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

Ion channels are not static in biological membranes. Here we present a single molecule optical approach to unravel the link between lateral membrane diffusion and ion channel function. The main advantage of this technique over other methods is that fluorescent labeling of proteins which might interfere with their lateral movement and function is not required.

The method can be applied to any membrane protein channel where free or restricted diffusion is important for organization and function. To begin, transfer 380 microliters of the DPhPC stock solution into a glass vial, and overlay the lipid stock solution with argon or nitrogen 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.

Dry the lipid sample under a stream of nitrogen and remove the remaining organic solvent from the lipid sample under vacuum using an oil-free vacuum pump overnight. Dissolve the lipid film in a hexadecane silicone oil solution by adding equal volumes of hexadecane and silicone oil using a one milliliter pipette to a final lipid concentration of 9.5 milligrams per milliliter. Place some glass coverslips into a stainless steel coverslip holder and clean them in a glass beaker for around 10 minutes with acetone in an ultrasonic cleaner.

Rinse the coverslips with double deionized water and dry them under a stream of nitrogen. Further, clean and hydrophylize a coverslip in a plasma cleaner with oxygen for five minutes. Mount a plasma-treated coverslip on a spin coater and coat the coverslip with a sub-micrometer thick film of agarose by slowly adding 140 microliters of heated 0.75%low melting agarose with a 200 microliter pipette at 3, 000 RPM for 30 seconds.

Immediately attach the spin-coated coverslip with the thin layer of the agarose hydrogel to the underside of the PMMA chamber. Ensure that the agarose hydrogel points upward. Fix the edges of the coverslip to the PMMA micro-machined device with transparent adhesive tape, and place the device on a hot plate heated to 35 degrees Celsius.

Carefully pour 200 microliters of 2.5%agarose solution into the inlet of the chamber without creating air bubbles. Immediately cover the wells of the PMMA chamber with around 60 microliters of the lipid oil solution to initiate lipid monolayer formation at the agarose oil interface to avoid dehydration of the spin-coated agarose in the wells of the PMMA chamber. Place the device on a hot plate at 35 degrees Celsius for about two hours.

Place around 20 microliters of lipid hexadecane silicone oil solution in each of the several micro-fabricated wells in a droplet incubation chamber. Prepare a microcapillary glass needle with a tip opening diameter of around 20 micrometers using a vertical or horizontal micropipette puller. Fill the needle with around five microliters of aqueous injection solution containing 8.8 millimolar HEPES, seven micromolar of a fluorescent dye, Fluo-8, 400 micromolar EDTA, 1.32 molar potassium chloride and 30 nanomolar TOM core complex or alternatively 20 nanomolar OMPF.

Mount the needle with the aqueous injection solution on a piezo-driven nanoinjector and inject 100 to 200 nanoliters of aqueous droplets into the wells in the droplet incubation chamber filled with lipid hexadecane silicone oil solution using the nanoinjector. Allow the formation of a lipid monolayer at the droplet oil interface for about two hours by maintaining the PMMA and droplet incubation chambers on a hot plate heated to 35 degrees Celsius. Manually transfer individual aqueous droplets from the wells of the droplet incubation chamber into the wells of the PMMA chamber using a single-channel microliter pipette with a 10 microliter disposable polypropylene tip.

Allow the droplets to sink onto the lipid monolayers at the hydrogel-oil interfaces to form a lipid bilayer between the droplets and the agarose hydrogel. Mount the PMMA chamber with the droplet interface bilayer or DIB membranes on the sample holder of an inverted light microscope, and assess the membrane formation using a 10x Hoffman modulation contrast objective. If DIB membranes have formed, mount the PMMA chamber on the sample holder of a TIRF microscope equipped with a conventional light source for epifluorescence illumination, a 488 nanometer laser, and a back-illuminated electron multiplying CCD camera to achieve a pixel size of around 0.16 micrometers.

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. Fine focus the same edge of the DIB membrane at high magnification with a 100x NA 1.49 apochromatic oil TIRF objective, again under epifluorescence illumination with a high intensity light source using a GFP filter set. Change the filter setting from GFP to the quad band TIRF filter set, switch on the 488 nanometer laser, and set the intensity of the laser on the objective lens.

To visualize single ion channels, adjust the TIRF angle and EM CCD camera gain so that the open ion channels in the DIB membrane appear as high contrast fluorescent spots on a dark background, and the signal to background ratio reaches a maximum. Ensure that the spots corresponding to the calcium ion 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. Check that the 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.

Finally, 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 and the state of channel activity, acquire sufficiently long and well-sampled trajectories. Cryo EM structure of Neurospora crassa TOM-CC is shown here.

Mitochondria from an N.crassa stain containing a TOM 22 with a 6-His tag were solubilized in DDM and subjected to nickel NTA affinity chromatography and anion exchange chromatography. SDS-PAGE of isolated TOM-CC crystal structure and SDS-PAGE of purified E.coli OMPF used for comparison are shown here. The fluorescent amplitude trace in the corresponding trajectory of TOM-CC indicate that the open-closed channel activity of TOM-CC correlates with the lateral membrane mobility of the complex.

The amplitude trace displays three permeability states:fully open state corresponding to moving channels, intermediate permeability state, and closed channel state corresponding to non-moving channels. TOM-CC in the intermediate state wobbles around its mean position by about plus/minus 60 nanometers. The fluorescent amplitude trace in the corresponding trajectory of OMPF are shown here.

OMPF reveals only one intensity level in comparison to TOM-CC, regardless of whether it is in motion or trapped. The trajectory segments corresponding to the time periods of trapped molecules are marked in gray. One important thing to notice is that this method analyzes single membrane proteins in an intact membrane environment.

The dynamics of membrane proteins will remain a horizon for scientific studies in the foreseeable future. We expect our method to contribute to this field significantly.