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WO2025160387A1 - Microscope pour l'éclairage de bicouche autonome - Google Patents

Microscope pour l'éclairage de bicouche autonome

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Publication number
WO2025160387A1
WO2025160387A1 PCT/US2025/012941 US2025012941W WO2025160387A1 WO 2025160387 A1 WO2025160387 A1 WO 2025160387A1 US 2025012941 W US2025012941 W US 2025012941W WO 2025160387 A1 WO2025160387 A1 WO 2025160387A1
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WO
WIPO (PCT)
Prior art keywords
membrane
objective
microscopy device
excitation
bilayer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/012941
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English (en)
Other versions
WO2025160387A9 (fr
Inventor
Gonzalo PEREZ-MITTA
Roderick Mackinnon
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Rockefeller University
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Rockefeller University
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Publication of WO2025160387A1 publication Critical patent/WO2025160387A1/fr
Publication of WO2025160387A9 publication Critical patent/WO2025160387A9/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure

Definitions

  • Integral membrane proteins encode 20-30% of the proteomes of organisms across all domains of life (see Almen, M. S., et al., BMC Biol., 2009; Wallin, E. et al., Protein Sci., 1998).
  • Receptors, transporters, and channels compose most IMPs in humans and are about 60% of all therapeutic drug targets, exemplifying the importance of these proteins in physiology and disease (Overington, J. P., et al., Nat. Rev. Drug Discov., 2006).
  • many dynamic features of IMPs remain enigmatic and controversial.
  • IMPs in the plasma membrane switch stochastically between diverse motions when observed under a microscope; the same protein can transition from a random walk to a linear motion or a complete stop (Krapf, D. et al., Curr. Top. Membr., 2015; Ritchie, K. et al. Biophys. J., 2005).
  • IMPs can interact with themselves and other proteins and adopt non-homogeneous distributions across membranes (see Gebmeier, B. F., et al., Proc. Natl. Acad. Sci. U. S. A., 2006; Saka, S. K. et al., Nat. Commun., 2014).
  • the origin of such features needs to be addressed to develop good dynamical models of cell membranes.
  • SB supported bilayer
  • FB freestanding bilayer
  • SB methods require forming a membrane on top of a solid (or gel) substrate (see Ramm, B., et al. Nat. Phys., 2021 ; Casuso, I. et al., Nat. Nanotechnol., 2012; Kiessling, V., et al., Front. Mol. Neurosci., 2017; Parperis, C., et al., Academic Press, 2021).
  • these methods are inadequate for the study of IMPs because proteins are strongly affected by the presence of the substrate (see Rojko, N.
  • FB methods where the membrane is suspended in solution, avoid immobilizing proteins, but current implementations of FBs have relied mostly on confocal optics that are not compatible with SPT (see Heinemann, F., et al., ChemPhysChem, 2011; Spindler, S., et al., Nano Lett., 2018; Weib, K. et al. Biophys. J., 2013).
  • the invention relates to a microscopy device, comprising a microscope having an objective and an imaging platform positioned in the field of view of the objective, an experimental chamber positioned on the imaging platform, the experimental chamber comprising a lower cavity, a sample holder having at least one opening, positioned over the lower cavity, a membrane positioned within the opening, the membrane and the sample holder forming a first sealed chamber with the lower cavity, and an excitation energy source configured to illuminate the membrane via an excitation objective.
  • the microscopy device further comprises at least one perfusion port fluidly connected to the lower cavity, the perfusion port configured to control a flow of a fluid into the lower cavity.
  • the sample holder further comprises an upper cavity positioned over the sample holder, forming a second sealed chamber with the sample holder.
  • the microscopy device further comprises at least one electrode positioned in the upper or lower cavity, configured to measure a current across the membrane.
  • the microscopy device comprises a first electrode positioned in the upper cavity and a second electrode positioned in the lower cavity.
  • the microscopy device further comprises at least one upper perfusion port fluidly connected to the upper cavity, configured to control a flow of a fluid into the upper cavity.
  • the microscopy device further comprises at least one lower perfusion port fluidly connected to the lower cavity, the lower perfusion port configured to introduce the fluid at a different pressure from a pressure of the upper perfusion port, in order to deform the membrane.
  • the microscopy device further comprises at least one manometer fluidly connected to at least one of the upper or lower perfusion ports, configured to measure a pressure within the upper or lower cavity.
  • the experimental chamber further comprises an illumination port in the lower cavity, the excitation objective positioned within the illumination port.
  • the microscopy device further comprises a linear actuator fixedly attached to the experimental chamber, configured to move the experimental chamber relative to the objective.
  • the microscopy device further comprises a linear actuator fixedly attached to the excitation objective, configured to move the excitation objective relative to the experimental chamber.
  • the excitation objective is a long working distance objective.
  • the excitation objective is oriented at an angle relative to the membrane of 30 degrees to 50 degrees. In one embodiment, the angle is about 39 degrees.
  • the microscopy device further comprises a window positioned in the bottom of the experimental chamber.
  • the microscopy device further comprises at least one electrode positioned in the lower cavity, configured to measure a current across the membrane.
  • the membrane is a planar lipid bilayer.
  • the invention relates to a method of imaging proteins in a membrane, comprising forming a membrane comprising one or more proteins on a sample holder, positioning the sample holder in an experimental chamber, positioning an excitation objective in the experimental chamber, illuminating at least a portion of the membrane with an excitation energy source via the excitation objective, and imaging the illuminated portion of the membrane with a microscope to produce an image of a protein of the one or more proteins.
  • the method further comprises the step of moving the sample holder relative to the microscope.
  • the sample holder is moved with a micromanipulator.
  • the method further comprises the step of moving the excitation objective relative to the membrane.
  • the method further comprises deforming the membrane.
  • the membrane is deformed by changing a pressure of a fluid above or below the membrane.
  • the method further comprises measuring a current across the membrane via at least one electrode.
  • the method further comprises splitting the image of the protein with a camera splitter.
  • the excitation energy source comprises a laser
  • the step of illuminating at least a portion of the membrane comprises expanding the laser, combining the expanded laser, focusing the combined laser through a water immersion and the excitation objective.
  • the method further comprises scanning the laser.
  • the scanning is performed by feeding the combined beam into a galvo-galvo scanner conjugated to the back focal plane of the excitation objective.
  • Figure 1 A depicts an image of a representative planar bilayer observed under transillumination. The boundaries between the bilayer, the Plateau-Gibbs border (torus), and the FEP partition can be seen.
  • the bilayer composition is POPE: POPG (3:1 by weight). Scale bar: 50 mm.
  • Figure IB depicts a schematic of an amplified-view of a technical illustration of the imaging chamber.
  • Planar bilayers are formed across perforated fluoroethylene-propylene copolymer films (FEP partitions) mounted on 3D-printed sample holders (Cup). Cups attach to the experimental chamber, shown in yellow, delimiting the top and bottom sides.
  • the chamber provides access to the XO at 39 deg with respect to the optical plane of the bilayer, a transillumination source, and perfusion adaptors that can be connected to a perfusion system (bottom perfusion) or to a manometer to regulate the curvature of the bilayer.
  • a different set of perfusion adaptors top perfusion
  • Ag/AgCl electrodes are added to the top and bottom sides for electrophysiological recordings.
  • Figure 1C depicts representative fluorescent images taken with the FBM under epifluorescence (top) and focused-laser illumination (bottom) of fluorescently labeled hTRAAK. Comparison between both illumination sources shows that resolved diffraction-limited particles can only be observed using focused-laser (FL) illumination. Insert shows a magnified view of the FL illumination. Scale bars: epi, 50 mm, FL, 50 mm, insert 10 mm. hTRAAK was labeled with LD655-NHS.
  • Figure ID depicts a scheme of the FBM depicting the relation between the main components of the system.
