WO2025160387A1 - Microscope for freestanding bilayer illumination - Google Patents

Abstract

The Freestanding-Bilayer Microscope (FBM) utilizes scanned focused laser illumination that achieves precise illumination of fluorescent probes at a freestanding lipid bilayer formed in an experimental chamber positioned on the imaging platform of the FBM.

Description

MICROSCOPE FOR FREESTANDING BILAYER ILLUMINATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/624,684 filed January 24, 2024, the contents of which are incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Integral membrane proteins (IMPs) 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). However, many dynamic features of IMPs remain enigmatic and controversial. For example, 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). Simultaneously, IMPs can interact with themselves and other proteins and adopt non-homogeneous distributions across membranes (see Lillemeier, 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.

[0003] Explaining these phenomena requires curtailing some of the intrinsic complexities of the cellular environment. This can be achieved with experimental setups that allow the study of IMPs in membranes of controlled composition with single-particle tracking (SPT) (see Wallin, E. et al., Protein Sci., 1998; Kusumi, A. et al., Curr. Opin. Cell BioL, 1996; Jaqaman, K. et al., Trends Cell Biol., 2012). SPT permits the study of the diverse aspects of IMP dynamics, such as anomalous diffusion, dynamic heterogeneity, and protein-protein interactions, by obtaining the full information contained in particle positions over the course of a time-lapse experiment (see Metzler, R., et al., Phys. Chem. Chem. Phys., 2014; Scott, S. et al. Phys. Chem. Chem. Phys., 2023; Vink, J. N. A., et al., Biophys. J. 1970-1983).

[0004] Existing methods to study membrane proteins in isolation from their cellular environments can be classified into supported bilayer (SB) and freestanding bilayer (FB) techniques. 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). Despite being easier to implement, these methods are inadequate for the study of IMPs because proteins are strongly affected by the presence of the substrate (see Rojko, N. et al. Biophys. J., 2014; Muller, D. J. et al., J. Mol. Biol., 2003). 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).

[0005] Thus, there is a need in the art for novel compositions and methods for illumination of freestanding bilayers. This invention addresses this unmet need.

SUMMARY OF THE INVENTION

[0006] In one embodiment, 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.

[0007] In one embodiment, 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. [0008] In one embodiment, the sample holder further comprises an upper cavity positioned over the sample holder, forming a second sealed chamber with the sample holder.

[0009] In one embodiment, the microscopy device further comprises at least one electrode positioned in the upper or lower cavity, configured to measure a current across the membrane.

[0010] In one embodiment, the microscopy device comprises a first electrode positioned in the upper cavity and a second electrode positioned in the lower cavity.

[0011] In one embodiment, 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.

[0012] In one embodiment, 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.

[0013] In one embodiment, 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.

[0014] In one embodiment, the experimental chamber further comprises an illumination port in the lower cavity, the excitation objective positioned within the illumination port.

[0015] In one embodiment, the microscopy device further comprises a linear actuator fixedly attached to the experimental chamber, configured to move the experimental chamber relative to the objective.

[0016] In one embodiment, 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.

[0017] In one embodiment, the excitation objective is a long working distance objective.

[0018] In one embodiment, 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. [0019] In one embodiment, the microscopy device further comprises a window positioned in the bottom of the experimental chamber.

[0020] In one embodiment, the microscopy device further comprises at least one electrode positioned in the lower cavity, configured to measure a current across the membrane.

[0021] In one embodiment, the membrane is a planar lipid bilayer.

In one embodiment, 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.

[0022] In one embodiment, the method further comprises the step of moving the sample holder relative to the microscope.

[0023] In one embodiment, the sample holder is moved with a micromanipulator.

[0024] In one embodiment, the method further comprises the step of moving the excitation objective relative to the membrane.

[0025] In one embodiment, the method further comprises deforming the membrane.

[0026] In one embodiment, the membrane is deformed by changing a pressure of a fluid above or below the membrane.

[0027] In one embodiment, the method further comprises measuring a current across the membrane via at least one electrode.

[0028] In one embodiment, the method further comprises splitting the image of the protein with a camera splitter.

[0029] In one embodiment, the excitation energy source comprises a laser, and 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. [0030] In one embodiment, the method further comprises scanning the laser.

[0031] In one embodiment, the scanning is performed by feeding the combined beam into a galvo-galvo scanner conjugated to the back focal plane of the excitation objective.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0033] 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.

[0034] 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) are added to the top chamber along with the DO. Ag/AgCl electrodes are added to the top and bottom sides for electrophysiological recordings.

[0035] 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.

[0036] 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).

