Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

1. Protein production

NOTE: This section describes the procedure for isolating OmpF from Escherichia coli BE BL21(DE3) omp6^31,32, which lacks LamB and OmpC, and TOM core complex from Neurospora crassa (Figure 1)^28,29. The latter requires mitochondria isolated from a N. crassa strain²⁸ containing a 6xHis-labeled form of the TOM subunit Tom22 (Figure 1A), which can be isolated as described²⁸. The following protocols usually yield 1-2 mg of N. crassa TOM-CC and 10 mg of E. coli OmpF. If the amount is to be adjusted, it is important that the protein/detergent ratios are precisely maintained. Unless otherwise specified, all steps should be performed at 4 °C.

1. Isolation of TOM core complex
   1. Solubilize purified N. crassa mitochondria (2 g of protein) obtained by differential sedimentation from approximately 1.5 kg (wet weight) hyphae, according to Bausewein et al.³⁰, at a protein concentration of 10 mg/mL in 20 mM Tris-HCl (pH 8.5), 1% (w/v) DDM, 20% (v/v) glycerol, 300 mM NaCl, 20 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min at 4 °C.
      CAUTION: PMSF is toxic. Wear appropriate personal protective equipment.
   2. Transfer the solubilized mitochondria into ultracentrifuge tubes and separate the non-solubilized membranes from solubilized membrane proteins by ultracentrifugation at 130,000 x g for 40 min at 4 °C and filtration with standard-grade filter paper.
   3. Equilibrate a prepacked Ni-NTA column (volume of 5 mL) with about 5 column volumes (CV) of buffer A1 (Table 1) using an automated protein purification system. Run the Ni-NTA column at a constant flow rate of 1 mL/min during all steps. Use a lower volume column (1 mL) if the proteins have been isolated from less than 2 g of mitochondria.
   4. Load the solubilized protein sample onto the Ni-NTA column at a flow rate of 1 mL/min.
   5. Wash the Ni-NTA column with 5 CV of buffer A1 (Table 1) to remove unbound proteins.
   6. Elute His-tagged TOM-CC with 30% buffer A2 (Table 1; 300 mM imidazole) and collect the protein peak as it appears in the 280 nm chromatogram (Figure 1B).
   7. For further purification of TOM-CC, equilibrate the prepacked anion exchange column (1 mL) with 5 CV each of buffer B1, B2, and B1 (Table 1) using the automated protein purification system. Run the anion exchange column at a constant flow rate of 1 mL/min during all steps.
   8. Load the TOM-CC peak fraction (step 1.1.6) on the anion exchange column (flow rate of 1 mL/min).
   9. Remove the unbound proteins by washing the column with 5 CV of buffer B1 (Table 1) and a linear salt gradient of 0%-20% buffer B2 (Table 1).
   10. Elute TOM-CC with a linear salt gradient of 20%-35% buffer B2, and collect the protein peak fraction as it appears in the 280 nm chromatogram (Figure 1C).
   11. Assess the sample purity by SDS-PAGE (Figure 1D) and determine the protein concentration using a commercially available protein assay, according to the manufacturer's protocol (see Table of Materials).
   12. Freeze the protein samples in liquid nitrogen and store them at -80 °C until further use.
       CAUTION: Liquid nitrogen should be handled in well-ventilated areas. Wear appropriate personal protective equipment.
        
2. Isolation of OmpF
   1. Recover the E. coli strain BE BL21(DE3) omp6, lacking LamB and OmpC³², from a frozen glycerol stock under sterile conditions, and streak onto a Luria-Bertani (LB) agar plate (Table 2). To do this, gradually spread the sample evenly over the entire plate.
   2. Allow the sample to soak into the agar for 5 min, before inverting the plate with the lid closed and incubating overnight at 37 °C.
   3. Select a single E. coli colony, inoculate 7.5 mL of LB medium (Table 2) with this single colony using a sterile toothpick, and leave to grow overnight (14 h) with agitation (170 rpm) at 37 °C.
   4. Transfer 2 x 1 mL of E. coli cells with sterile pipettes into 2 x 500 mL of LB medium (Table 2), and leave to grow overnight (~14 h) at 37 °C with agitation (~170 rpm) in a shaker.
   5. Harvest the cells by centrifugation at 5,000 x g for 20 min at 4 °C, freeze the pellet in liquid nitrogen, and store at -80 °C until further use.