  • 473 nm, 561 nm and 660 nm lasers are fed to three beam expanders (BE) and optically conjugated to a galvo-galvo scanner (GG), an apodization mask (AM), and to the back focal plane of an excitation objective (XO) through three optical relays (L1 -L3).
  • Photons emitted from the sample are collected by a detection objective (DO).
  • DO detection objective
  • FIG. 2A through Figure 2C depicts representative data from single-Particle-Tracking benchmark of the FBM.
  • Figure 2A Left depicts representative SPT data for the M2R receptor diffusing in a freestanding bilayer (top) and in a Quartz supported bilayer (bottom).
  • M2R receptor was reconstituted in POPE:POPG (3: 1) vesicles for FBM experiments and in DOPC:POPG (7:3) for SBs experiments.
  • POPE:POPG 3: 1) vesicles for FBM experiments and in DOPC:POPG (7:3) for SBs experiments.
  • vesicles were allowed to fuse into previously formed POPE: POPG (3: 1) bilayers, followed by labeling with LD655-labeled NbALFA and extensive washing.
  • SBs M2R-containing vesicles were allowed to burst and fuse onto solid substrates followed by washing.
  • M2R was labeled with LD655-CoA through AcpS site-directed labeling.
  • SB experiments were performed by TIRF illumination achieved with FBM optics, exchanging the FBM chamber for a prism-containing chamber.
  • Figure 2B depicts a plot of Diffusion (D) vs. anomalous diffusion (a) coefficients plots.
  • FIG. 3A through Figure 3E depicts representative data on simultaneous imaging and electrical recordings of ion channels. Determination of hTRAAK open probability (Po).
  • Figure 3 A depicts representative tension activation curves of hTRAAK showing its characteristic mechanosensitivity. The lateral tension of the bilayer was calculated from changes in the bilayers’ capacitance (grey) upon application of increasing pressure steps from 1 to 8 mmH20 (Perez-Mitta, G., et al., Proc.
  • FIG. 3B depicts a representative plot of the electrophysiology of hTRAAK.
  • Left Potassium currents through hTRAAK channels, measured at voltage steps of 500 ms from -80 mV to 80 mV. Only odd voltage steps are shown for clarity.
  • hTRAAK was reconstituted into POPE:POPG (3:1) vesicles and fused into bilayers of the same composition.
  • Right current-voltage characteristic showing the average ⁇ SD of the currents at each voltage. The trace at 50 mV is highlighted for illustrative purposes, and black arrows indicate the interval for the calculation of the average.
  • Figure 3C depicts representative imaging of hTRAAK.
  • FIG. 3D depicts representative images for detection of hTRAAK tracks analyzed using SPT software. Bottom: Equations used to calculate Po, where i is the single-channel current for hTRAAK and LR is the reciprocal of the labeled fraction of hTRAAK channels.
  • Figure 4A through Figure 4C depicts representative data of single particle tracking of hTRAAK in phase-separated bilayers.
  • Figure 4A depicts a representative image of hTRAAK in DphPC: DSPC: cholesterol (2: 1 : 1 molar ratio)
  • DphPC DSPC: cholesterol (2: 1 : 1 molar ratio)
  • Left frame from a video overlapping hTRAAK (red) and DSPE-AF488 (green) channels.
  • DSPE partitions into the ordered phase.
  • Right: hTRAAK trajectories obtained from SPT analysis show the preference of hTRAAK for the disordered phase.
  • Scale bar 10 mm.
  • hTRAAK was labeled with LD655-NHS.
  • Figure 4B depicts representative images from a sequence of a single hTRAAK diffusing along the boundary between the two phases.
  • Scale bar 2 mm
  • Figure 4C depicts a graph plotting the diffusion coefficient (D) distributions of hTRAAK. D were measured by SPT analysis in POPE:POPG (3: 1 weight ratio, grey) and in DphPC: DSPC: cholesterol (2: 1 :1 molar ratio, red) bilayers showing no significant difference between the two lipid compositions
  • Figure 5A through Figure 5C depicts views of the optomechanical model of the FBM.
  • Figure 5A depicts a top-front view. A magnified view of the excitation optics and the experimental chambers is shown in the box.
  • Figure 5B depicts a top view.
  • Figure 5C depicts a cross-sectional view of the excitation optics and experimental chamber.
  • BE Beam expanders
  • BC Beam (laser) combiner
  • GG Galvo-Galvo scanner
  • L1-L3 Optical relays
  • AM Apodization mask
  • MM movable mirror
  • mM micromanipulator
  • MA manometer
  • MCS multi-camera splitter
  • XO excitation objective
  • DO detection objective
  • FN1 Upright microscope from Nikon (model FN1).
  • Figure 6A through Figure 6C depicts images comparing different modes of illumination of a POPE: POPG (3:1 by weight) bilayer with Rhodamine-DOPE (0.01% w/w).
  • Figure 6A depicts a representative image of epifluorescence illumination.
  • Figure 6B depicts a representative image of a focused-laser illumination with a Gauss (left) and Bessel (right) beam.
  • Figure 6C depicts a representative image of a scanned focused-laser illumination using a scanning amplitude of 50 (left) and 100 (right) mV. Images from Figure 6 A and Figure 6B were taken on the same bilayer, and Figure 6C was on a different one. Scale bars: 10 mm.
  • Figure 7A through Figure 7B depicts a schematic of the optomechanical models of FBM and TIRF chambers.
  • Figure 7A depicts a schematic of the FBM chamber. Amplified views of the FBM chamber depicting the exchangeable cup that separates the top and bottom chamber and the FEP partition where the freestanding bilayer is formed.
  • Figure 7B depicts a schematic of the TIRF chamber. Amplified view of the TIRF chamber used for the SB experiments depicting the quartz coverslip on which SBs were formed and the TIRF cup that contains the imaging buffer. The coverslip is coupled to a Pellin-Broca prism through immersion oil. Arrows are used to represent the directions of the incoming excitation (XO) and detection optics (DO).
  • XO incoming excitation
  • DO detection optics
  • Figure 8A through Figure 8B depicts representative data from single-particle-tracking (SPT) experiments.
  • Figure 8A depicts a graph of the classification of the data on M2R shown in Figure 2B. Results show a significant difference between the dominant type of motion in the FBM (Brownian) and SBs (immobile).
  • Figure 8B depicts a representative graph plotting diffusion coefficients (D) for DOPE-Cy5, Gai and M2R in the FBM. The mean, 25;75 percentile (Box) and 5;95 percentile (bars) are shown overlaying the data. Gai and M2R were labeled with LD655 through Sfp site-directed labeling and with NbALFA-LD655, respectively.
  • Figure 9A through Figure 9C depicts representative data from single-particle-tracking (SPT) experiments.
  • Figure 9A depicts a representative plot of diffusion coefficients (D) vs. anomalous coefficient (a) for GIRK2.
  • Figure 9B depicts a representative plot of diffusion coefficients (D) vs. anomalous coefficient (a) for hTRAAK.
  • Figure 9C depicts a graph of the classification of the tracks shown in Figure 9A and Figure 9B for GIRK2 and hTRAAK. The results indicate that both proteins remain mobile in FBM experiments.
  • GIRK2 and hTRAAK where labeled with NbALFA-LD655 and LD655-NHS, respectively.
  • Figure 10A through Figure 10C depicts representative experiments demonstrating that planar protein aggregates are observed in FBs after proteoliposome fusion.
  • Figure 10A depicts a representative image showing that aggregates of 1-10 mm are routinely observed after vesicle fusion for M2R. Scale bars: 10 mm.
  • Figure 10B depicts a representative image showing that aggregates of 1-10 mm are routinely observed after vesicle fusion for GIRK2. Scale bars: 10 mm.
  • Figure 10C depicts representative images from a time sequence showing the fusion of two planar aggregates of GIRK2. Scale bars: 4 mm.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.
  • Described herein is a freestanding bilayer microscope (FBM) device configured for single-molecule imaging of membrane proteins.