[0037] Figure 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. For FBM experiments, vesicles were allowed to fuse into previously formed POPE: POPG (3: 1) bilayers, followed by labeling with LD655-labeled NbALFA and extensive washing. For 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. Scale bars: FBM: 5 mm, SB: 2 mm. Right: MSD analysis of the tracks on the left. Fit of the data indicates Brownian diffusion only for the FBM (top). Figure 2B depicts a plot of Diffusion (D) vs. anomalous diffusion (a) coefficients plots. Data points were obtained from MSD analysis of 7620, 11307, 17731, and 6568 tracks for the FBM (green), mica-supported (grey), quartz-supported (blue), and pegylated-quartz-supported (red) membranes. The distribution of points for the FBM experiments (D ~2 mm2/s and a=l±0.25) indicated that only FBM permits unconstrained diffusion of M2R. Figure 2C depicts representative histrograms of D for M2R, Gail and DOPE- Cy5. Experiments were performed using the FBM, observing unconstrained Brownian diffusion of all species. The distributions of D were fit to log-normal distributions (r2=0.99) and showed a weak dependence on size. Gai was labeled with LD655 through Sfp site-directed labeling. Insert: Fit of the mean of distributions to a Saffman-Delbruck (r2 = 0.95) and Einstein-Smoluchowski (r2 = 0.68) model of diffusion. [0038] Figure 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. Natl. Acad. Sci. 2023;120, e2221541120). Figure 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. frame of a video showing scanned focus-laser illumination of the hTRAAK containing bilayer from panel A. The dotted line shows the perimeter of the bilayer used to calculate its area. Scale bar: 20 mm. Figure 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 3E depicts a representative box blot of Po calculation for different bilayers. The data are shown along a box plot showing the median (0.017), 25;75 percentile (box, 0.016;0.031), and 5;95 percentiles (whisker, 0.052) (N=6).

[0039] 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) 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

[0040] 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. Abbreviations stand for 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).

[0041] 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.

[0042] 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).

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Figure 11 depicts a representative graph plotting the number of hTRAAK particles detected per frame during a FBM video. Each frame lasts 30 ms. An exponential fit to the data is shown as a line (r2=0.99).

DETAILED DESCRIPTION

[0047] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0049] As used herein, each of the following terms has the meaning associated with it in this section.

[0050] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0051] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0052] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in 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.

[0053] Described herein is a freestanding bilayer microscope (FBM) device configured for single-molecule imaging of membrane proteins. 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. In some embodiments, 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.

[0054] Referring now to Fig. IB, shown is an exemplary freestanding bilayer microscope (FBM) device 100. 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the sample holder 204 is configured to hold the membrane. In some embodiments, the experimental chamber 102 further comprises a second sealed chamber 208 in a top cavity positioned above the sample holder 204. In some embodiments, the bottom surface of the experimental chamber 102 comprises a transparent window. In some embodiments, 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. In some embodiments, the excitation objective is configured to direct illumination to the membrane from the illumination system 104.

[0055] In some embodiments, 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. In some embodiments, 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. In some embodiments, the ports 212, 214 may comprise one or more valves. In some embodiments, 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. In some embodiments, the one or more manometers may be utilized to regulate the curvature of the bilayer. In some embodiments, the lower and/or upper ports (212 and 214) are configured to allow fluid access to the first and/or second sealed chamber, thereby perfusing the membrane positioned in the sample holder. In some embodiments, 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). It should be appreciated that 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. In some embodiments, 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.

[0056] In some embodiments, 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).

[0057] In some embodiments, the sample holder 204 is configured to hold a membrane. In some embodiments, the membrane is a bilayer, for example a planar lipid bilayer. In some embodiments, the membrane comprises fluorinated ethylene propylene (FEP). In some embodiments, 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. In some embodiments, the membrane comprises pores/perforations. In some embodiments, 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. In some embodiments, the membrane further comprises a lipid solution. In some embodiments, the membrane comprises an organic solvent. In some embodiments, 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).

[0058] In some embodiments, and parallel to the detection optics plane, one or more access ports (lower ports 212 and/or upper ports 214) 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). In the embodiments and examples disclosed herein, no bottom perfusion was done, and the chamber was connected solely to the manometer. In some embodiments 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 (or second sealed chamber 208) 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. For the perfusion of solution on top of the sample holder 204, 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. [0059] In some embodiments, 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. In some embodiments, 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. In some embodiments, the experimental chamber may be coupled to the imaging platform such that the experimental chamber 102 may be translatable in space. For example, the imaging platform may comprise a micromanipulator, or linear actuator. In some embodiments, to translate the chamber in space, 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. In some embodiments, 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.

