Name: Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling (Video) | JoVE Journal
Uploaded: 2026-03-20T13:10:00.000+00:00
Duration: 7 min 51 s
Description: Learn single-antibody labeling to visualize nanoscale antibody-epitope interactions in T cell membranes using single-molecule localization microscopy. This protocol enables direct observation of therapeutic antibody binding in native cellular environments.

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Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM146786 and the College of Liberal Arts and Sciences, University of Illinois Chicago. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All authors declare that they have no conflicts of interest to disclose.

The organization of plasma membrane proteins plays a vital role in regulating cell function. Conventional fluorescence microscopy techniques are limited by the diffraction limit of light, masking their nanoscale organization. SMLM overcomes this limitation by achieving spatial resolutions of 10-20 nm47, enabling the characterization of their nanoscale assemblies10,48,49. Clustering artifacts in SMLM arise...

The plasma membrane contains a diverse array of proteins that regulate cell mobility, adhesion, sensing, and intercellular communication. Understanding the nanoscale distribution of plasma membrane proteins is important, as protein function is often governed not only by expression levels but also by their spatial organization on the membrane1,2. Notably, many therapeutic antibodies target membrane proteins3,4, making nanoscale investigations essential for understanding how antibody interactions are influenced by the local membrane protein organization. However, precise mapping of membrane protein distribution, single-molecule profiling of their abundance, and real-time monitoring of their dynamic interactions with binding partners remain challenging.
Widely used single-molecule localization microscopy (SMLM) techniques, such as direct stochastic optical reconstruction microscopy (dSTORM)5,6 and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT)7, use immunofluorescence (IF) staining for sample labeling. They achieve lateral resolutions of ~ 10 nm and have provided extensive insights into the nanoscale organization of therapeutic membrane targets, including human epidermal growth factor receptor 2 (HER2)8,9, CD2010, T cell receptors11,12,13, programmed death-ligand 114, B cell receptors15,16,17, and CD1917,18. For example, dSTORM studies using the therapeutic monoclonal antibody Trastuzumab to directly detect HER2, a key receptor implicated in breast cancer, have revealed distinct clustering states among HER2-positive patients8, while different therapeutic antibodies have been shown to modulate HER2 clustering9. DNA-PAINT combined with lattice light sheet microscopy has elucidated the oligomeric states and interaction profiles of CD20 in B-cells with therapeutic monoclonal antibodies rituximab, ofatumumab, and obinutuzumab10. Although conventional SMLM provides valuable insights into nanoscale organization, its full potential is limited by traditional IF staining approaches. The quality of the SMLM image is dependent on the IF staining, which in turn is governed by the specificity and affinity of the labeling antibody. Traditional SMLM uses high-affinity antibodies that typically achieve superior IF staining and image quality. However, most therapeutic antibodies interact with membrane receptors of live cells with moderate affinity to minimize off-target side effects19. For instance, bispecific T cell engagers (TCEs) exhibit a lower affinity for CD3, which prevents TCR internalization20,21,22. Such interactions cannot be accurately captured by conventional IF techniques.
We have previously developed a strategy that directly leverages single-molecule antibody-epitope interactions, termed single-antibody labeling (SAL). SAL enables high-density mapping of antibody-epitope interactions while preserving native binding equilibria23. While we have previously reported a protocol for intracellular cytoskeletal and mitochondrial proteins24, SAL on membrane proteins has not been demonstrated. In this protocol, we provide a step-by-step workflow for applying SAL to membrane proteins, demonstrated through the labeling of the tetraspanin CD81, a reported therapeutic target candidate25,26,27,28, in both suspension and adherent cells. Using fluorescently labeled anti-CD81 antibodies to directly probe the spatial distribution of CD81 on U2OS and Jurkat T cells, we demonstrate the application of SAL nanoscale studies of membrane proteins. We termed this strategy as membrane SAL (mSAL). Finally, we describe a clustering-based strategy to reduce the isolated single-molecule detections outside of the cell.
In essence, SAL uses time-lapse single-molecule labeling to capture individual antibody-epitope interactions, and subsequent single-emitter deconvolution yields a super-resolution image based on SMLM. By adjusting antibody concentration and selecting an appropriate non-illuminating interval (NII) between consecutive image frames, SAL matches the antibody on-rate to the single-molecule detection rate, enabling single-molecule localization. SAL requires longer NIIs due to the inherently slow binding kinetics of antibodies, which prolong the image acquisition durations (~3-17 h). SAL is best suited for applications that require precise nanoscale mapping of epitopes and the evaluation of individual antibody-epitope interaction dynamics. Despite its lower throughput, SAL offers unique insight into epitope organization and antibody engagement under near-physiological conditions, making it particularly valuable for characterizing therapeutic antibodies in situ.