  • FBM bilayer microscope
  • the disclosed device allows for the imaging of planar bilayer membranes, as opposed to supported bilayers, thereby allowing for unconstrained free diffusion devoid of artifacts caused by the presence of a substrate.
  • the disclosed device allows for the use of single-particle tracking methods to study membrane proteins, allowing for a more thorough study of membrane proteins and their interactions within cells.
  • the FBM device 100 generally comprises a microscope having an objective, an imaging platform positioned in view of the objective, an experimental chamber 102 mounted on the imaging platform and configured to hold a membrane, and an illumination system 104 configured to illuminate the membrane.
  • the experimental chamber 102 comprises a body 202 having a cavity therein, wherein the cavity is partially enclosed by a bottom surface of the body 202 and one or more side walls.
  • the experimental chamber 102 further comprises a sample holder 204 (also referred to as “cup”) configured to be positioned within the cavity of the body 202 at a depth less than that of the cavity, thereby forming a lower cavity below the sample holder 204.
  • a first sealed chamber 206 is formed in the lower cavity, enclosed by the bottom surface of the lower cavity and the sample holder 204.
  • the sample holder 204 is configured to hold the membrane.
  • the experimental chamber 102 further comprises a second sealed chamber 208 in a top cavity positioned above the sample holder 204.
  • the bottom surface of the experimental chamber 102 comprises a transparent window.
  • the experimental chamber 102 further comprises an illumination port 210 in the lower cavity, providing access into the lower cavity and configured to fit an excitation objective such that the membrane may be illuminated.
  • the excitation objective is configured to direct illumination to the membrane from the illumination system 104.
  • the experimental chamber 102 further comprises one or more lower ports 212 fluidly connected to the first sealed chamber 206 and configured to control the delivery of fluid into the first sealed chamber 206.
  • the experimental chamber 102 further comprises one or more upper ports 214 fluidly connected to the second sealed chamber 208 and configured to control the delivery of fluid into the second sealed chamber 208.
  • the ports 212, 214 may comprise one or more valves.
  • the ports 212, 214 may further comprise one or more manometers configured to monitor fluid pressure within the first and/or second sealed chambers 206 and 208.
  • the one or more manometers may be utilized to regulate the curvature of the bilayer.
  • the lower and/or upper ports are configured to allow fluid access to the first and/or second sealed chamber, thereby perfusing the membrane positioned in the sample holder.
  • the fluid may be an aqueous solution (such as potassium chloride or sodium chloride) or a buffer solution (such as phosphate buffer, (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid) (HEPES), or Tris).
  • HEPES phosphate buffer
  • Tris Tris
  • changing a pressure of the fluid in the first or second sealed chambers (above or below the membrane) may change the curvature of the membrane positioned in the sample holder.
  • the lower and upper ports 212, 214 are fluidly connected to perfusion lines, wherein the perfusion lines may be fluidly connected at an opposing end to a fluid source, a pump, or a perfusion system.
  • the experimental chamber 102 further comprises at least one electrode positioned within the first sealed chamber 206 and configured to measure a current across the membrane positioned on the sample holder 204. In some embodiments, the experimental chamber 102 further comprises at least one electrode within the second sealed chamber 206 and configured to measure a current across the membrane. In some embodiments, each of the first and second sealed chamber comprise at least one electrode configured to measure the current across the membrane. In some embodiments, the electrodes may be Ag/AgCl electrodes configured to record currents across the membrane to monitor the formation of particle bilayers via an increase in capacitance, and to record the activity of ion channels in the bilayer (Fig. IB).
  • the sample holder 204 is configured to hold a membrane.
  • the membrane is a bilayer, for example a planar lipid bilayer.
  • the membrane comprises fluorinated ethylene propylene (FEP).
  • FEP fluorinated ethylene propylene
  • the membrane may comprise any other material, the material preferably having the characteristics of low autofluorescence, good bilayer forming properties, low refractive index, and transparency.
  • Other materials that may be suitable include other fluoropolymers (such as polytetrafluoroethylene (PTFE)), polymers such as polyethylene terephthalate (PET), polycarbonate (PC), or glass.
  • the membrane comprises pores/perforations.
  • the pores may have a diameter ranging between about 50 pm and 1000 pm, between about 100 pm and 500 pm, between about 100 pm and 300 pm, between about 150 pm and 250 pm, or about 200 pm.
  • the membrane further comprises a lipid solution.
  • the membrane comprises an organic solvent.
  • the organic solvent may be decane or any other alkane liquid at room temperature, or natural oils such as squalene. The interfacial forces between the organic solvent, water and the membrane material lead to the formation of a lipid bilayer at the center of a pore, held in place by a Plateau-Gibbs border of lipid and solvent (torus) (see e.g. Fig. 1A).
  • one or more access ports may allow for buffer perfusion and pressure regulation (see Fig. IB).
  • the access ports 212, 214 may control pressure using a hydrostatic manometer, for example a manometer made from a modified 30 ml syringe and another micromanipulator (see Figs. 5A - 5C).
  • a 3- way valve may be added to the tubing connecting the chamber and the manometer to introduce an electrode, for example an Ag/AgCl electrode when performing electrophysiological experiments.
  • the top part of the chamber may be coupled to the sample holder 204 (or cups), which may be produced via any suitable manufacturing process, for example 3D printing or injection molding (Fig. IB).
  • the sample holder 204 may be mounted to the experimental chamber 102 and/or the membrane may be mounted to the sample holder 204 with vacuum grease (high vacuum grease, Dow Corning) to prevent leaks during electrical measurements.
  • the center of the sample holder 204 may be aligned to the illumination port 210 which may be, for example a cylindrical or conical hole traversing the entire chamber and configured to transmit illumination through the experimental chamber 104 (see Fig. 7A).
  • the bottom of the chamber may be sealed for example with a glass coverslip or any other transparent material known to one of skill in the art.
  • one or more perfusion lines may be mounted with wax on the experimental chamber 102 providing access to the sample holder 204 (see Fig. IB). In some embodiments, these lines may be connected to a perfusion system, which in some embodiments comprises a custom-made manual perfusion system.
  • the experimental chamber 102 and any component parts thereof may be manufactured using any suitable method of manufacturing, including but not limited to 3D printing with resin, injection molding, etc.
  • the experimental chamber 102 and any component parts thereof may comprise any material known to one of skill in the art, for example, but not limited to, plastics, metals, metal alloys, polylactic acid (PLA), polycarbonate, polyether ether ketone (PEEK), polyethylene, and the like.
  • the chamber may be designed to contain and/or orient the excitation objective (XO) through a cylindrical or conically shaped illumination port 210 (Figs. IB and 7A) and may comprise a gasket or a silicon rubber fdm to prevent leakage.
  • the gasket may in some embodiments be a 50A Durometer material or any other water-resistant material known to one of skill in the art.
  • the gasket or the film may be perforated at the center, for example with a 3 mm punch, to allow the access of the excitation objective of the illumination system 104.
  • the experimental chamber may be coupled to the imaging platform such that the experimental chamber 102 may be translatable in space.
  • the imaging platform may comprise a micromanipulator, or linear actuator.
  • the chamber and/or imaging platform may be coupled (see Figs. 1, 5A and 7A) to a right-angle adaptor plate or otherwise fixedly attached to a micromanipulator or other multi-axis precision linear actuator.
  • the linear actuator may have a resolution of 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.5 mm, 0.25 mm, 0.1 mm, or any other suitable resolution.
  • the illumination system 104 comprises an illumination source, one or more optical components, and an excitation objective (XO).
  • the excitation objective may be a long working distance objective.
  • the illumination source may be one or more lasers.
  • the one or more lasers may emit light at wavelengths ranging between 400 nm and 700 nm.
  • three lasers may be utilized having wavelengths of 473 nm, 561 nm, and 660 nm.