[0060] Referring now to Fig. 5 A, shown is an exemplary illumination system 104 for use with the FBM device 100. Generally, the illumination system 104 comprises an illumination source, one or more optical components, and an excitation objective (XO). In some embodiments, the excitation objective may be a long working distance objective. In some embodiments, the illumination source may be one or more lasers. In some embodiments, the one or more lasers may emit light at wavelengths ranging between 400 nm and 700 nm. In some embodiments, three lasers may be utilized having wavelengths of 473 nm, 561 nm, and 660 nm. In some embodiments, the illumination is a single laser beam formed via combining the laser beams from the more lasers. In some embodiments, the laser beam may have a patterned intensity profde. For example, the laser beam may comprise concentric rings of consecutive high intensity and low intensity light. In some examples, the laser beam is a Bessel beam or a Gaussian beam. In some embodiments, the one or more lasers may pass through one or more optical components before arriving at the objective. In some embodiments, 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. In some embodiments, 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.

[0061] In some embodiments, 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). In some embodiments, and to achieve a focused illumination, 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). In some embodiments, 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.

[0062] In some embodiments, and to define a small area of illumination within the bilayer, a galvo-galvo scanner (GG) and an apodization mask (AM) may be conjugated to the back focal plane of the excitation objective 304. By scanning the focused laser oblique to the bilayer at a higher frequency than acquisition, a virtual light sheet that produces a homogeneous illumination field may be created (Fig. 6C). The AM contains concentric rings of different diameters that crop the incoming expanded laser to produce a Bessel beam after focusing it into the bilayer (Gao, L., et al., Nat Protoc. 2014). This creates a smaller and more focal point (Fig. 6B). Finally, a movable mirror (MM) 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. In some embodiments, 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.

[0063] 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. In some embodiments, 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). To permit frequent and rapid access to the top chamber, water immersion objectives with long working distances may be used. Because SPT requires both sufficient resolution and sufficient emitted light intensity, in some embodiments a 25X objective lens with high numerical aperture may be used to detect single molecules at fast frame rates. In some embodiments, the objective lens may have a numerical aperture ranging between 0.8 and 1.33.

[0064] Some embodiments of the disclosed system include methods of microscopy of proteins in a membrane. In some embodiments, 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. In some embodiments, 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.

[0065] 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.

EXPERIMENTAL EXAMPLES

[0066] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0067] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore specifically point out certain embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : FBM: Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

[0068] 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.

[0069] FBM, Freestanding Bilayer Microscope, is developed herein as a method to perform in vitro single-molecule imaging of membrane proteins in membranes. It was shown that the use of planar freestanding bilayers, as opposed to supported bilayers, leads to unconstrained free diffusion devoid of artifacts caused by the presence of the substrate. The use of the FBM was demonstrated by performing single-particle-tracking of the integral membrane protein M2R, as well as the lipidated Gail and fluorescent DOPE lipids. From the tracking results, it was found that over 90% of the tracks underwent Brownian diffusion. The distribution of diffusion coefficients for the three species was determined; it was found that they fit to a log-normal distribution. By fitting the median of the distributions to the estimated hydrodynamic radius of each molecule, it was found that the data correspond better to a Safmann-Delbruck model than to an Einstein-Smoluchowski model, which implies a dependence of diffusion on radius weaker than (radius)-l (Saffman, P. G., et al., Biophysics 1975;72, 3111-3113).

[0070] 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. By using the FBM, it was determined that the Po of hTRAAK at the basal tension of PBs is 2.5%, with an estimated increase to 6.3% at tensions up to 1.2 kBT/nm2, which is about 60% of the lytic tension needed to rupture a typical lipid membrane. These Po values imply that hTRAAK works within a range of low Po and are consistent with previous results (Brohawn, S. G., et al., Proc. Natl. Acad. Sci. 2014; 111, 3614-3619).

[0071] It was also shown that the FBM can be used to study the influence of lipid composition on the diffusion and distribution of membrane proteins. A ternary mixture of lipids that phase separate at room temperature was used to demonstrate this. Incorporation of TRAAK into these bilayers did not change the amount of lipid phase separation, but there was a striking preference of TRAAK for the liquid disordered (Ld) phase. No particles of TRAAK were detected within the ordered phase. This phenomenon suggests a mechanism of exclusion and enrichment of TRAAK that could play an important role in the much more complex plasma membrane of cells.

[0072] 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.

[0073] Finally, these results show that the diffusion of membrane proteins in FBs is different than in cell membranes. Diffusion coefficients in FBs are larger than in cell membranes by about ten-fold, and a larger fraction of the diffusion is Brownian. There are many hypotheses to explain smaller diffusion coefficients, higher immobile fractions, and anomalous diffusion in cell membranes (Banks, D. S., et al., Biophys. J. 2005;89, 2960-2971; Kusumi, A., et al., Annu. Rev. Cell Dev. Biol. 2012;28, 215-250; Krapf, D., Curr. Top. Membr. 2015;75, 167-207). 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.

[0074] The methods are now described.

M2R-ALFA and Al -M2R expression and purification

[0075] 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.

[0076] 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). At the C terminus of the hM2R gene, the PPX recognition site, eYFP gene, and HislO-tag were included for purification purposes. A1-M2R was expressed as described for M2R-ALFA.

