Name: Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2025-12-12T13:30:00.000+00:00
Duration: 8 min 39 s
Description: Learn single-protein tracking in lipid bilayers using fluorescence microscopy with single-molecule sensitivity. This protocol reveals hidden protein states and dynamics for membrane protein research.

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This work was supported by the Air Force Office of Scientific Research FA9550-20-1-0324, FA9550-23-1-0583 (J.A.B. and G.P.N.), and FA9550-18-1-0395 (J.A.B.).
G.P.N. acknowledges the following funding agencies: 
(1) NEXTGENERATIONEU (NGEU) funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) - A Multiscale integrated approach to the study of the nervous system in health and disease (DD n. 1553, 11.10.2022) 
(2) NEXTGENERATIONEU (NGEU) funded by the Ministry of University and Research (MUR), National Recovery and Resilience Plan (NRRP), project CN_00000041 - National Center for Gene Therapy and Drugs based on RNA Technology (DD n.1035, 17.06.2022).

The authors have no conflicts of interest to declare.

In this article, we present a single-particle tracking method for directly observing individual membrane proteins as they move through a solid-supported lipid bilayer membrane and interact with one another. AQP4 was chosen as a representative example because of its ability to form dynamic self-assemblies regulated by its different isoforms32,33,34. Above, we outline a reliable method for sample preparation and key parameters to track single-membrane proteins at millisecond time scales. The steps include: (1) preparing SUVs, (2) determining the appropriate level of fluorescent labeling, (3) preparing solid supports, (4) creating transmembrane protein samples within a solid-supported lipid bilayer, (5) preparing antifade agents, (6) constructing a simple single-molecule microscope, (7) collecting single-molecule tracking data, and (8) analyzing the tracking data using ImageJ.
The described step-by-step method is conceptually straightforward; however, each critical step has its own pitfalls and limitations. Among these, sample preparation is the most challenging (steps 1-5). Therefore, several details should be kept in mind. The first is how to create a solid-supported lipid bilayer. There are three main methods: (1) Langmuir-Blodgett (LB), (2) SUV fusion and rupture, and (3) spin-coating35,36,37,38,39. Each method has its advantages and disadvantages. For example, the Langmuir-Blodgett method provides greater control over the properties and chemical composition of bilayers, including asymmetric compositions between the two leaflets, and allows the formation of multilayers in a controlled manner. However, using an LB-trough is technically demanding, time-consuming, and costly to acquire35. Spin-coating is a relatively simple technique for creating lipid bilayers; however, it tends to produce multilayers and requires a large excess of material, often an impractically large amount39. Vesicle fusion and rupture are straightforward processes that require only a small amount of material. However, creating a hydrophilic surface is crucial, and some lipid compositions form bilayers via vesicle fusion more easily than others (e.g., in our lab, we have found that vesicles with a net negative charge form bilayers through vesicle fusion only with the addition of divalent cations and extended incubation times)40. Besides establishing a hydrophilic surface (step 3), other important considerations when forming a solid-supported bilayer through vesicle fusion include washing the newly formed bilayer both before and after incorporating the membrane protein. The pre-insertion wash removes excess SUV and larger multilamellar vesicles (MLVs), while the final wash after membrane insertion clears unincorporated protein. Insufficient washing can introduce background signals during tracking studies.
We have screened numerous detergents to determine if they are suitable for transmembrane protein insertion into solid-supported lipid bilayers, including C12E9, Tween-20, TritonX-100, and many others. Our preferred detergent is DOTM because it is easy to remove and is the most gentle on the membrane (less likely to disrupt the membrane integrity). The amount of protein added is critical. Too much, and it will be impossible to observe single tracks. When adapting this procedure to other transmembrane proteins, the amount will have to be adjusted through trial and error. However, the quantities given in this procedure are an excellent starting point. The amount of detergent must be kept at or below the critical micellar concentration; otherwise, the bilayer will be solubilized and removed from the quartz substrate.
Insertion of membrane proteins into the supported lipid bilayer needs to be optimized for each set of experimental conditions. However, there are a few common methods to achieve this, including the use of proteoliposomes and the direct incorporation of membrane proteins into a preformed bilayer41. A proteoliposome is a liposome, such as an SUV, with a protein embedded in it. These proteoliposomes can fuse with a preformed solid-supported lipid bilayer or be incorporated simultaneously with the SUVs during bilayer formation42. The fusion causes rupture, releasing the protein into the membrane. It is simple to implement, but it results in a random orientation of the membrane proteins. This can lead to strong interactions with the solid support, resulting in different diffusion properties depending on the protein&#39;s orientation3. This protocol describes a method that utilizes direct incorporation, yielding an orientational preference for AQP4 and other membrane proteins with large hydrophilic domains on one side of the protein2,5,43,44.
Direct observation is a powerful tool used in scientific discovery. Dynamic single-molecule imaging is especially important in membrane-protein biophysics, where observing the motion and interactions of individual proteins is used to test hypotheses or generate new ones that better describe how a protein behaves. The protocol and methods described here are ideal for two-dimensional solid-supported lipid bilayers but can be extended to more complex environments with multiple components2, live cells34,45,46,47, and three dimensions48. Single-molecule tracking opens the door to many other experiments, including two-color single-molecule tracking49, single-molecule FRET50,51, and dynamic super-resolution imaging such as Stochastic Optical Reconstruction Microscopy (STORM) and Point Accumulation Imaging Nanoscale Topography (PAINT). By expanding the instrument to include temperature control, the thermodynamic properties of membrane proteins can also be explored26,43,52.