  • the illumination is a single laser beam formed via combining the laser beams from the more lasers.
  • the laser beam may have a patterned intensity profde.
  • the laser beam may comprise concentric rings of consecutive high intensity and low intensity light.
  • the laser beam is a Bessel beam or a Gaussian beam.
  • the one or more lasers may pass through one or more optical components before arriving at the objective.
  • the one or more optical components may serve to expand, combine, and focus the one or more lasers into a beam with the desired intensity profde.
  • the one or more optical components may include any optical component known to one of skill in the art, for example, but not limited to, lenses, concave lenses, convex lenses, biconcave lenses, biconvex lenses, beam splitters, mirrors, polarizers, waveplates, prisms, objectives, filters, screens, diffractors, diffusers, gratings, galvo-galvo scanners, apodization masks, and the like.
  • Fig. ID depicts a schematic of an exemplary optical set-up for the illumination system 104.
  • the illumination source may be a scanned focused laser illumination that achieves precise illumination of fluorescent probes (proteins) at the bilayer (Figs. 1C, ID, and 6B). Focused illumination allows observation of single particles on the bilayer at typical frame rates of 50 Hz (Fig. 1 C).
  • a plurality of laser beams are expanded and combined and then focused through a water immersion and long working distance objective at the bilayer and positioned at an angle with respect to the plane of the bilayer (Figs. ID, 5D - 5D).
  • the objective may be positioned at an angle ranging between 10 degrees to 60 degrees, between 20 degrees and 50 degrees, between 25 degrees and 45 degrees, between 30 degrees and 45 degrees, between 35 degrees and 45 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, or about 40 degrees with respect to the bilayer.
  • a galvo-galvo scanner and an apodization mask (AM) may be conjugated to the back focal plane of the excitation objective 304.
  • GG galvo-galvo scanner
  • AM apodization mask
  • a movable mirror may be placed behind the AM to control the angle of illumination (Fig. 1A) by changing the point of illumination on the back focal plane of the excitation objective on the vertical axis.
  • the movable mirror may increase the angle distribution by ⁇ 34 degrees, ⁇ 10 degrees, ⁇ 15 degrees, ⁇ 20 degrees, ⁇ 25 degrees, ⁇ 30 degrees, ⁇ 35 degrees, ⁇ 40 degrees, ⁇ 45 degrees, ⁇ 50 degrees, ⁇ 55 degrees, or ⁇ 60 degrees.
  • the light emitted by fluorophores in the bilayer may be collected by any suitable microscope, including but not limited to a simple microscope, an upright microscope, an inverted microscope, a fluorescence microscope, an epifluorescence microscope, a laser scanning microscope, a confocal microscope, a super-resolution microscope, or a digital microscope.
  • a commercial upright microscope may be used which may in some embodiments be connected to a splitter, for example a 4-camera splitter (see Fig. 5A - 5D).
  • a splitter for example a 4-camera splitter (see Fig. 5A - 5D).
  • water immersion objectives with long working distances may be used.
  • a 25X objective lens with high numerical aperture may be used to detect single molecules at fast frame rates.
  • the objective lens may have a numerical aperture ranging between 0.8 and 1.33.
  • the membrane may be a bilayer, for example a planar lipid bilayer.
  • the method may comprise the step of forming the bilayer, for example over a hole in a substrate such that the bilayer is suspended in the hole in the substrate and enclosing the bilayer within a sealed cavity comprising a fluid on the top and/or bottom of the bilayer.
  • One or more proteins may then be introduced to the bilayer, and the substrate may be mounted within a fixture disposed within the viewing area of a microscope.
  • the fixture may comprise a cavity for introducing an excitation energy source to the membrane, for example a laser or other light source.
  • the excitation energy source may be movable with respect to the membrane, either by moving the substrate or a part of the fixture or the whole fixture relative to the excitation energy source, or by holding the fixture and the substrate stationary and moving the excitation energy source relative to the fixture and the membrane.
  • the fixture may be formed of multiple parts, and a method may comprise assembly of the fixture and/or positioning the excitation energy source such that the excitation energy illuminates one or more proteins in the membrane.
  • a method may comprise deforming the membrane via perfusion, for example via the controlled increase or decrease of pressure on the top or bottom of the membrane via controlled introduction of perfusion fluid to a sealed chamber enclosing the membrane on one or both sides.
  • Perfusion fluid may be introduced via one or more ports.
  • Example 1 FBM: Freestanding bilayer microscope for single-molecule imaging of membrane proteins.
  • Integral membrane proteins are fundamental elements of cell signaling and energy metabolism. Yet, they remain incredibly difficult to study due to their low expression levels and the complexity of their native environments. Current techniques cannot fully address the dynamics of proteins in membranes; how they interact with themselves and others, and how their motion and distribution are affected by the environment.
  • FBM Freestanding Bilayer Microscope
  • the FBM is based on planar bilayers (PB), a proven, effective method for the fully functional reconstitution of different membrane proteins utilized in different laboratories worldwide. This offers versatility as the FBM setup can easily be combined with other PB-based assays. This was demonstrated by simultaneous imaging and electrical recordings of the mechanosensitive channel hTRAAK. hTRAAK is located in the Nodes of Ranvier of myelinated neurons of mammals, and it has been shown to play an important role in thermal and mechanical nociception (Noel, J. et al., EMBO J. 2009;28, 1308-1318). Determining the open probability, Po, of this channel has been challenging owing to its flickery single-channel kinetic behavior.
  • PB planar bilayers
  • the FB microscope will be an important tool to elucidate the mechanisms of membrane protein organization and dynamics. For example, the information contained in single-particle tracking can be used to study protein-protein interactions in the membrane. Combined with the unique compositional control offered by the planar bilayers, this could lead to new insights into signaling mechanisms that depend on dynamic complex formation, such as G-protein coupled receptors, receptor tyrosine kinases, or immune receptor pathways. Furthermore, the possibility of combining the FBM with smFRET offers the possibility to study structural changes of proteins as a function of interactions with other species in a reaction-diffusion setting, something that is today only possible with simulations.
  • the FBM provides a good starting point to test these hypotheses experimentally.
  • the FBM is a reductionist system that will permit controlled, stepwise building in of complexity so that users may eventually understand the properties of diffusion in cell membranes, and ultimately the signaling processes that rely on cell membrane diffusion.
  • An ALFA-tag was introduced at the C terminus of the human muscarinic 2 receptor (hM2R) gene, followed by a 3C PreScission Protease (PPX) recognition site, the eYFP gene, and a polyhistidine tag (HislO-tag) (M2R-ALFA).
  • the ALFA-tag was used for binding with an antiALFA nanobody (ALFANb) conjugated to a fluorophore for single molecule visualization, while the eYFP and HislO-tags were inserted for purification purposes (Gotzke, H., et al., Nat. Commun. 2019; 10, 4403).
  • the gene was then cloned into a pFastBac vector for protein expression with the Bac-to-Bac Baculovirus System.
  • Spodoptera frugiperda Sf9 insect cells were transfected with isolated bacmid DNA to produce a recombinant baculovirus stock.
  • the virus was amplified through three rounds of progressive infection of Sf9 cells to create a high-titer stock used for large-scale expression of M2R-ALFA.
  • Six liters of Sf9 cells were cultured to a density of 3 million cells per mL and infected with 20 mL of baculovirus stock.
  • the cells were grown for 48 hours post-infection at 27°C shaking at 120 rpm, after which cells were pelleted by centrifugation at 3,500g for 15 minutes at 4°C. Cell pellets were resuspended in lx phosphate- buffered saline (lx PBS; Thermo Fisher Scientific), and centrifuged at 3,500g for ten minutes at 4°C. Pellets were flash-frozen in liquid nitrogen and stored at -80°C until use.