<table><tbody><tr><td>anti-CD81 Alexa Fluor 488 Antibody</td><td>Thermo Fisher Scientific</td><td>MA5-44132</td><td>Imaging antibody</td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Bovine serum albumin</td><td>MilliporeSigma</td><td>A7906-100G</td><td></td></tr><tr><td>Chambered cover glass, 8-well</td><td>Cellvis</td><td>C8-1-N</td><td></td></tr><tr><td>DMEM media</td><td>Thermo Fisher Scientific</td><td>11960069</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>Fetal bovine serum</td><td>MilliporeSigma</td><td>F0926-500ML</td><td></td></tr><tr><td>Glutaraldehyde, 10 %</td><td>Electron Microscopy Sciences</td><td>16120</td><td></td></tr><tr><td>Gold colloids, 100 nm</td><td>Ted Pella</td><td>15711&amp;minus;20</td><td>Fiducial marker</td></tr><tr><td>Immersion oil&amp;nbsp;</td><td>Cargille Laboratories</td><td>16245</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;equipped with TIRF module</td><td>Nikon Instruments</td><td>Eclipse Ti2-E</td><td>Microscope</td></tr><tr><td>Jurkat E6.1 cell line</td><td>MilliporeSigma</td><td>88042803-1VL</td><td>Suspension cells</td></tr><tr><td>Laser Launch, 6 line</td><td>Oxxius</td><td>L6CC-CS8-1511</td><td>Excitation laser</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>Microcentrifuge tube, 1.5 mL&amp;nbsp;</td><td>VWR</td><td>89000-028</td><td></td></tr><tr><td>Microscopy imaging software</td><td>Nikon Instruments</td><td>NIS-Elements Advanced Research</td><td>Image acqisition software</td></tr><tr><td>Objective, 100x/1.49 CFI APO TIRF&amp;nbsp;</td><td>Nikon Instruments</td><td>MRD01991</td><td>Microscope objective</td></tr><tr><td>Paraformaldehyde, 16 %</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td>Antibiotics</td></tr><tr><td>Poly-L-Lysine solution, 0.01%&amp;nbsp;</td><td>MilliporeSigma</td><td>P4707-50ML</td><td></td></tr><tr><td>Prime 95B sCMOS Camera</td><td>Teledyne Vision Solutions</td><td>01-PRIME-95B-R-M-16-C</td><td>Camera</td></tr><tr><td>RPMI 1640 media</td><td>Thermo Fisher Scientific</td><td>11875-093</td><td>Media for suspension cells</td></tr><tr><td>Sodium azide</td><td>Thermo Fisher Scientific</td><td>19038-1000</td><td></td></tr><tr><td>Sodium borohydride</td><td>MilliporeSigma</td><td>213462-25G</td><td></td></tr><tr><td>T25 culture flasks</td><td>Thermo Fisher Scientific</td><td>169900</td><td></td></tr><tr><td>TetraSpeck microspheres</td><td></td><td></td><td>Fiducial marker</td></tr><tr><td>Tissue culture dish, 100 mm&amp;nbsp;</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>U2OS cell line</td><td>ATCC</td><td>HTB-96</td><td>Adherent cells</td></tr></tbody></table>

single-molecule localization microscopy of membrane proteins using single-antibody labeling

Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling

The plasma membrane defines the cell shape and serves as the interface that governs intercellular communication. Membrane proteins constitute a major class of therapeutic targets; therefore, super-resolving the cell membrane through its constitutive proteins holds great promise in advancing cell biology and antibody therapeutics. In this regard, single-molecule localization microscopy (SMLM) enables nanoscale visualization of protein organizations on biological structures. Despite its importance, applying SMLM to plasma membrane proteins poses unique challenges. In this protocol, we present an effective approach using time-lapse single-antibody labeling (SAL) termed membrane SAL (mSAL). We provide detailed step-by-step instructions, including optimization of the antibody concentration, laser power density, duration of non-illumination intervals, image reconstruction, and density-based cluster analysis, to resolve nanoscale membrane protein distribution and membrane morphology. We use the tetraspanin protein CD81 as the model membrane protein to demonstrate the capability of mSAL on both adherent and suspension mammalian cells. In addition to super-resolving the cell membrane and distributions of membrane proteins, our technique enables the investigation of the pharmacodynamics of therapeutic antibodies interacting with their membrane targets in the native membrane environment.

The mSAL technique described in this protocol enables visualization of single antibody binding events to their corresponding plasma membrane protein targets, producing a super-resolution map of membrane protein distribution. CD81 is used here as a representative example, though the approach is readily adaptable to other membrane proteins.
Immobilizing Jurkat T cells with sufficient spacing between cells ensures membrane epitope accessibility by antibodies and glass surface for fiducials to set...