Under natural biological conditions, membrane proteins are surrounded by an extensive network of interactions, which includes other membrane proteins, proteins in the intra- and extracellular fluids, various types of lipids and cholesterol, substrates or ligands, solution interactions, and interactions between extracellular and intracellular matrices. A classic biophysical approach to better understand specific and collective interactions starts with a single component, gradually introducing additional components one at a time until the complexity reflects the native environment-a &#34;Divide and Conquer&#34; approach to biophysics. A modern molecular-scale method for studying these interactions is through direct observation using time-lapse single-molecule fluorescence imaging to track individual proteins as they move and interact with their environment. In this paper, we describe the fundamental steps of this approach, including the formation of lipid bilayers on quartz coverslips and the insertion of a large membrane protein into these pre-made bilayers in a manner suitable for single-protein imaging. This will be followed by a &#39;work-horse&#39; protocol for tracking single proteins in supported bilayers.
This article describes how to create solid-supported lipid bilayers using small unilamellar vesicles (SUVs) that rupture on hydrophilic quartz coverslips. The quartz surfaces are prepared by either an acid wash or ultraviolet (UV) ozone cleaning. The lipid used to create the SUVs should, when possible, match the main chemical composition of the biological membrane of interest (e.g., aquaporin-4 (AQP4) is found in the plasma membrane of astrocytes and the major components are POPC (46.4%), POPE (8.5%), PS (5.6%), cholesterol (30%), PI (2.5%), and SM (5.6%))1,2. To prevent interaction with the solid substrate, additives such as polyethylene glycol (PEG) grafted to a lipid lift the bilayer off the surface3,4. The amount of each additive must be carefully adjusted to ensure that protein diffusion is not obstructed, while also providing enough support to lift the membrane from the substrate surface. Additives like PEGylated lipids typically exhibit two phases: a &#39;brush phase&#39; and a &#39;mushroom phase&#39;. The brush phase occurs when the PEG is at high concentration, causing the polymer to stand upright to reduce steric hindrance. The mushroom phase occurs at low concentrations, when the polymers are sufficiently separated to exist as random coils. In general, the brush phase hampers diffusion, but the mushroom phase offers little support. The concentration between the brush and mushroom phases is optimal, providing support with minimal impact on diffusion3,4.
Some membrane proteins will spontaneously insert into solid-supported lipid membranes, such as P450s and cytochrome P450 reductase5, while others will need to be inserted using detergents, which is a delicate process. Careful washing is necessary to remove these detergents, as well as any unincorporated proteins that can interfere with single-protein tracking - a process that will be described in detail. The main focus of this article is the preparation of a sample for single-molecule tracking (SMT) using time-lapse fluorescence imaging. This requires an objective with high magnification and numerical aperture (NA) -- typically a 100x and 1.45 NA oil immersion objective6,7. It also requires a fast, highly sensitive camera (EMCCD, CMOS, or ICCD camera) and laser excitation. Laser excitation needs to cover a large enough field of view to measure long protein trajectories, which is often accomplished using total internal reflection to create an evanescent field of about 50 &#956;m x 100 &#956;m or epi-illumination with beam shaping optics (Gaussian to top-hat) about 80 &#956;m x 80 &#956;m8 or a highly inclined and laminated optical sheet (HILO) with a field similar to TIRF. Total internal reflection fluorescence (TIRF) microscopy has the advantage of reducing background signal. A simple, relatively inexpensive TIRF microscope design is presented below. By adding temperature controls, it is possible to measure several thermodynamic properties such as enthalpy, entropy, and Gibbs free energy9. The technique also requires fluorescence labeling of the membrane protein at a degree of labeling that ensures most membrane proteins carry a fluorescent tag.
The transmembrane protein of interest in this article is AQP4. AQP4 is a water channel polarized in the endfeet of astrocytes and other areas of the central nervous system&#39;s vasculature10. It can transport up to 3 billion water molecules per second and is involved in water homeostasis, cell migration, brain edema, learning, and memory formation through synaptic plasticity11. Dysregulation of AQP4 function has been linked to various neurological conditions, including brain edema, neuromyelitis optica, and epilepsy12,13,14,15. AQP4 is a 32-37 kD protein, depending on its isoform, and has a tetrameric quaternary structure with eight hydrophobic membrane domains connected by joining loops, an intracellular N-terminal domain, and a large intracellular C-terminal domain16. There are two main isoforms of AQP4, M1 and M23; the difference arises from the initiation at the methionine 1 residue or the methionine 23 residue. Research has shown that the M1 isoform can form dyads or triads with itself, depending on its palmitoylation state9. The M23 isoform assembles into large protein aggregates known as Orthogonal Arrays of Particles (OAPs). When co-expressed, the M1 isoform limits OAP growth by forming a peripheral protein coating around its exterior17,18,19. The dynamics of OAP aggregation may be a key factor in modulating water permeability and homeostasis in the brain. In this article, we describe the necessary steps to track individual AQP4s in a membrane and observe single protein-protein interactions.