  • lx phosphate- buffered saline lx PBS; Thermo Fisher Scientific
  • a short peptide tag, Al was cloned at the N terminus of the hM2R gene to enable postpurification AcpS phosphopantetheinyl transferase (PPTase) catalyzed site-specific protein labeling (A1-M2R) (Zhou, Z., et al., ACS Chem. Biol. 2007;2, 337-346).
  • PPTase AcpS phosphopantetheinyl transferase
  • A1-M2R site-specific protein labeling
  • the PPX recognition site, eYFP gene, and HislO-tag were included for purification purposes.
  • A1-M2R was expressed as described for M2R-ALFA.
  • M2R constructs (M2R-ALFA and A1-M2R) were purified following the same protocol (Falzone, M. E., et al., Proc. Natl. Acad. Sci. 2023;120, e2301121120). All purification steps were carried out at 4°C unless specified otherwise.
  • Sf9 cells were lysed by osmotic shock, by resuspending the pellets in the following buffer for 30 minutes: 10 mM Tris HC1 pH 7.4, 10 mM MgC12, 5 units per m benzonase nuclease, 5 mM 0-mercaptoethanol (0ME), 2 mM phenylmethylsulfonyl fluoride (PMSF), lx of 3-7 TIU/mg aprotinin saline solution, and 0.1% v/v of protease inhibitor cocktail (PIC) stock (1 mg/mL leupeptin, 1 mg/ML pepstatin A, 1 M benzamidine HC1).
  • PIC protease inhibitor cocktail
  • lysis slurry was centrifuged at 30,000g for 20 minutes. Once the supernatant was removed, cell pellets were resuspended in high salt buffer (20 mM HEPES pH 7.4, 750 mM NaCl, 10% (v/v) glycerol, 5 mM 0ME, 5 units per mL benzonase nuclease, 5 pM atropine, lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of PIC stock) and homogenized using a dounce homogenizer.
  • high salt buffer (20 mM HEPES pH 7.4, 750 mM NaCl, 10% (v/v) glycerol, 5 mM 0ME, 5 units per mL benzonase nuclease, 5 pM atropine, lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of
  • IMPs were extracted by solubilizing the membrane with l%:0.05% n-dodecyl-0-D-maltopyranoside: cholesterol hemisuccinate (DDM:CHS) for one hour at room temperature, followed by one hour at 4°C. After extraction, insoluble material was pelleted by centrifugation at 30,000g for 30 minutes. The supernatant was bound to TALON resin and washed with buffers (20 mM HEPES pH 7.4, 0. l%:0.01% DDM:CHS, 1 pM atropine, ImM 0ME, 1 pM atropine, 0.1% v/v of PIC stock) containing a gradual increase in NaCl concentration.
  • buffers (20 mM HEPES pH 7.4, 0. l%:0.01% DDM:CHS, 1 pM atropine, ImM 0ME, 1 pM atropine, 0.1% v/v of PIC stock
  • the resin was washed with five column volumes (CV), referring to the volume of resin used, of wash buffer containing 750 mM and 587 mM NaCl, respectively.
  • CV column volumes
  • the resin was washed with five CVs of wash buffer with 424 mM NaCl and 20 mM imidazole.
  • M2R was eluted with 261 mM NaCl and 200 mM imidazole wash buffer.
  • the elution fractions were pooled and subsequently diluted tenfold with the following dilution buffer in order to lower the imidazole concentration and introduce the superagonist iperoxo: 20 mM HEPES pH 7.4, 100 mM NaCl, 0.1%:0.01% DDM:CHS, 0.1 mM TCEP, 12 pM iperoxo.
  • the protein was then bound to GFPNb resin for 1 hour.
  • the protein was buffer exchanged by washing the resin on the gravity column with 20 CV of buffer containing the same components as the dilution buffer, except for an increased iperoxo concentration of 50 pM. PPX was added to the resin.
  • the protein was nutated overnight to ensure complete digestion and to change the ligand bound to M2R from the antagonist atropine to iperoxo.
  • the cleaved M2R was eluted by collecting the flow through of the GFPNb resin by gravity.
  • A1-M2R was labeled with two-fold excess LD655-CoA dye in the presence of 10 mM MgC12, 2 mM TCEP, and a 1 :5 ratio of AcpS (expressed and purified in-house) to A1-M2R.
  • the enzyme catalyzed fluorophore conjugation reaction was allowed to proceed in the dark for 2 hours at room temperature, followed by 2 hours at 4°C. A labeling efficiency of -10% was attained using this method.
  • M2R-ALFA was fluorescently labeled through binding with NbALFA-LD655 after vesicle fusion into freestanding bilayers.
  • M2R constructs were dephosphorylated through a 30-minute room temperature treatment with 200 units of lambda protein phosphatase (NEB), 10 units of calf intestinal phosphatase (NEB), 5 units of antarctic phosphatase (NEB), and 1 mM manganese chloride. Proteins were concentrated in 30 kDa MWCO concentrators prior to their respective purification by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column and running buffer (20 mM HEPES pH 7.4, 100 mM KC1, 50 mM NaCl, 10 pM iperoxo, 100 pM TCEP, 0.1 %:0.01 % DDM:CHS). M2R reconstitution in lipid vesicles was done immediately after gel filtration. Purified M2R that was not reconstituted was flash frozen with 10% glycerol and stored at -80°C.
  • the short peptide tag, S6 was inserted in an internal loop between amino acids 112 and 113 of the human Gai l gene (Zhou, Z., et al., ACS Chem. Biol. 2007;2, 337-346).
  • the S6 tag was inserted for site-specific protein labeling via the Sfp PPTase enzyme catalyzed peptide tag modification.
  • the Gail-S6 gene was cloned into the pFastBac vector. Genes for the wild-type human G
  • a HislO-eYFP linked to a PPX recognition site was inserted.
  • Recombinant baculovirus for each subunit was prepared separately as described above.
  • For large-scale expression of the heterotrimeric G protein six liters of Trichoplusia ni High Five insect cells were cultured to a density of 2 million cells per mL. Each liter of cells was coinfected with an experimentally determined ratio of Gail, G
  • the cells were grown for 36-48 hours and then harvested by centrifugation at 3,500g for 15 minutes at 4°C. Cell pellets were resuspended in lx PBS and centrifuged at 3,500g for 10 minutes. Pellets were flash-frozen and stored at -80°C until further use.
  • the extraction was centrifuged at 35,000g for one hour. The supernatant was bound to GFPNb resin and then washed with 10 CVs of wash buffer (20 mM Tris HC1 pH 8, 100 mM NaCl, 1% Na-cholate, 3 mM MgC12, 20 pM GDP) on column.
  • wash buffer (20 mM Tris HC1 pH 8, 100 mM NaCl, 1 % Na cholate, 50 mM MgC12, 30 pM GDP, 10 mM NaF, 30 pM A1C13) was used to elute Gail-S6 from the Gbg complex bound to the GFPNb resin.
  • the resin was moved to room temperature, and the elution buffer was warmed in a 30°C water bath for 15 minutes; GDP was added to the elution buffer afterward.
  • the resin and elution buffer were incubated together, nutating for 30 minutes at room temperature, and Gail-S6 alone was collected by gravity flow.
  • the eluate containing Gail-S6 was labeled using two-fold molar excess LD655-CoA dye, Sfp enzyme that was purified inhouse (1 :2 ratio of enzyme to protein), and 10 mM MgC12. A labeling efficiency of -10% was obtained.
  • Gail was dephosphorylated through a 30 minute room temperature treatment with 200 units of LPP, 10 units of CIP, 5 units of AP, and 1 mM manganese chloride.
  • the protein was concentrated and purified using a Superdex 200 Increase 10/300 GL column (20 mM Tris HC1 pH 7, 150 mM KC1, 10 mM DTT, 0.4 mM TCEP, 1% Na cholate, 2 mM MgC12, 20 pM GDP). Fractions containing LD655-Gai were collected and reconstituted into lipid vesicles.