[0077] 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). After mixing for 30 minutes, the 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. 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. 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. Next, 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. [0078] 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.

[0079] 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.

[0080] 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.

Gan expression and purification

[0081] 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|31 and Gy2 subunits were also cloned into the pFastBac vector. At the N terminus of Gy2, 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|31 , Gy 2 baculovirus (8 mL of Gv2, 12 mL of G|31, and 15 mL of Gail) to achieve expression of the heterotrimeric G protein subunits in stoichiometric proportions. 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.

[0082] All purification steps were carried out at 4°C unless specified, and stock guanosine 5’- diphosphate disodium salt (GDP) aliquots were made fresh before use. Cell pellets were resuspended in buffer containing 20 mM Tris HC1 pH 8, 100 mM NaCl, 5 mM DTT, 3 mM MgC12, 20 pM GDP, 2 mM PMSF, DNase I, lx of 3-7 TIU/mg aprotinin saline solution, and 0.1% v/v of PIC stock. After mixing for 15 minutes, the cell slurry was manually homogenized. Membranes were extracted in 1% Na chocolate for 1.5 hours. 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. Gai elution 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

[0083] 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.

[0084] All purification steps were carried out at 4°C. The lysed cells were mixed with a five-fold excess volume of buffer (50 mM Tris HC1 pH 8.5, 150 mM KC1, 1 mM EDTA, DNase I, lx of 3-7 TIU/mg aprotinin saline solution, 0. 1% v/v of PIC stock) relative to the wet pellet volume for 15 minutes. The membranes were manually homogenized and extracted using 2% DDM for 1.5 hours. The extraction mixture was centrifuged at 39,000g for 30 minutes. The supernatant was bound to GFPNb resin for one hour. The resin was washed with 10 CV of 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.

[0085] 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.

GIRK-ALFA expression and purification

[0086] At the C terminus of hGIRK2, 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. After 12 hours, 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.

[0087] 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). Membranes were extracted with 1 ,5%:0.3% DDM:CHS for 2 hours. The extraction was centrifuged at 39,000g for 30 minutes to pellet the insoluble fraction, and the supernatant was bound to GFPNb resin for one hour. 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). 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.

Anti-ALFA Nanobody expression and purification

[0088] 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. The next morning, 20 mL of small-scale culture was added to one liter of LB supplemented with 50 pg/mL of kanamycin for a 1 :50 dilution. Once the cells reached an optical density (OD) at 600 nm of about 0.6, the cells were induced with 0.5 mM IPTG and allowed to grow overnight at 16°C. Cells were harvested by centrifuging at 3,500 xg for 15 minutes at 4°C. Cell pellets were resuspended in lx PBS, and centrifuged at 3,500 xg for 10 minutes at 4°C. Flash frozen pellets were stored at -80°C.

[0089] All purification steps were carried out at 4°C unless otherwise specified. 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. 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. 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.

[0090] To prepare fluorescent NbALFA for binding ALFA tagged IMPs on the bilayer, the cysteines of purified NbALFA were non-specifically labeled with maleimide LD655 (LD655- MAL). Thawed NbALFA was incubated with 0.1 mM of freshly made TCEP at room temperature for 10 minutes. The reduced protein was buffer exchanged into IxPBS (not containing calcium and magnesium) at pH 7-7.5 using a PD-10 desalting column to remove excess TCEP, which has been found to interfere with high efficiency bioconjugation by reacting with maleimides to form other products. The NbALFA was mixed with five-fold molar excess LD655-MAL and reacted overnight in the dark at 4°C. To separate the labeled nanobody from excess dye, 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.

Proteoliposome (PLs) reconstitution

[0091] 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.

[0092] For FBM experiments, 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. After removal from the desiccator, the lipid film was rehydrated in buffer (20 mM HEPES pH 7.4, 100 mM KC1, 50 mM NaCl, 10 pM iperoxo, 100 pM TCEP) to a stock concentration of 10 mg/mL. The POPE:POPG (3: 1) lipids were sonicated to clarity in a water bath sonicator. 1% n-Decyl-P-maltoside (DM) was added to the lipids to foster protein insertion into vesicles. The lipids were nutated for 30 minutes at room temperature and sonicated again. To create a protein to lipid ratio (PLR) of 1 :20, M2R-ALFA and POPE:POPG lipids were combined to a final working concentration of 0.25 mg/mL and 5 mg/mL, respectively. The mixture was nutated for one hour at room temperature. To remove the detergent, biobeads (Bio-Beads SM-2 Adsorbent, Bio-Rad) equilibrated in 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. After detergent removal, the proteoliposomes were harvested, flash frozen and stored at -80°C.

[0093] A1-M2R was reconstituted into a 7:3 weight ratio of l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC): POPG lipids for SB experiments. 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.