<table><tbody><tr><td>1/4 Waveplate</td><td>Thorlabs</td><td>WPQ05M-633</td><td>make sure to match the laser</td></tr><tr><td>100X Oil Objective</td><td>Olympus</td><td>12-563-659</td><td>1.45 N/A apochromatic</td></tr><tr><td>16:0-18:1 PC</td><td>Avanti Polar Lipids</td><td>850457</td><td>POPC Phospholipids</td></tr><tr><td>18:0 PEG2000 PE</td><td>Avanti Polar Lipids</td><td>880210</td><td>Ammonium Salt</td></tr><tr><td>300mm Lens</td><td>Thorlabs</td><td>AC254-300-A-ML</td><td></td></tr><tr><td>633 nm He:Ne Laser</td><td>Melles Griot</td><td>05-LHP-927</td><td>Can be changed to other sources, and to other wavelengths</td></tr><tr><td>633nm band-pass filter</td><td>Chroma Tech</td><td>633/10X</td><td>Match to the laser being used</td></tr><tr><td>655nm Long-Pass Filter</td><td>Chroma Tech</td><td>ET655lp</td><td>Change to match the wavelengths of the dye/laser</td></tr><tr><td>Andor Solis I</td><td>Andor&amp;nbsp;</td><td>Andor Solis I</td><td>Software for image capture</td></tr><tr><td>ATTO 655&amp;nbsp;</td><td>ATTO-TEC</td><td>AD 655</td><td>NHS-Ester modification, any dye with a the modification that is suitable for single molecule work can be used. ATTO is recommended but Cy5/7 can be used</td></tr><tr><td>Bio-beads SM-2 Resin</td><td>BioRad</td><td>1523922</td><td></td></tr><tr><td>Calcium Chloride</td><td>Millipore Sigma</td><td>C4901</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C3155-50MG</td><td>from bovine liver, aqueous soltion, &amp;ge;30000 units/mg</td></tr><tr><td>Chloroform</td><td>Fisher Scientific&amp;nbsp;</td><td>C547-1</td><td>HPLC or spectroscopic grade</td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667-500MG</td><td></td></tr><tr><td>Coverslips</td><td>ESCO Optics</td><td>R525000</td><td>25mm Quartz</td></tr><tr><td>Dichroic Beamsplitter</td><td>Semrock</td><td>FF545/650-Di01-25x36</td><td>Change to match the wavelengths of the dye/laser</td></tr><tr><td>EMCCD Camera</td><td>Andor&amp;nbsp;</td><td>iXon Ultra 888</td><td></td></tr><tr><td>Falcon Tubes 15mL&amp;nbsp;</td><td>Greiner Bio-one</td><td>188261</td><td>cellestar cell culture tubes</td></tr><tr><td>Glucose Oxidase</td><td>Sigma-Aldrich</td><td>G2133-50KU</td><td>from Aspergillus niger, typer VII, &amp;ge;100000 units/g solid (without added oxygen)</td></tr><tr><td>Heat Gun</td><td>Harbor Freight</td><td>56434</td><td>Used in the heating of the parafilm, generally any heat gun could be used</td></tr><tr><td>HEPES</td><td>Fisher