      NOTE: The wet weight of the cell pellet is typically 5 g per 1 L of culture. At this point, the protocol can be paused.
   6. Thaw and resuspend the cells (2 g) in 20 mL of lysis buffer C1 (Table 2), and pass the suspension three times at 1,000 psi through a precooled (4 °C) high-pressure cell disruption system, with a maximum sample volume of 35 mL, according to the manufacturer's instructions.
      CAUTION: The use of a French press can lead to serious injuries. Never exceed the pressure limit of the cell used. Wear appropriate personal protective equipment.
   7. Remove unbroken cells from the lysate by centrifugation at 4,000 x g for 15 min, and collect the supernatant.
   8. Collect the membranes by ultracentrifugation at 100,000 x g for 1 h.
   9. Resuspend the membrane pellet in 10 mL of buffer C2 (Table 2), and mix with an equal volume of SDS buffer C3 (Table 2) using a ball-bearing glass homogenizer.
      CAUTION: β-mercaptoethanol in buffer C3 is toxic. Follow all relevant safety regulations.
   10. Incubate the suspension in a water bath at 50 °C for 30 min.
   11. Centrifuge the sample at 100,000 x g at 20 °C for 1 h.
   12. Resuspend the membrane pellet in 10 mL of SDS buffer C4 (Table 2) using a ball-bearing glass homogenizer, and incubate the suspension in a water bath at 37 °C for 30 min.
       NOTE: If the pellet cannot be resuspended, add more SDS buffer C4 up to a volume of 20 mL.
   13. Centrifuge the sample at 100,000 x g at 20 °C for 30 min, and collect the supernatant.
   14. Mix the supernatant with octyl polyoxyethylene (octyl POE) to a final detergent concentration of 0.5% (w/v), and dialyze the sample twice against buffer C5 (Table 2) at 4 °C for 24 h. For this purpose, use dialysis tubing with a cut off of 20 kDa, according to the manufacturer's instructions, and place the sample in the dialysis tubing or device.
       NOTE: The cut off of the dialysis tubing needs to be adjusted should proteins with other molecular masses be dialyzed.
   15. Assess the sample purity by SDS-PAGE and determine the protein concentration using a commercially available protein assay, according to the manufacturer's protocol (Table of Materials).
       NOTE: Samples heated to 95 °C show monomeric OmpF. Samples not heated show OmpF in its trimeric form (Figure 1F).
   16. Shock-freeze dialyzed OmpF in aliquots in liquid nitrogen, and store at -20 °C until further use.
       ​CAUTION: Liquid nitrogen should be handled in well-ventilated areas. Wear appropriate personal protective equipment.

2. Optical single-channel recording of ion channels in DIB membranes

NOTE: This section describes the procedure for preparing DIB membranes in microfabricated polymethyl methacrylate (PMMA) chambers² to monitor lateral protein movement and ion flux through single ion channels¹⁷. The dimensions and exact drawings for manufacturing the chamber can be found in Lepthin et al.². Figure 2 gives an overview of the assembly of the PMMA chamber² and the formation of the DIB membranes. Unless otherwise specified, all steps are performed at room temperature (RT). Figure 3 shows a schematic representation of a DIB membrane and how Ca²⁺ flux through a single channel protein is used to monitor both movement in the membrane and the open-closed state of the channel.

1. Preparation of lipids
   1. Remove the lipid stock solution containing 1,2-diphytanoyl-sn-glycero-3-phosphocholine (25 mg/mL DPhPC) dissolved in chloroform from the freezer at -20 °C, and warm to RT. For this purpose, it is generally sufficient to warm up the sample slowly by hand for a few minutes.
      CAUTION: Chloroform is toxic. Wear appropriate personal protective equipment and perform all procedures involving chloroform under a fume hood.
   2. Transfer 380 µL of DPhPC stock solution (25 mg/mL DPhPC) into a glass vial. Avoid pipettes with rubber sealings. Instead, use microliter glass syringes with stainless steel plungers.
   3. After handling the lipid stock solution, overlay the lipid stock solution with Ar or N₂ gas to prevent lipid oxidation. Use the lowest possible gas flow to avoid evaporation of the organic solvent in which the lipids are dissolved or splashing of the solvent sprays from the vial. Make sure that the lids of the glass vials containing lipids with organic solvent are coated with polytetrafluoroethylene (PTFE) on the inside.