[0094] 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.

[0095] For FBM experiments, 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. Following a one-hour nutation at room temperature, 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. For the last two buffer exchanges, equilibrated biobeads were included in the buffer. Proteoliposomes were aliquoted, flash frozen and stored at -80°C.

[0096] 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. After one hour of incubation at room temperature, 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.

Freestanding bilayer Microscope (FBM) set-up. Detection optics

[0097] Data acquisition was done with up to 4 sCMOS cameras (Orca-Fl ash4.0 V3, Hamamatsu) connected to a commercial upright microscope (FN1, Nikon Instruments), with four 10 mm spacers between the arm and the head, (FN-S10, Nikon Instruments) through a camera splitter (Multicam LS image splitter, Cairn research) (Figure 5). The camera splitter was equipped with emission filters (ET460/50m, ET525/50m, ET605/70m, and ET700/75m, Chroma Technology) and dichroic mirrors (T495LPXR, T565LPXR and T660LPXR, Chroma Technology) for simultaneous fluorescent recordings of different fluorophores. 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). For epifluorescence measurements, a tunable light source (SPECTRA X Light engine, Lumencor) with excitation filters (395/25, 440/20 ,270/24, 510/25, 550/15, 575/25 and 640/30, Chroma Technology) was attached to the microscope head and controlled by computer through the Nis- Elements software. For all the experiments shown in this work, a 25X, 1.1 Numerical aperture (NA), 2 mm working distance (WD) objective was used (CFI75, Apochromat Multi-Photon LWD 25X, Water immersion, Nikon Instruments).

Freestanding bilayer Microscope (FBM) set-up. Excitation optics (Focus-Laser illumination) [0098] For the focus-laser illumination, three solid state continuous wave lasers (473 nm, 561 nm, and 660 nm GEM lasers, Novanta Photonics) were expanded using pairs of convergent lenses in a Keplerian design (C240TME-A, 8 mm focal length, AC127-025-AB, 25 mm focal length, Thorlabs) (BE in Figure 5), and combined, using an array of dichroic (T4951pxr, T6001pxr, Chroma Technologies), broadband dielectric mirrors (BB111-E01, Thorlabs), and right-angle mirrors (MRA10-E02, Thorlabs) mounted on a custom-made support (BC in Figure 5). 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). An inhouse written software in Lab View (National Instruments) 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. Methods 2011 ;8, 417-423), which was itself conjugated to the back focal plane of the excitation objective, XO, (TL20X-MPL, 20X, 0.6NA, 5.5 WD, Thorlabs) by relay optics (LI to L3) using a 4f configuration. LI (fl=75mm, f2=100mm, Thorlabs), L2 (fl=150mm, f2=100mm, Thorlabs), and L3 (fl=125 mm, f2=125 mm, Thorlabs) were made with 30 mm cage components (Thorlabs) mounted on custom-made adaptor posts for the case of LI and adaptor plates for L2 and L3. 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 [0099] 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 partition fabrication

[0100] FEP sheets (Fluorinated Ethylene Propylene Copolymer - Film, 0.075 mm, Goodfellow) were cut into 1x1 cm squares and indented at the center, either with a weak CO2 laser or a tungsten needle. By sparking the partitions between a high-frequency generator (BD10AS, Electro Technic Products) and a sharp needle, the damaged region on the partition melted, leaving a round and smooth hole (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.

Planar bilayers (PBs) formation

[0101] Before FBM experiments, a small amount (0.5 ml) of lipid solution was added to both sides of the partition and left to dry for ~10 minutes. After mounting a cup on the experimental chamber, the entire chamber was filled with imaging buffer (IB, 150 mM KC1, 10 mM Hepes pH 7.4, 2.5 mM Protocatechuic acid, 5 nM Protocatechuic acid dehydrogenase) followed by the addition of the perfusion adaptors. The perfusion lines were purged of air by perfusing 1-2 ml of IB, after which the system was allowed to equilibrate for 5 minutes. Then, FEP partitions were carefully mounted onto the cups with vacuum grease, and the top electrode was inserted into the top chamber, leading to a sharp increase in current. PBs were then made by applying a small amount (1-10 nl) of lipid across the hole of the bilayer. To do this, 1 ml of lipid was first loaded on a pipette and then quickly ejected into the air, leaving a residual lipid solution on the pipette. The pipettes were then inserted into the buffer on the cup, and the bilayer was formed by pushing an air bubble on top of the partition’s hole. The formation of the bilayer was clear from imaging (Figure 1 A) and electrical recordings (quick reduction in the current followed by a progressive increase in capacitance). All experiments were done at room temperature. Experiments were done using POPE:POPG (3: 1 weight ratio) lipids, except for the phase separation experiments.

Proteoliposome (PL) fusion

[0102] Unless noted otherwise, 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.