Bioreagents</td><td>BP310-500</td><td></td></tr><tr><td>Low Autofluorescence Immersion Oil</td><td>Thorlabs</td><td>MOIL-30</td><td></td></tr><tr><td>Methonal</td><td>Fisher Scientific&amp;nbsp;</td><td>A452-1</td><td>HPLC or spectroscopic grade</td></tr><tr><td>Micro Centrifuge Tube</td><td>VMR</td><td>525-1126</td><td>1.5 mL&amp;nbsp;</td></tr><tr><td>Micro Pipetters</td><td>Ependorf</td><td></td><td>Various; 1000 &amp;micro;L, 100 &amp;micro;L and 10 &amp;micro;L are suggested</td></tr><tr><td>&amp;Oslash;1.5&quot; Lens Tubes</td><td>Thorlabs</td><td>SM1.5L10</td><td>The external thread is shaved down for sample preperation</td></tr><tr><td>Optical Table</td><td>Newport</td><td>RPR Reliance</td><td>The size of tabel is dependent on available space</td></tr><tr><td>Optical Tissues</td><td>Thorlabs</td><td>MC-50E</td><td>Cleaning various components</td></tr><tr><td>Parafilm</td><td>Millipore Sigma</td><td>P7669</td><td>Verify nothing fluorescent off gases during the process</td></tr><tr><td>Piezo Actuator</td><td>Newport</td><td>PZA12</td><td></td></tr><tr><td>Pneumatic Vibration Isolators</td><td>Newport</td><td>I-2000 Series</td><td>used to stabilize the optical table for any unevenness&amp;nbsp;</td></tr><tr><td>Refrigerated Bath</td><td>Fisher Scientific&amp;nbsp;</td><td>Isotemp 3016</td><td></td></tr><tr><td>Shutter Controller</td><td>Thorlabs</td><td>SC10</td><td>Linked to the imaging software</td></tr><tr><td>Sodium Chloride</td><td>Millipore Sigma</td><td>S988</td><td></td></tr><tr><td>Steritop Filter</td><td>Millipore Sigma</td><td>S2GPT05RE</td><td></td></tr><tr><td>Switchbox Driver and Controller&amp;nbsp;</td><td>Newport</td><td>PZC200</td><td>Stage controller</td></tr><tr><td>Syringe Filter</td><td>J.T. Baker</td><td>SF01-98</td><td>PES 25mm 0.2 &amp;micro;M</td></tr><tr><td>TEC Element</td><td>Thorlabs</td><td>TEC3-2.5</td><td>Used to connect the Peltirer heat pump to the sample holder and objective collar</td></tr><tr><td>Temperature contoller</td><td>Meerstetter Engineering</td><td>TEC-1091</td><td></td></tr><tr><td>Temperature Detector</td><td>Thorlabs</td><td>TH100PT</td><td></td></tr><tr><td>Thermistor</td><td>Thorlabs</td><td>TH10K</td><td></td></tr><tr><td>Tube-O-Dialyzer</td><td>G Biosciences</td><td>786-611</td><td>4K MWCO</td></tr><tr><td>UV Ozone Cleaner</td><td>Novascan</td><td>PSDP_UV</td><td>Pro Series - Digital&amp;nbsp;</td></tr><tr><td>Zoom Optics&amp;nbsp;</td><td>Thorlabs</td><td>SM1NR1</td><td></td></tr></tbody></table>