   4. Dry the lipid sample (step 2.1.2) under a stream of N₂, and remove the remaining organic solvent from the lipid sample overnight under vacuum (2.0 mbar) using an oil-free vacuum pump.
   5. Dissolve the lipid film in hexadecane/silicone oil solution by adding equal volumes of hexadecane and silicone oil (500 µL each) using a microliter glass syringe to a final lipid concentration of 9.5 mg/mL.
      ​NOTE: The lipid solution is stable at RT for several weeks.
2. Preparation of agarose hydrogel
   1. Prepare approximately 1 mL of low melting agarose solution (0.75% [w/v]) in double deionized water, and heat to 85 °C in a heating block for 20 min to be used for spin coating glass coverslips.
      NOTE: The agarose solution can be kept at RT for several weeks and can be reheated several times.
   2. Prepare low melting 2.5% (w/v) agarose solution in 0.66 M CaCl₂ and 8.8 mM HEPES (pH 7.2), and heat to 85 °C in a heating block for 20 min. Make sure that the agarose is well melted.
      NOTE: The agarose solution can be kept for several weeks at RT and can be reheated several times, just like the agarose (step 2.2.1) used for spin coating.
3. Polymethyl methacrylate (PMMA) chamber assembly and lipid monolayer formation at the hydrogel hexadecane/silicone oil interface
   1. Place some glass coverslips (40 mm x 24 mm x 0.13 mm) into a stainless-steel coverslip holder, and clean them in a glass beaker for 10 min with acetone in an ultrasonic cleaner. Use just enough acetone to submerge the coverslips and cover the beaker loosely with a glass plate to create a pressureless, closed system that minimizes the escape of vapors during the cleaning process.
      ​CAUTION: Acetone is a highly flammable solvent with a low flash point. Vapor/air mixtures are explosive. Operate the ultrasonic cleaner in a well-ventilated area. Wear appropriate personal protective equipment and observe official safety precautions (e.g., no open flames and no sparks).
   2. Rinse the glass coverslips with double-deionized water and dry them under a stream of N₂.
      NOTE: Coverslips cleaned in this way can be stored for several weeks.
   3. Further clean and hydrophilize a coverslip in a plasma cleaner with oxygen (0.5 mbar) for 5 min.
      NOTE: Carry out this step immediately before spin coating with agarose solution, as the hydrophilic effect of plasma cleaning wears off over time.
   4. Mount a plasma-treated coverslip on a spin coater (Figure 2, step 1).
   5. Coat the coverslip with a submicrometer-thick film of agarose by slowly adding 140 µL of heated 0.75% (w/v) low-melting agarose (step 2.2.1) at 3,000 rpm for 30 s, using a 200 µL pipette (Figure 2, step 1).
      NOTE: The thickness of the agarose film can be determined by atomic force microscopy (AFM), as described¹⁷.
   6. Immediately attach the spin-coated coverslip with the thin layer of the agarose hydrogel to the underside of the PMMA chamber². Ensure that the agarose hydrogel points upward (Figure 2, step 2).
   7. Fix the edges of the coverslip to the PMMA micro-machined device with transparent adhesive tape.
   8. Place the PMMA device on a hot plate heated to 35 °C (Figure 2, step 3).
   9. Carefully pour 200 µL of 2.5% agarose solution (step 2.2.2) into the inlet of the chamber with a pipette (Figure 2, step 3), without creating air bubbles so that the thin hydrogel applied by spin coating can equilibrate with the buffer of the 2.5% agarose solution and remain hydrated. Ensure that the 2.5% agarose solution is spread around the spin-coated agarose hydrogel but not over it (Figure 3A), which can be avoided by gently pressing the PMMA chamber onto the heating plate.
   10. Immediately cover the wells of the PMMA chamber (Figure 2, step 4) with a total of 60 µL of lipid/oil solution (step 2.1.5), to initiate lipid monolayer formation at the agarose-oil interface and to avoid dehydration of the spin-coated agarose in the wells of the PMMA chamber. Keep the device on a hot plate at 35 °C for about 2 h.
       NOTE: The circular wells in the PMMA chamber have a diameter of 0.5 mm and a depth of 1.8 mm, according to previously published work².
