demonstrating that the double-lifetime behavior is independent of the experimentand pore diffusion properties.
Movie S1. Electropores at ∼300 mV, recorded at 62 Hz. 137 × 137 μm. Image correction: FT, MoPS.
Movie S1
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Movie S2. An electropore diffuses in the Ld phase in a DPhPC/DPPG/cholesterol (molar ratio, 1:1:1) membrane at 160 mV; recorded at 99 Hz. 28 × 28 μm. Imagecorrection: FT, MoPS, BMS.
Movie S2
Movie S3. Electropores trapped in Ld pools in a DPhPC/bSM/cholesterol (molar ratio, 1:1:1) membrane at 130 mV; recorded at 49 Hz. 40 × 40 μm. Imagecorrection: FT, MoPS, BMS.
Movie S3
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Movie S4. A large pore causes the bilayer to significantly reduce in size at 200 mV; recorded at 33 Hz. 137 × 137 μm. Image correction: raw image.
Movie S4
Movie S5. Rupture at 200 mV, recorded at 10 Hz. 137 × 137 μm. Image correction: FT, MoPS.
Movie S5
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