tracking single proteins in lipid bilayers using fluorescence microscopy

Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Direct observation is a fundamental method for scientific discovery and understanding. Time-lapse single-molecule fluorescence imaging allows the observation of individual proteins as they move and interact with their surroundings, making states that would normally be hidden in ensemble measurements visible. Single-protein tracking is especially suited for studying membrane proteins, where its two-dimensional nature enables fast, wide-field imaging with single-fluorophore sensitivity. Using artificial lipid bilayers that mimic membranes where transmembrane or peripheral membrane proteins are found (such as plasma membranes or membranes of intracellular organelles from various cell types) permits measurement of specific interactions in near-native environments. It also allows for the gradual addition of complexity, one component at a time, while suppressing background interference from fluorescence and Raman scattering. By adding temperature control, it becomes possible to determine the thermodynamic properties of single-particle interactions as well. In this work, we describe a protocol for sample preparation, data collection, single-molecule fluorescence tracking, and analysis of membrane protein trajectories in lipid bilayers using Aquaporin-4. This work serves as a foundational example and can be adapted to different proteins and bilayer compositions.

As described in protocol section 2, lysines were labeled using NHS-esters conjugated to a fluorescent dye molecule (NHS-dye). After conjugation to the protein, measure the DOL with a UV-Vis spectrometer. For this experiment, the DOL of AQP4 tetramers was 4.12. Next, determine the probability that each AQP4 tetramer will have at least one dye molecule using the Poisson distribution provided below.
<img alt="Poisson distribution formula, P(n, DOL), mathematical equation for probability calculations." src="/files/ftp_upload/69095/69095eq7.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 7">     (2)
Where P is the probability that each AQP4 is labeled, using equation 2, the probability that each AQP4 tetramer has a fluorescent label attached to it is 98%. The distribution of labels per tetramer is shown in Figure 2 and reflects the brightness distribution observed in the sample.
The signal-to-noise ratio achieved in this example data was 8-9x above background for a single fluorophore and upwards of 15-20 for the brightest particles. This variance comes from the position in the excitation beam and the DOL described in protocol step 2.
Within the generated XML file, the columns of interest are J, K, L, and M, which correspond to the particle number ID, the frame in which the particle was detected, the X position of the particle, and the Y position of the particle, respectively (see Supplemental File 6). In the second sheet of the file, the step-size distribution and average diffusion coefficient are calculated, where the step-sizes are the distances a particle moves between frames, and the average diffusion is given by equation 3:
<img alt="Diffusion calculation: Average displacement formula equation." src="/files/ftp_upload/69095/69095eq8.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 8">     (3)
Where Dave is the average diffusion coefficient, and Δt is the frame rate. This equation assumes Brownian motion, which is generally valid for small displacements and can be confirmed by analyzing the mean-squared displacement versus time tag from a sample of individual tracks3,30,31.
A histogram of step-sizes shows a distribution of particles diffusing, which can be attributed to different-sized OAPs (Figure 5).
<img alt="Laboratory distillation setup; glass apparatus for liquid separation and purification." class="xfigimg" src="/files/ftp_upload/69095/69095fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Lipid drying apparatus. The apparatus consists of a 10 mL round-bottom flask with a vacuum distillation connector. Nitrogen enters at low pressure through a 0.2 µm syringe filter with a needle attached to a yellow synthesis cap, which is connected to a vacuum distillation connector. Pressure is released through Tygon tubing attached to a syringe with a cut-off needle. <a href="https://www.jove.com/files/ftp_upload/69095/69095fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Probability histogram; number of fluorescent labels; statistical distribution; data analysis result." class="xfigimg" src="/files/ftp_upload/69095/69095fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Expected distribution of fluorescent labels on each AQP4 unit using equation 2. The most probable number of fluorophores is 4, and 98% of all AQP4s will have