Imaging with the FBM

[0103] Focused-laser illumination was turned on after perfusion for hTRAAK and Gail, whilst for M2R and GIRK2, illumination was preceded by the addition of 1 nM labeled NbALFA and another 5 ml of perfusion. The illuminated area was adjusted for every bilayer by changing the input voltage amplitude on the GG. To create a square illumination, two sine waves (10-100 mV) were used, one for the horizontal and one for the vertical scan, at frequencies of 400 and 2500 Hz, respectively (Figure 6C). Videos were recorded, typically at a frame rate of 50 Hz. Single particle tracking was done using uTrack software to obtain the diffusion coefficient (D) and the anomalous coefficient (a) for every track (Jaqaman, K. et al., et al., Nat. Methods 2008;5, 695- 702). Further analysis of the data was done using custom-written software in Matlab (Mathworks). D obtained from the analysis (in pixel s2/frame) were converted to mm2/s using the effective pixel size (0.26 mm for the 25X objective) and the time difference between consecutive frames.

[0104] Analysis of the tracks (Figure 8A) to identify the fraction of immobile particles was done by selecting a diffusion cutoff based on the 1% lowest diffusion coefficients (0.5 mm2/s) of the distribution for M2R measured with the FBM. Particles with diffusion coefficients (D) lower than this threshold were considered immobile, whilst particles with higher D were considered Brownian. Particles with an anomalous coefficient (a) higher than 1.25 were considered directed. Brownian and confined-Brownian were not distinguished in this analysis. To analyze the single tracks for Figure 2A, videos were cropped around a particle, and single particle tracking was performed using Trackmate (Fiji) (Tinevez, J.-Y., et al., Methods 2017; 115, 80-90). Coordinates were then extracted to calculate the mean squared displacement for different increments of time (MSD vs. t) and then fitted using equation 1.

Quartz coverslip preparation

[0105] For quartz-supported bilayer experiments, 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. After cleaning, 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.

Mica coverslip preparation

[0106] 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.

Formation of Supported bilayers (SBs)

[0107] To prepare SB from M2R containing liposomes onto the different substrates, we follow a published protocol with a few modifications (Ramm, B., et al., J. Vis. Exp. 2018). Coverslips (prepared as described in the previous sections) were placed inside a 37°C incubator in a pipette box containing wet tissue paper to keep the humidity constant. Proteoliposomes were diluted to 0.1 mg/ml in M2R reconstitution buffer without TCEP before adding 75 ml of the sample onto the coverslips. After 1 minute, an extra 150 ml of buffer was added. After another 2 minutes, the coverslips were washed by adding another 200 pl of buffer, followed by careful mixing, removing, and adding more buffer. This procedure was repeated 3 times.

SBs TIRF experiments

[0108] A separate chamber for TIRF experiments on microscopy coverslips was exchanged for the FBM chamber, keeping the rest of the microscope the same (Figure 7B). The TIRF chamber was designed to hold a Pellin-Broca prism with a 20 mm square aperture (ADB-20, Thorlabs) on top of which the samples (coverslips) were mounted using 10 ml of immersion oil (Type F immersion liquid, Leica Microsystems). After assembling the coverslip, a 3D-printed ring (TIRF - Cup) was secured with vacuum grease and filled with IB for imaging (Figure 7B). Imaging was done as described before for FBM experiments, except that longer exposures were used (120 ms, for mica and Quartz and 130 ms for Pegylated Quartz).

[0109] hTRAAK determination ofV₀

[0110] For Po determination experiments, POPE:POPG (3: 1 weight ratio) 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. Following the fusion of the PLs, 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. To calculate the number of hTRAAK channels in the bilayer, 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.

[0111] Phase-separation experiments

[0112] 4.4 mg of l,2-diphytanoyl-sn-glycero-3 -phosphocholine (DphPC, Avanti Polar Lipids), 2 mg of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids) and Img of Cholesterol (Co, Avanti Polar Lipids) in chloroform were mixed to a final 2: 1 : 1 molar ratio. At this point, 250 ng of l,2-distearyl-sn-glycero-3-phosphoethanolamine-N-(TopFluor AF488) (ammonium salt) was added to indicate the ordered phase. 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. After imaging with 2 channels, 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.

[0113] The experimental results are now described

Diffusion of Integral Membrane proteins

[0114] To benchmark the performance of the FBM, the diffusion of the human M2 Muscarinic receptor (M2R), an archetypical G-protein coupled receptor (GPCR) in this system was compared against total internal reflection fluorescence (TIRF) experiments performed in supported bilayers (SB) (Kruse, A. C., et al., Nat. Rev. Drug Discov. 2014). SBs are relatively easy to implement in commercial microscopes, and thus they are extensively used as lipid membrane models (Loose, M., et al., Science 2008; Richter, R. P., et al., Langmuir 2006; Casteliana, E. T., et al., Surf. Sci. Rep. 2006).