  • Unreconstituted protein was flash-frozen with 10% glycerol and stored at -80°C. hTRAAK expression and purification
  • the human mechanosensitive potassium channel TRAAK (hTRAAK) gene was cloned into a modified pPICZ-B vector to create a fusion protein construct containing a C terminal PPX cleavage site, the GFP gene, and HislO-tag.
  • the hTRAAK protein construct was then transformed into P. pastoris strain SMD11-63H and expressed in large scale cultures as described previously (Brohawn, S. G., et al., Science 2012;335, 436-441). Cell pellets were flash frozen in liquid nitrogen and subsequently disrupted by milling 5 times for 3 minutes at 25 Hz. Lysed cells were frozen and stored at -80°C until they were ready for purification.
  • the resin was washed with 10 CV of wash buffer (20 mM HEPES pH 8, 150 mM KC1, 0.1% DDM).
  • wash buffer (20 mM HEPES pH 8, 150 mM KC1, 0.1% DDM).
  • PPX was added to the resin in a 1 :20 protease to protein ratio, and the protein was digested overnight.
  • the flow through containing cleaved TRAAK was collected from the resin and was concentrated using a 10 kDa MWCO concentrator.
  • TRAAK was non-specifically labeled using amine-reactive LD655-NHS dye.
  • the protein was buffer exchanged using a PD-10 column into lx PBS without calcium and magnesium at pH 8. 50-fold molar excess LD655-NHS dye was added to the protein solution; protected from light, the reaction was allowed to proceed overnight at 4°C. The protein was labeled with an efficiency of 15%.
  • Labeled TRAAK was concentrated using a 10 kDa MWCO concentrator and subsequently purified using a Superdex 200 Increase 10/300 GL column (20 mM TrisHCl pH 7.4, 150 mM KC1, 1 mM EDTA, 0.03% DDM). Fractions containing LD655-TRAAK were reconstituted.
  • a PPX cleavage site, eGFP gene, and His 10 tag were preceded by an ALFA-tag.
  • the fusion protein construct was cloned into the pBacMam vector and the BacMam system was used to create a high titer recombinant baculovirus stock using Sf9 cells.
  • Four liters of the HEK293S GnTI- strain from ATCC (CRL-3022) were cultured to a density of 3.5 million cells per mL and infected with 100 mL of the high titer GIRK2-ALFA recombinant baculovirus.
  • the cells were induced with 10 mM sodium butyrate. After shaking at 37°C for an additional 40-48 hours, the cells were harvested by centrifugation at 3,500g for 15 minutes at 4°C. Cell pellets were resuspended in lx PBS and centrifuged at 3,500g for ten minutes at 4°C. The supernatant was discarded. Cell pellets were flash frozen and stored at -80°C.
  • GIRK2-ALFA Purification of GIRK2-ALFA was carried out at 4°C unless noted otherwise.
  • Cell pellets were first mixed in resuspension buffer (25 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 1 mM MgC12, 1 mM CaC12, 2 mM DTT, 2 mM PMSF, DNase I, lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of PIC stock) for 15 minutes.
  • the cell slurry was homogenized (Dounce), and the lysate was centrifuged at 39,000 xg for 15 minutes. The supernatant was discarded.
  • Pellets were suspended in buffer (25 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 1 mM MgC12, 1 mM CaC12, 2 mM DTT, lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of PIC stock) and homogenized (Dounce).
  • buffer 25 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 1 mM MgC12, 1 mM CaC12, 2 mM DTT, lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of PIC stock
  • DTT lx of 3-7 TIU/mg aprotinin saline solution, 0.1% v/v of PIC stock
  • Membranes were extracted with 1 ,5%:0.3% DDM:CHS for 2 hours. The extraction was centr
  • the resin was washed on column with 20 CVs of wash buffer (20 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 2 mM DTT, 0.05%:0.01% DDM:CHS).
  • wash buffer (20 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 2 mM DTT, 0.05%:0.01% DDM:CHS).
  • PPX was added to the resin slurry for overnight digestion.
  • the flowthrough containing cleaved GIRK2-ALFA was collected from the GFPNb resin by gravity flow.
  • the protein was concentrated using a 100 kDa MWCO concentrator and purified of contaminants using a Superose 6 Increase 10/300 GL column with the buffer: 20 mM Tris HC1 pH 7.5, 150 mM KC1, 50 mM NaCl, 10 mM DTT, ImM Na2EDTA pH 7.4, 0.025%:0.005% DDM:CHS.
  • GIRK2-ALFA was reconstituted into liposomes after gel filtration.
  • the GIRK2-ALFA construct was fluorescently labeled with NbALFA-LD655 after vesicle fusion into freestanding bilayers.
  • the NbALFA was expressed as an N terminal Hisl4-SUMO fusion in E. coli cells (Gotzke, H., et al., Nat. Commun. 2019; 10, 4403).
  • the recombinant plasmid was transformed into One Shot BL21 Star (DE3) cells.
  • a single colony was used to inoculate small-scale cultures: 50 mL of lysogeny broth (LB) with 50 pg/mL of kanamycin. The cells were grown overnight ( ⁇ 18 hours) at 37°C shaking at 225 rpm.
  • a cell pellet was suspended in lysis buffer (20 mM HEPES pH 7.9, 300 mM NaCl, 2 mM PMSF, DNasel, and 1 mM TCEP, 2x of 3-7 TIU/mg aprotinin saline solution, 0.2% v/v of a protease inhibitor mixture stock [0.1 g/mL trypsin inhibitor, 1 mg/mL pepstatin A, 1 mg/mL pepstatin A, 1 M benzamidine HC1, 0.5 M AEBSF] and mixed for 15 minutes.
  • lysis buffer (20 mM HEPES pH 7.9, 300 mM NaCl, 2 mM PMSF, DNasel, and 1 mM TCEP, 2x of 3-7 TIU/mg aprotinin saline solution, 0.2% v/v of a protease inhibitor mixture stock [0.1 g/mL trypsin inhibitor, 1 mg/mL pepstatin A, 1 mg/mL
  • the cells were lysed by sonication and the lysate was clarified by centrifuging at 16,500 rpm for 40 minutes at 4°C.
  • the supernatant was bound to equilibrated Ni-NTA resin for one hour and washed with wash buffer (20 mM HEPES pH 7.9, 300 mM NaCl, and 1 mM TCEP) containing no imidazole followed by 20 mM imidazole.
  • wash buffer (20 mM HEPES pH 7.9, 300 mM NaCl, and 1 mM TCEP) containing no imidazole followed by 20 mM imidazole.
  • ALFANb was eluted off the resin with 400 mM imidazole containing wash buffer.
  • the Hisl4- SUMO tag was cleaved of the protein by adding ULP1 (prepared in-house) to the elution.
  • the NbALFA-protease solution was placed in 8 kDa MWCO dialysis tubing and dialyzed overnight at room temperature against a buffer composed of 20 mM HEPES pH 7.9, 300 mM NaCl, 0.5 mM TCEP, and 2 mM DTT.
  • the insoluble precipitate was removed by centrifuging the protein for 10 minutes at 3,500g.
  • the supernatant was run through Ni-NTA resin equilibrated in wash buffer containing 10 mM imidazole in order to collect cleaved NbALFA.
  • the digested protein was concentrated with a 10 kDa MWCO concentrator and purified using a Superdex 75 10/300 GL column (20 mM HEPES pH 7.9, 150 mM NaCl, 1 mM TCEP). Purified NbALFA was stored at -80°C.
  • the NbALFA was mixed with five-fold molar excess LD655-MAL and reacted overnight in the dark at 4°C.
  • the protein was run on the Superdex 75 10/300 GL column equilibrated with 20 mM HEPES pH 7 and 150 mM NaCl. A labeling efficiency of ⁇ 80% was achieved; NbALFA-LD655 was stored at -80°C.