a fluorescent label. <a href="https://www.jove.com/files/ftp_upload/69095/69095fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Optical assembly diagram; components include tube ring, Parafilm gasket, quartz coverslip, lens tube." class="xfigimg" src="/files/ftp_upload/69095/69095fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Sample holder assembly. The assembly is made of a 1" SM1 lens tube with the male threads removed and beveled to prevent interference with the microscope objective. Includes a quartz coverslip and a parafilm gasket. The entire assembly is secured with an SM1 retaining ring. <a href="https://www.jove.com/files/ftp_upload/69095/69095fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy setup for optical excitation, includes EMCCD camera, diagram, XYZ stages, lens system." class="xfigimg" src="/files/ftp_upload/69095/69095fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Microscope. On the right side is a simplified diagram of the microscope; the green line shows the excitation light coming from the laser. Starting from the right, the laser light passes through a ¼ waveplate, then is focused by a 300 mm lens. The light is reflected by a long pass dichroic mirror onto the objective, where it is focused onto the back aperture. By using a translation mirror to shift the excitation beam, the beam is positioned at the edge of the objective's aperture, creating an evanescent field in the sample. The in-focus emitted light is collected by the objective and is represented by the red line. The emitted light then passes through the dichroic, then a long pass filter, and is focused by a 300 mm lens onto the camera, providing a 1.7x optical magnification (170x total magnification from the objective and tube lens). On the left side, is an image of the microscope. Note that additional turning mirrors are used to correctly position the laser. Simultaneously, the detection arm is arranged to maximize the collection of photons from the fluorescently labeled transmembrane proteins. <a href="https://www.jove.com/files/ftp_upload/69095/69095fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Step size probability graph, &lt;D&gt;=0.0143 µm²/s, data analysis, probability distribution." class="xfigimg" src="/files/ftp_upload/69095/69095fig5.jpg" data-altupdatedat="1783396841000" data-alt="Figure 5"> 
Figure 5: Histogram of step sizes. The blue line with red circles is a histogram of all the step sizes. The black line is a fit for multiple diffusion coefficients. The step-size probability distribution is given by: <img alt="Static equilibrium equation P(Δr)=Σf(Δr͞i/2DiΔt)e^(-Δr͞i²/4DiΔt), mathematical formula." src="/files/ftp_upload/69095/69095eq9.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 9"> where fi is the fractional population of each type of diffusing particle, assuming each undergoes Brownian motion. The histogram and fit show a heterogeneous distribution of slower- and faster-moving particles, attributed to a distribution of multiple OAP sizes. <a href="https://www.jove.com/files/ftp_upload/69095/69095fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Spreadsheet calculator used to determine mol% compositions, total lipid amounts, and optional fluorescent/FRAP components for SUV preparation. <a href="https://www.jove.com/files/ftp_upload/69095/Lipid%20Blend%20Calculator.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental File 2: Raw single-molecule fluorescence microscopy time-lapse image stack used in trajectory tracking (source file for TrackMate tutorial). <a href="https://www.jove.com/files/ftp_upload/69095/0_1_raw_cropped.tif" target="_blank">Please click here to download this File.</a>
Supplemental File 3: Animated preview of raw single-molecule fluorescence data showing intensity and background prior to processing. <a href="https://www.jove.com/files/ftp_upload/69095/0_1_raw_cropped.gif" target="_blank">Please click here to download this File.</a>
Supplemental File 4: Animation of filtered particle paths (post-thresholding) showing representative AQP4 trajectories over time. <a href="https://www.jove.com/files/ftp_upload/69095/0_1_overlayParticles.gif" target="_blank">Please click here to download this File.</a>
Supplemental File 5: Animated overlay showing full-field particle tracking of AQP4 molecules using TrackMate output. <a href="https://www.jove.com/files/ftp_upload/69095/0_1_overlayALL.gif" target="_blank">Please click here to download this File.</a>
Supplemental File 6: Spreadsheet showing extracted trajectory coordinates and calculated step-size distributions used for diffusion analysis. <a href="https://www.jove.com/files/ftp_upload/69095/Reprentative%20Trajectoies%20and%20Step%20Sizes.xlsx" target="_blank">Please click here to download this File.</a>