[0115] 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.

2018;e58139). 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. To perform TIRF experiments, 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. For FBM experiments, 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). This was done to avoid the increase in background produced by proteoliposomes partitioning into the torus. 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.

[0116] Videos for both SB and FBM experiments contained well-resolved diffraction-limited particles that were detected and analyzed using single-particle-tracking software (Jaqaman, K., et al., Nat. Methods 2008;5, 695-702). Particle trajectories (tracks) were obtained after particle detection and assignment across successive frames. Figure 2A shows a characteristic example of a track for an FBM (top) and an SB (bottom) experiment. To analyze the tracking data, the mean squared displacement (MSD) of the track was plotted against increasing lag times (t) and the data was fitted to the logarithmic anomalous diffusion equation,

Log(MSD)=Log(D)+a Log(r)

Equation 1

[0117] where D is the generalized diffusion coefficient and a is the anomalous coefficient, which takes a value of 1.0 ± 0.25 for normal (Brownian) diffusion (Sungkawom, T., et al., Nature 2017;550, 543-547; Metzler, R., et al., Phys. Chem. Chem. Phys. 2014;16, 24128-24164). It was found that particles in SB are immobile, as evidenced by the shape of the MSD curve and the fitting results (D=0.002pm2/s, a=-0.05). Comparatively, particles in the FB are mobile, and the fit indicates Brownian diffusion (D=0.8pm2/s, a=0.92). A similar analysis was performed on thousands of tracks (Figure 2B). The results, shown as D vs. a plots, reported a stark difference in the distribution of diffusion and anomalous coefficients; most particles undergo normal diffusion in the FBM whilst most particles are immobile in SBs regardless of the substrate material (Figure 2B and Figure 8A). These FBM experiments were reproduced with two other membrane proteins with different folds and oligomeric structures, the dimer TRAAK and the tetramer GIRK2, finding similar results (Figure 9), i.e. most particles (>80%) underwent Brownian diffusion with diffusivities ~1.0 pm2/s.

[0118] Unconstrained Brownian diffusion was also observed for phospholipids (1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE-Cy5) and the fluorescently labeled membrane- associated guanine-nucleotide-binding protein Gai (Supp. Videos 7-8). Analysis and classification of the tracks yielded histograms of diffusion coefficients that follow log-normal distributions (r2=0.99) (Figure 2C). It was found that Gai diffuses faster than M2R and the lipid DOPE faster than either protein. The relation between the diffusion coefficients of the three species is compatible with a model of diffusion that depends weakly on the hydrodynamic radius. Particularly, this data provides a better fit for a Saffman-Delbruck (SD) model (r2= 0.95) than for the Einstein- Smoluchowski (ES) model (r2=0.68) (Figure 2C) (Saffman, P. G., et al., Biophysics 1975;72, 3111-3113).

[0119] Besides the substrates tested with SPT, 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.

1992; 1103, 307-316). In all cases tested, proteins in SBs were immobilized in contrast to FBs.

[0120] It should be noted that after the fusion of M2R-containing liposomes, the presence of large aggregates of protein in the bilayer was observed (Figure 10A). These aggregates have somewhat round shapes and lie in the plane of the bilayer without noticeable height. Similar aggregates for the ion channel GIRK2 (Figure 10B) and TRAAK were observed (Figure 4). Proteins exchanged between these aggregates and the rest of the bilayer. They also fused with each other in ways reminiscent of descriptions of protein condensates (Figure 10C).

Applications of the FBM on ion channels. Estimation of open probability with multiple channel recordings

[0121] In FB experiments, both sides of the bilayer are isolated, which permits coupling singleparticle imaging experiments with functional measurements that require transport across the bilayer. This property of the FBM was used to determine the open probability of an ion channel in bilayers containing multiple channels using simultaneous imaging and electrical recordings. To perform these experiments, the human mechanosensitive potassium channel TRAAK (h TRAAK) was chosen. hTRAAK in freestanding bilayers was studied, showing tension sensitivity curves measured with a freestanding bilayer tensiometer that was developed and which is fully compatible with the FBM (Perez-Mitta, G., et al., Proc. Natl. Acad. Sci. 2023;120, e2221541120) (Figure 3A).

[0122] Determining ion channel open probability (Po) 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). However, 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.

[0123] The exact number of channels that contribute to the current is the main unknown in the effort to determine the Po of a channel from macroscopic experiments. Using the FBM, the number of hTRAAK channels (N) in the bilayer was determined and the current through those channels (I) was simultaneously measured (Figure 3B, C and D). Knowing N and I, hTRAAK Po was calculated from the simple relation, I = i N Po, where i is the current through a single channel (Sorum, B., et al., Proc. Natl. Acad. Sci. 2021 ; 118, e2006980118). It was determined that the median of the channel Po at the bilayer basal tension (0.2-0.3 kBT/nm2) to be 1.7% (IQR=1.6;3.1) (Figure 3E). Furthermore, increasing the tension of the bilayer by about 4-fold increased the channel activity by only 2.5-fold.