  • Membrane Proteins were reconstituted into liposomes immediately after labeling (if applicable) and purification. A given protein was reconstituted using a specific mixture of phospholipids. The following general protocol to dry lipid films was repeated for all lipid combinations used to reconstitute the proteins discussed in this paper. The desired ratio of lipids in chloroform was mixed in a glass vial, and the chloroform was evaporated under a steady stream of argon gas. The lipid film was solubilized in a small amount of pentane and the solvent was again evaporated under a steady stream of argon. The lipid film was thoroughly dried by storage in a vacuum desiccator overnight.
  • M2R-ALFA was reconstituted into a 3: 1 weight ratio of 1- palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE): l-palmitoyl-2-oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol) (POPG) lipids.
  • POPE palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine
  • POPG l-palmitoyl-2-oleoyl-sn- glycero-3-phospho-(l'-rac-glycerol)
  • PLR protein to lipid ratio
  • biobeads Bio-Beads SM-2 Adsorbent, Bio-Rad
  • Bio-Rad Bio-Rad
  • 20 mM HEPES pH 7.4, 100 mM KC1, 50 mM NaCl, 10 pM iperoxo, and 100 pM TCEP were added to the protein- lipid mixture so that the dry volume of beads was roughly one-third the volume of vesicles.
  • the mixture was moved to nutate at 4°C and biobeads were changed every 8-12 hours for a total of 3- 4 changes.
  • the proteoliposomes were harvested, flash frozen and stored at -80°C.
  • A1-M2R was reconstituted into a 7:3 weight ratio of l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC): POPG lipids for SB experiments.
  • DOPC dioleoyl-sn-glycero-3- phosphocholine
  • the same reconstitution process and buffers were used as described above for M2R-ALFA.
  • the protein was protected from light during reconstitution to prevent bleaching of the LD655 fluorophore conjugated to the receptor.
  • Gail-S6 was reconstituted into a 3: 1 weight ratio of POPE:POPG lipids.
  • the lipids were brought to a concentration of 10 mg/mL using 20 mM Tris HC1 pH 8, 150 mM KC1, 2 mM MgC12 and sonicated to clarity in a water bath sonicator, followed by addition of 1% DM and room temperature incubation for 30 minutes. After more sonication, a mixture with 0.405 mg/mL Gai 1-S6 and 5 mg/mL lipids was made for a PLR of 1 : 12. The mixture was nutated for one hour at room temperature in the dark. Dialysis was then used to remove the detergent.
  • the mixture was placed in 10 kDa MWCO dialysis tubing, and the buffer (20 mM Tris HC1 pH 8, 150 mM KC1, 20 mM MgC12, and 5 mM DTT) was exchanged five times every 12 hours, adding fresh DTT each time.
  • Proteoliposomes were harvested, flash frozen and stored at -80°C.
  • GIRK2-ALFA was reconstituted into a 3 : 1 weight ratio of POPE:POPG lipids. Lipids dried overnight were made 20 mg/mL using buffer (20 mM Tris HC1 pH 7.5, 150 mM KC1, and 50 mM NaCl). Lipids were sonicated to clarity, and 1% DM was added. After lipids were incubated at room temperature for 30 minutes, they were sonicated again and combined with 2 mg/mL GIRK2-ALFA in equal parts to create a final PLR of 1 : 10.
  • the vesicles were placed in dialysis tubing of 50 kDa MWCO and dialyzed in 10 mM K2HPO4 pH 7.4, 150 mM KC1, and 1 mM K2EDTA.
  • the buffer was exchanged five times every 12 hours, adding fresh DTT for each change.
  • equilibrated biobeads were included in the buffer.
  • Proteoliposomes were aliquoted, flash frozen and stored at -80°C.
  • TRAAK was also reconstituted into a 3: 1 weight ratio of POPE:POPG. Lipids were dissolved at 10 mg/mL in 20 mM Tris HC1 pH 7.4 and 150 mM KC1 buffer. The lipids were sonicated to clarity. 1% DM was added, and the lipid mixture was nutated at room temperature for 30 minutes, followed by further sonication. A PLR of 1 : 10 was achieved by combining channel and lipids for a final concentration of 0.5 mg/mL and 5 mg/mL, respectively.
  • the vesicles were dialyzed in 30 kDa MWCO tubing using 10 mM Tris HC1 pH 7.4 and 150 mM KC1 buffer containing equilibrated biobeads. The buffer was exchanged every 12 hours for a total of three times. The vesicles were then removed from dialysis tubing and mixed directly with biobeads for 6 hours prior to being harvested, flash frozen, and kept at -80°C until use.
  • Cameras were connected to a workstation (Dual Xenon 8-core, 128Gb of memory, a Titan XP video card, and 2 hard drives, 4x NVME Itb and 8x HDD 8tb) through a Camera Link communication protocol (V3 Firebird Camlink Board). Data acquisition and storage were done with commercial software (NIS- Elements AR 5.11, Nikon Instruments). For transmitted light, a manually controlled quartz halogen lamp was housed in the microscope (FN-LH, Nikon Intruments) ( Figure ID).
  • SPECTRA X Light engine Lumencor
  • excitation filters 395/25, 440/20 ,270/24, 510/25, 550/15, 575/25 and 640/30, Chroma Technology
  • CFI75 Apochromat Multi-Photon LWD 25X, Water immersion, Nikon Instruments
  • the combined beam was fed into a 4 mm galvometer-galvometer scanner (LSKGG4/M, Thorlabs) ( Figure ID, Figure 5).
  • a laser shutter was placed before the GG scan head for fast laser gating (LS6, Uniblitz Electronics).
  • the lasers were operated by SMD12 PSU (Novanta Photonics) controllers connected to the workstation through a PXI serial interface module (PXI- 8430/4, National instruments) housed in a PXI chassis (NI PXI-1033, National instruments).
  • the GG scan head was, in turn, operated by a GG controller (Thorlabs) driven by a user-defined signal (DC or sine wave) through a BNC analog output (BNC-2110, National Instruments) also housed in the PXI chassis by an analog output module (PXI-6723, National Instruments).
  • BNC-2110 National Instruments
  • PXI-6723 National Instruments
  • An inhouse written software in Lab View was used to control the lasers, the shutter, and the GG.
  • the output of the GG was conjugated to an apodization mask (Annular Mask, Photo Sciences) (Planchon, T. A., et al., Nat.
  • L2 and L3 adaptor plates were, in turn, mounted on a modified breadboard (MB2530/M, Thorlabs) designed to raise and orient the plane of excitation optics orthogonal to the detection optics plane ( Figure 5).
  • All custom- made parts, as well as the optomechanical models of the microscope, were designed using computer-assisted design software (Inventor Profesional, AutoDesk). All machined parts were fabricated in Aluminum 6061 by a manufacturing service (Xometry) following our designs.
  • Electrophysiology set-up For electrical recordings, the voltage across the lipid bilayer was clamped with an amplifier in whole-cell mode (Axopatch 200B, Axon Instruments) by two Ag/AgCl electrodes placed on the cup and in the manometer line (see previous section). The analog current signal was filtered at 1 kHz (low-pass, Bessel) and digitized at 10 kHz (Digidata 1550B digitizer, Molecular Devices). Digitized data were recorded on a computer using the software pClamp (Molecular Devices) and analyzed using Clampfit (Molecular Devices).
  • FEP sheets Fluorinated Ethylene Propylene Copolymer - Film, 0.075 mm, Goodfellow
  • FEP sheets Fluorinated Ethylene Propylene Copolymer - Film, 0.075 mm, Goodfellow
  • BD10AS Electro Technic Products
  • Figure 1 A Changing the duration and intensity of the spark led to hole sizes ranging from 100 to 500 mm.
  • Perforated partitions were sonicated for 30 seconds and stored in ethanol for up to five days.