This article provides a detailed description of how to create samples for single-protein tracking in solid-supported lipid bilayers. It also explains a straightforward fluorescence microscope with single-molecule sensitivity and a fast frame rate. Finally, we outline the procedure for extracting single-protein trajectories.

Direct observation is a fundamental method for scientific discovery and understanding. Time-lapse single-molecule fluorescence imaging allows the ...

Proteins operate in a complex state space and much of it is hidden. We use single molecule imaging to reveal these states. When tracking single membrane proteins in a lipid bilayer, the main challenges are sample preparation, photobleaching, and time resolution.To begin, remove the lipid stalk solutions from the freezer and allow them to reach room temperature. Take a clean 10 milliliter flask out of the oven and let it cool to room temperature. Add 900 microliters of spectroscopic grade chloroform, and 100 microliters of spectroscopic grade methanol to the cooled round bottom flask.Then add the appropriate amounts of each lipid to the flask and swirl to mix thoroughly. Now place a vacuum distillation connector on top of the round bottom flask and attach it to the nitrogen drying line. Then turn on the nitrogen gas and adjust the pressure until the flow is just barely felt on the back of the hand.Pipette one milliliter of buffer for every five micromoles of total lipid into the round bottom flask. Place a glass stopper on the flask and seal it with paraffin film. Position the flask and a clamp on a ring stand and submerge its bottom in a 60 degree Celsius sonicater bath.Confirm that the solution becomes opaque and that no lipids remain attached to the inner wall of the flask. After the one hour incubation, turn on the sonicater and set the amplitude to its highest level. Move the flask around within the bath to locate the spot with the strongest agitation where the solution visibly bumps and sprays inside the flask.Sonicate the solution for 30 minutes. Observing the transition from opaque to transparent and slightly opalescent. Remove the lipid solution from the flask and transfer it to a micro centrifuge tube.Centrifuge the tube at 100, 000 G for one hour at four degrees Celsius. After centrifugation, remove and transfer the supernatant to a new centrifuge tube. Place the 25 millimeter quartz cover slips into a beaker and add equal volumes of nano pure water, 30%hydrogen peroxide and concentrated nitric acid.Heat the cover slips in the prepared solution for 30 minutes until bubbling begins. Check the solution every 10 minutes and swirl gently to prevent the cover slips from sticking together. Observe the cover slips sliding apart and separating during swirling.Once separated, gradually reduce the swirling speed to maintain their separation and allow the bubbling solution to coat the cover slips evenly. Then rinse the cover slips thoroughly with purified water while gently swirling to remove all chemical residue. Alternatively, clean the quartz cover slips using n-hexane followed by methanol, wiping with lens tissue for each solvent.Place the cover slips inside a UV ozone cleaner with the surface to be treated facing the lamp. Allow oxygen to flow into the chamber at five pounds per square inch for five minutes. Then turn off the oxygen flow.Now turn on the ultraviolet lights for 15 minutes, then allow the cover slips to rest for at least 10 minutes to permit the ozone to dissipate. Place a freshly cleaned cover slip into a 25 millimeter sample holder. Using a one quarter inch SM one lens tube, cut an eight