4. Preparation of lipid-coated aqueous droplets in hexadecane/silicone oil solution
   1. Place 20 µL of lipid hexadecane/silicone oil solution (step 2.1.5) to each of several microfabricated wells in a droplet incubation chamber (Figure 2, step 4). Suitable containers for the lipid hexadecane/silicone oil solution are small recesses of 40 mm x 30 mm x 3.5 mm in a thin PMMA plate².
   2. Prepare a microcapillary glass needle with a tip opening diameter of 20 µm using a vertical or horizontal micropipette puller (e.g., used for making patch pipettes or sharp glass electrodes). Estimate the opening diameter of the microcapillary glass needle under a binocular stereomicroscope at low magnification, in a zoom range between 7.5x and 35x.
      NOTE: The settings for the preheating mode before the glass capillary is pulled, the heating mode for pulling, and the traction force should be determined experimentally in advance, according to the manufacturer's specifications of the pulling device and the type of capillary.
   3. Fill the microcapillary glass needle with 5 µL of aqueous injection solution containing 8.8 mM HEPES (pH 7.2), 7 µM Fluo-8, 400 µM EDTA, 1.32 M KCl, and 30 nM TOM core complex, or alternatively 20 nM OmpF using a microcapillary pipette tip. Use only double-distilled water previously treated with a chelating resin that binds multivalent metal ions (Ca²⁺) to prepare the aqueous injection solution.
   4. Mount the microcapillary glass needle with the aqueous injection solution on a piezo-driven nanoinjector.
   5. Inject 100-200 nL of aqueous droplets (Figure 2, step 4) containing 8.8 mM HEPES (pH 7.2), 7 µM Fluo-8, 400 µM EDTA, 1.32 M KCl, and 30 nM TOM core complex (step 1.1.12), or alternatively 20 nM OmpF (step 1.2.16) into the wells in the droplet incubation chamber filled with lipid hexadecane/silicone oil solution (see 2.1.5) using the nanoinjector. Make sure that the droplets do not touch each other by placing them several millimeters (>10 mm) apart into the wells. Otherwise, if lipid monolayers have not yet formed at the oil/buffer interface, they will merge into larger droplets.
      NOTE: If no class capillaries and no nanoinjectors are available, the droplets can alternatively be prepared manually with single-channel microliter pipettes for volumes between 0.1 µL and 0.5 µL, as described². In this case, however, the droplet volumes are not as accurate.
   6. Allow the formation of a lipid monolayer at the droplet/oil interface for about 2 h by maintaining the PMMA and droplet incubation chambers (Figure 2, step 4) on a hot plate heated to 35 °C.
5. Preparation and imaging of single ion channels in DIB membranes
   1. Manually transfer individual aqueous droplets from the wells of the droplet incubation chamber under a stereomicroscope into the wells of the PMMA chamber (Figure 2, step 5), using a single-channel microliter pipette with a 10 µL disposable polypropylene tip. Allow the droplets to sink onto the lipid monolayers formed at the hydrogel-oil interfaces for about 5 min to form a lipid bilayer (Figure 3) between the droplets and the agarose hydrogel.
   2. Mount the PMMA chamber with DIB membranes on the sample holder of an inverted light microscope and assess membrane formation using a 10x Hoffman modulation contrast objective (Figure 2, step 6). DIB membrane formation is indicated by a clear white ring at the interface between the droplet and the hydrogel (Figure 4A). DIB membranes that have been broken do not show this ring (Figure 4B).
      NOTE: Alternatively, the DIB membranes can be visualized at 10x magnification by phase contrast or differential interference contrast (DIC) microscopy.
   3. If DIB membranes have formed, mount the PMMA chamber on the sample holder of a TIRF microscope (Figure 2, step 7) equipped with a conventional light source for epifluorescence illumination, a 488 nm laser (P_max = 100 mW), and a back-illuminated electron-multiplying CCD camera (512 x 512 pixels; >95% QE) to achieve a pixel size of ~0.16 µm.
   4. Focus the edge of a DIB membrane with a 10x magnification objective under epifluorescence illumination with a high-intensity light source using a GFP filter set.
   5. Fine focus the same edge of the DIB membrane at high magnification with a 100x/N.A. 1.49 apochromat oil TIRF objective, again under epifluorescence illumination, with the high-intensity light source using a GFP filter set that allows visualizing weak background fluorescence of the fluorescent dye Fluo-8 in the droplet (Figure 4C).