  • PBs Planar bilayers
  • PLs containing the IMPs of interest were mixed with an equal volume of IM KC1, briefly sonicated (1 second), added on top of the bilayer, and left to sink for 2 minutes before adding 1 ml of 3M KC1 to induce the fusion of vesicles on the bilayer. Unfused PLs were washed away by perfusing 5 ml of IB. For PLs containing Gail KC1 induction was not necessary as we found that G-proteins can diffuse from liposomes into the freestanding bilayer.
  • quartz coverslips were plasma-cleaned with oxygen as a processing gas for 0.5-1 minute. Pegylation of Quartz coverslips was done as explained in Chandradoss et al. (Chandradoss, S. D., et al., J. Vis. Exp. 2014;50549). Briefly, quartz coverslips were cleaned by dipping them in different cleaning solutions, in the order: acetone, IM KOH, and piranha (H2SO4:H2O2 3:1), followed by washes with deionized water.
  • coverslips were placed in a staining jar with 100 ml of methanol, 5 ml acetic acid, and 3 ml of 3 -aminopropyl trimethoxysilane (APTES). After 30 minutes, the solution was replaced by fresh methanol, and this was repeated 3 times. The coverslips were then dried with Nitrogen gas and incubated with a 0.1 M sodium bicarbonate (pH 8.5) solution with 0.6 mM biotinylated NHS-ester PEG (5,000 Da) and 25 mM NHS-ester mPEG (5,000 Da) for 3-5hs. Finally, coverslips were rinsed with deionized water, dried with Nitrogen gas, and stored at - 20°C until use. We should note that pegylated quartz coverslips showed fewer fluorescent particles than the other methods, implying that pegylation prevented SB formation to some degree.
  • APTES 3 -aminopropyl trimethoxysilane
  • Mica coverslips were prepared by coupling a mica sheet to a quartz coverslip with optical glue as described elsewhere (Matysik, A., et al., J. Vis. Exp. 2014;e52054). Briefly, coverslips were cleaned with 2% detergent (Hellmanex III, Hellma Analytics) and ethanol before gluing previously cut mica leaflets (5 mm x 5 mm, 1872-CA, SPI) on top of them with a low viscosity optical adhesive (NOA60, Norland products). After curing the adhesive with one hour exposure to UV light, another coverslip was glued on top of the mica with a high-viscosity optical adhesive (NOA63, Norland products), followed by another round of UV exposure. Coverslips were separated to expose a freshly cleaved mica surface just before performing SB experiments.
  • lipid bilayers were used to match the composition of hTRAAK liposomes. Liposomes were added on top of the bilayers and left to sink and fuse as described before, with the following differences: liposomes were diluted 50 times with IB followed by a 2-fold dilution with IM KC1 to a final concentration of 0.1 mg/ml, and hTRAAK vesicles were allowed to sink for only 30 s before inducing fusion with KC1. This is because hTRAAK-containing liposomes have a stronger proclivity to fuse than all the other proteins we studied.
  • the top chamber was perfused with 5 ml to remove unfused vesicles. Imaging using a scanned-focused laser was then performed as described above. Currents under voltage clamp were measured during the whole procedure.
  • videos were cropped to a region of the bilayer with homogeneous focus and illumination. Then, uTrack software was used to detect particles within the cropped videos. The number of detections was plotted as a function of time (frames) for each video to estimate the bleaching rate ( Figure 11). Then, the average number of particles from different frames was calculated up to a frame that showed less than 5% reduction on the number of particles.
  • the number of detections was divided by the area of the (cropped) frames, which was calculated from the coordinates of the farthest detected particles, to obtain the density of detections. This density was then multiplied by the area of the bilayer, measured using the ROI area measurement feature on the NIS-elements software, and by a correction factor (LR) for the labeling efficiency (15%) to obtain the total number of channels (N) on the bilayer. Potassium currents were measured at 50 mV and divided by N and the single channel current for hTRAAK at that voltage (3.6 pA) to obtain the Po.( Sorum, B., et al., Proc. Natl. Acad. Sci. 2021) hTRAAK tension titration experiments were done as described previously without any modification. ( Perez-Mitta, G., et al., Proc. Natl. Acad. Sci. 2023) We note an important assumption in the determination of Po is that incorporated TRAAK channels are functional.
  • the lipid mixture was dried under Argon gas and left under vacuum for at least 2 hours before resuspending into a mixture of Decane: Butanol (9: 1) to a final concentration of 22 mg/ml.
  • PBs were formed as described above.
  • hTRAAK trajectories were obtained using uTrack software and were overlayed on the first frame of the lipid channel using MatLab (MathWorks). Experiments were done in IB at room temperature.
  • SBs were formed on three different substrates to account for any variability of the chosen substrate on the system: mica, quartz, and pegylated quartz (Loose, M., et al., Science 2008;320, 789-792; Castellana, E. T., et al., Surf. Sci. Rep. 2006;61, 429-444). Fluorescently labeled M2R was reconstituted in liposomes of DOPC: POPG (7:3) (Ramm, B., et al., J. Vis. Exp.
  • Liposomes were added to the substrate (coverslip) and incubated at 37 degrees to induce bursting and bilayer formation, followed by extensive washing before imaging by TIRF illumination.
  • the FBM experimental chamber was replaced with a prism-containing chamber ( Figure 7B).
  • the coverslips were then mounted on the prism and imaged using the scanned focused laser of the FBM at total internal reflection. Videos were recorded at 8-10 Hz.
  • an ALFA-tag was introduced at the C-terminus of M2R to render it amenable to effective and non-invasive fluorescent labeling after fusion to the planar bilayer (Gbtzke, H., et al., Nat. Commun. 2019; 10, 4403).
  • M2R was reconstituted in POPE:POPG (3: 1) liposomes and fused into previously formed freestanding bilayers of the same lipid composition. After fusion, the top chamber was perfused with 5-10 chamber volumes to remove unfused vesicles, followed by the addition of 1 nM anti-ALFA nanobody (NbALFA) labeled with LD655. To remove excess NbALFA-LD655 the chamber was perfused with another 10 chamber volumes. The bilayers were then imaged using focused beam illumination, and videos were recorded at 50 Hz.
  • NbALFA anti-ALFA nanobody
  • FRAP was used to examine other solid support preparations (piranha-cleaned Quartz and SB formation on top of a Langmuir-Blodgett transferred lipid monolayer or over a transferred bilayer) to explore conditions that would possibly allow free diffusion (Kalb, E., et al., Biochim. Biophys. Acta BBA - Biomembr.
  • Determining ion channel open probability can usually be accomplished by measuring single channels or performing noise analysis on recordings with multiple channels, so called macroscopic current recordings (Alvarez, O., et al., Adv. Physiol. Educ. 2002;26, 327- 341).
  • macroscopic current recordings Alvarez, O., et al., Adv. Physiol. Educ. 2002;26, 327- 341.
  • the single-channel kinetics of TRAAK and its very low open probability which renders a linear relationship between current variance and mean, preclude a good estimation of Po by either of the aforementioned methods.
  • hTRAAK was purified, labeled, and reconstituted in POPE:POPG (3: 1, weight ratio) liposomes. After liposome fusion into FBs of the same lipid composition, videos of diffusing channels were recorded and SPT analysis was performed as described above. From this analysis, the density of detected particles was calculated within a region of the bilayer with homogeneous focus and illumination and the number of hTRAAK in the entire bilayer was extrapolated after correcting for the labeling efficiency and subsequent multiplying by the total bilayer area (Figure 3D).
  • Tinevez, J.-Y. et al. TrackMate An open and extensible platform for single-particle tracking. Methods 115, 80-90 (2017).

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Abstract

Le microscope à bicouche autonome (FBM) utilise un éclairage laser focalisé balayé qui réalise un éclairage précis de sondes fluorescentes au niveau d'une bicouche lipidique autonome formée dans une chambre expérimentale positionnée sur la plateforme d'imagerie du FBM.
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