millimeter diameter double layered par film gasket and position it in the center of the sample holder.Apply a 50 microliter droplet of the small Unilamellar vesicles to the center of the eight millimeter gasket. After sealing the chamber, incubate at 37 degrees Celsius for one hour. Post incubation, use a pipette to rinse away the solution.Add 50 microliters of fresh buffer to the bilayer and repeat this process a total of 10 times. After the final rinse, remove the buffer from the sample holder. Add a 50 microliter aliquot of the desired protein in detergent, ensuring the concentration is at or below the critical micella concentration.Incubate the sample at 37 degrees Celsius for at least one hour to allow protein incorporation, rinse the sample with buffer to remove any unincorporated protein and detergent. Set the vertical pixel shift speed to approximately 600 nanoseconds and increase the vertical clock voltage using overclock mode. Then configure the horizontal pixel readout to its maximum speed.Adjust the pre amplifier gain to two. Set the amplifier output for electron multiplication and set the electron multiplier gain to its highest level. Then set the exposure time to 25 miliseconds.Confirm that the actual frame rate exceeds the exposure time slightly. Now adjust the laser power until the signal clearly stands out from the background noise. Collect enough imaging data to yield a minimum of 1000 tracks per sample.For tracking analysis, crop all data sets to a consistent size and perform background correction using Image J.In Fiji, drag and drop the movie or stack TIFF file that needs to be analyzed into the interface. To enter the pixel calibration details, go to the analyze tab, select set scale, and apply the pixel to distance calibration specific to the instrument. Then save a copy of the dataset to preserve the original and track any changes made.Subtract the background from a selected region of interest and apply it to the saved copy of the original data. Go to the process tab and choose subtract background. In the plugins tab, select tracking, followed by trackmate to launch the trackmate dialogue window for tracking analysis.The degree of labeling for AQP four tetramers was calculated to be 4.12 using UV visible spectrometry and the Poisson distribution analysis indicated that 98%of Tetramers carried at least one fluorescent dye molecule. The histogram shows a wide distribution of step sizes consistent with diffusion of differently sized orthogonal arrays of particles, and the calculated average diffusion coefficient was 0.0143 square micrometers per second. In this video, we have identified key states important to aquaporin four regulation and their associated thermodynamic forces.This protocol eliminates guesswork in creating biomimetic membranes with purified transmembrane proteins that are suitable for time lapse single molecule tracking. Protein machines operated at inflection points time lapse single protein tracking enables direct observation of stochastic pathways, branches, and dead ends.

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Research

JoVE Journal

Biochemistry

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but  rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

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

Learn single-protein tracking in lipid bilayers using fluorescence microscopy with single-molecule sensitivity. This protocol reveals hidden protein states and dynamics for membrane protein research.

Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

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