[0124] For these experiments, 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).

Protein partitioning into phase-separated bi layers

[0125] Cell membranes contain hundreds of different kinds of lipids (Van Meer, G., et al., Nat. Rev. Mol. Cell Biol. 2008;9, 112-124). It has been shown that the interaction between different species of lipids can give rise to critical phenomena. For example, ternary mixtures of lipids of a lipid with a high Tm, a lipid with low Tm, and cholesterol can phase-separate in vitro, leading to the coexistence of liquid-ordered and liquid-disordered phases (Veatch, S. L. et al., Biophys. J. 2003;85, 3074-3083). Additionally, it has been observed that the protein distribution in membranes is not homogeneous, and some have proposed that inhomogeneous lipid composition could be a driving mechanism of this distribution (Sezgin, E., et al., Nat. Rev. Mol. Cell Biol. 2017; 18, 361-374). To test whether lipid composition and phase-separation can drive an inhomogeneous distribution of proteins, single-particle-tracking experiments were performed with hTRAAK in bilayers of DphPC:DSPC:cholesterol (2: 1 : 1), a mixture that phase separates at room temperature, forming liquid ordered and liquid disordered phases (Veatch, S. L., et al., Biophys. J. 2006;90, 4428-4436). A small amount of fluorescent lipid was added to label the ordered phase (DSPE-AF488). The data show a striking preference of hTRAAK for the disordered phase (Figure 4A). This preference is further evidenced by the distribution of tracks that avoid the ordered regions (Figure 4A, right panel). TRAAK molecules that approached the boundary between phases often moved along the perimeter before returning to the disordered phase (Figure 4B). It was also found that the diffusion coefficient distribution of TRAAK in the phase-separated bilayer does not differ significantly from the distribution in the binary mixture POPE:POPG (3: 1) (Figure 4C).

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[0185] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. 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.

2. The microscopy device of claim 1, further comprising 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.

3. The microscopy device of claim 1, wherein the sample holder further comprises an upper cavity positioned over the sample holder, forming a second sealed chamber with the sample holder.

4. The microscopy device of claim 3, further comprising at least one electrode positioned in the upper or lower cavity, configured to measure a current across the membrane.

5. The microscopy device of claim 4, wherein the at least one electrode comprises a first electrode positioned in the upper cavity and a second electrode positioned in the lower cavity.

6. The microscopy device of claim 3, further comprising at least one upper perfusion port fluidly connected to the upper cavity, configured to control a flow of a fluid into the upper cavity.

7. The microscopy device of claim 6, further comprising 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.

8. The microscopy device of claim 7, further comprising 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.

9. The microscopy device of claim 1, the experimental chamber further comprising an illumination port in the lower cavity, the excitation objective positioned within the illumination port.

10. The microscopy device of claim 1, further comprising a linear actuator fixedly attached to the experimental chamber, configured to move the experimental chamber relative to the objective.

11. The microscopy device of claim 1, further comprising a linear actuator fixedly attached to the excitation objective, configured to move the excitation objective relative to the experimental chamber.

12. The microscopy device of claim 1, wherein the excitation objective is a long working distance objective.

13. The microscopy device of claim 1, wherein the excitation objective is oriented at an angle relative to the membrane of 30 degrees to 50 degrees.

14. The microscopy device of claim 13, wherein the angle is about 39 degrees.

15. The microscopy device of claim 1, further comprising a window positioned in the bottom of the experimental chamber.

16. The microscopy device of claim 1, further comprising at least one electrode positioned in the lower cavity, configured to measure a current across the membrane.

17. The microscopy device of claim 1, wherein the membrane is a planar lipid bilayer.

18. 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.

19. The method of claim 18, further comprising the step of moving the sample holder relative to the microscope.

20. The method of claim 19, wherein the sample holder is moved with a micromanipulator.

21. The method of claim 18, further comprising the step of moving the excitation objective relative to the membrane.

22. The method of claim 18, further comprising deforming the membrane.

23. The method of claim 22, wherein the membrane is deformed by changing a pressure of a fluid above or below the membrane.

24. The method of claim 18, further comprising measuring a current across the membrane via at least one electrode.

25. The method of claim 18, further comprising splitting the image of the protein with a camera splitter.

26. The method of claim 18, wherein the excitation energy source comprises a laser, and 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.

27. The method of claim 26 further comprising scanning the laser.

28. The method of claim 27, wherein the scanning is performed by feeding the combined beam into a galvo-galvo scanner conjugated to the back focal plane of the excitation objective.