   6. Change the filter setting from GFP to the quad-band TIRF filter set.
   7. Switch on the 488 nm laser and set the intensity of the laser on the objective lens to a value between 8 mW and 10 mW.
      NOTE: Since there is often no quantitative information on the laser intensity at the objective lens, the laser intensity settings should be calibrated beforehand in separate measurements at the lens, with an optical laser power meter equipped with a photodiode sensor, according to the manufacturer's specifications.
      CAUTION: To ensure safe operation of the laser, the operator must be aware of the possible dangers of laser radiation and the accident prevention regulations.
   8. To visualize single ion channels, adjust the TIRF angle and EMCCD camera gain (e.g., EM gain multiplier setting: 285) so that open ion channels in the DIB membrane appear as high-contrast fluorescent spots on a dark background (Figure 4D-G), and the signal-to-background ratio, when visually inspected, reaches a maximum. Ensure that the spots, corresponding to Ca²⁺-flux through single ion channels, remain in focus and have a round shape, with high intensity in the center and gradually decreasing toward the periphery. Membranes without channels show only background fluorescence (Figure 4C).
   9. Before recording the movement and open-closed activity of individual channels over time, check that fluorescent spots are in focus, to ensure that the ion channels have reconstituted into the DIB membranes and are moving laterally in the membrane plane. If this is not the case, it could indicate that a fluorescent molecule is dipping in and out of the evanescent field generated by the laser near the membrane.
   10. Record a series of membrane images that allows proper tracking of the position and monitoring of the open-closed state of the individual ion channels. To determine the type of lateral mobility (e.g., free motion vs transient anchorage [for reference, see¹⁶]) and the state of channel activity (e.g., open or closed), acquire sufficiently long (e.g., 30 s to 1 min) and well-sampled trajectories (e.g., frame rate of 48 s^-1).
       NOTE: The observation of channeled, restricted, or hop diffusion¹⁶ requires that the sampling rate is sufficiently fast to measure unrestricted diffusion within confining boundaries. In addition, ensure that the data sampling time is sufficiently long to measure the transition from unconstrained to constrained diffusion (for details, see Jacobson et al.¹⁶).
6. Image and data processing
   ​NOTE: Open-source image processing packages³³ or self-written routines¹⁷ based on commercial software packages can be used to analyze the spatiotemporal dynamics of individual ion channels in DIBs. To be able to correlate lateral membrane mobility with the ion flux through the individual channels, no filter algorithms should be applied.
   1. Correct image time series for bleaching by applying standard procedures³⁴ using self-written routines, by fitting the average frame intensity of the full image to , where t is the frame index (time) and a_k is the fitting parameters. Then, correct the time series according to , where I(t) is the intensity. Alternatively, for initial data analyses, perform background corrections by using open source software with implemented rolling-ball algorithms³³ and a rolling-ball radius, depending on the spot and pixel size of the camera (e.g., 50 pixels).
   2. Determine the spatial positions and amplitudes of a fluorescence spot within a defined region of interest (ROI) (e.g., 30 x 30 pixels), by fitting I(t) at a given time t to a two-dimensional Gaussian function with planar tilt that accounts for possible local illumination gradients according to . Here, x = (x, y) is the ROI with the fluorescence intensity information, A and σ are the amplitude and widths of the Gaussian, p_k are parameters that characterize the background intensity of the ROI, and μ = (x₀, y₀) defines the position of the Gaussian. The ion flux through an individual channel is given by the parameter A. The trajectory of the channel is given by x, and allows for determining the lateral mobility of the channel. Alternatively, it is possible to determine the positions and intensities of individual spots using open-source platforms³³ and plug-ins as described^35,36. Estimate the positional accuracy³⁷ after conversion of the EMCCD camera readout into photon numbers³⁸.
   3. Plot the fluorescence amplitude A versus time and the corresponding trajectory x to determine a possible correlation between the channel diffusivity and the ion flux through the channel. Perform this with any graphics software. In case of free lateral channel movement, determine the lateral diffusion coefficient D by linear regression of the time delay τ, and calculate the mean square displacement of the spots from x according to .
      NOTE: Typical diffusion coefficients¹⁷ for TOM-CC and OmpF freely moving in DIB membranes range between 0.5 and 1.5 µm² s^-1. Molecules whose diffusion constant is less than 0.01 mm²·s^-1 are defined as immobilized¹⁷.