This protocol describes single-antibody labeling (SAL) to resolve the nanoscale spatial organization of plasma membrane proteins. By leveraging cumulative antibody-epitope interactions at the single-molecule level, membrane SAL (mSAL) maps local epitope distributions while simultaneously capturing antibody binding behavior in the native cellular environment.

The plasma membrane defines the cell shape and serves as the interface that governs intercellular communication. Membrane proteins constitute a major ...

My research focuses on the membrane of T cells and aims to reveal new nanoscale insights relevant to therapeutic targeting. Existing imaging methods do not typically directly visualize nanoscale antibody-epitope interactions in cells. This protocol overcomes that limitation by enabling direct observation within the cellular environment.After mounting the sample on the microscope and depositing gold colloids onto the surface, use bright field illumination to verify that enough colloids have been deposited within the region of interest. Confirm that the ideal density of 5 to 10 gold colloids is present within the region of interest before proceeding. For the optical configuration of the antibody probe fluorophore, open the imaging software and access the optical configuration settings.Select the appropriate dichroic mirror and set the laser excitation wavelength and power density. Adjust the camera integration time and select the emission filter. Then, set the camera pixel binning to achieve a pixel size of 150 to 160 nanometers.Now, create an optical configuration for the photo bleaching step by selecting a photo bleaching mode in the imaging software. Set the laser power to 100%and adjust the camera integration time to one second. Set the imaging parameters in the image acquisition software by defining the non-illuminating interval, number of frames, and frequency of the photo bleaching step.Next, prepare the imaging buffer by diluting the CD81 antibody to a concentration of 1 microgram per milliliter in 200 microliters of 5%BSA made in DPBS. The final recommended antibody concentration is 0.5 nanomolar for Jurkat T cells, or 2 nanomolar for U-2 OS cells. Add the prepared imaging buffer to the well containing the cells and start the image acquisition in the software to begin data collection.Use the live view function in the imaging software configured for membrane single-antibody labeling to monitor antibody binding in real time. Configure the imaging parameters in the image acquisition software to obtain at least a 10-frame acquisition and set the non-illuminating interval to five seconds. Start the acquisition and evaluate the sparsity of single molecule localizations in the recorded movie.If multiple overlapping antibody binding events are observed immediately after adding the imaging buffer, or if single-molecule localizations accumulate over time, abort the acquisition. Reduce the antibody concentration by twofold and repeat the acquisition on a new sample. Continue reducing the antibody concentration by twofold and reevaluating the number of events per unit area until the desired single-molecule localization density is achieved.If the event density is sparse, increase the antibody concentration in the imaging buffer by twofold. Repeat the acquisition on a new sample and continue increasing the antibody concentration by twofold while reevaluating the number of binding events per unit area until the desired single-molecule localization density is achieved. For non-illuminating interval optimization, configure the imaging parameters in the image acquisition software to obtain a 50-frame acquisition.After adding the antibody and starting acquisition, evaluate the density and sparsity of single-molecule localizations in the recorded movie. Determine the lowest non-illuminating interval that yields an optimal single-molecule event density. Finally, if spatially overlapping binding events occur as the image acquisition progresses, introduce a photo bleaching step at regular intervals.Adjust the photo bleaching frequency so that fluorescence from bound antibodies is removed before overlapping events accumulate. Set the file path in the MATLAB software to the folder that contains the reconstructed M cell comma-separated values file. Run the code by clicking Run in the MATLAB toolbar.When prompted, select the M cell CSV file and click Open to load it into the program. Run the analysis using the previously defined inputs as a starting point. Evaluating the scatter plot containing the unclustered localization data.Choose a minimum point value that is higher than the number of noise localizations but lower than the number of localizations on the cell. Evaluate the window that depicts the clustered data. Confirm that clusters are retained on the cell with the expected phenotype while localizations outside the cell are minimized.If the clustering parameters are too restrictive, decrease the minimum points value and increase epsilon. If the clustering parameters are too inclusive, increase the minimum points value and decrease epsilon. Finally, after adjusting the minimum points value and epsilon, rerun the code to obtain optimal clustering.Jurkat T cells were immobilized with sufficient spacing to preserve membrane integrity and allow antibody access to membrane epitopes. Gold colloids were visualized and used as fiducial markers for lateral drift correction during M cell image acquisition. Optimization of the non-illumination interval showed that a five-second interval produced antibody binding events with minimal spatial overlap compared to shorter or longer intervals at one nanomolar antibody concentration.Application of drift correction improved the reconstructed M cell image compared to the uncorrected reconstruction. Density-based clustering of M cell localizations reduced low-density background interactions and highlighted clustered CD81 binding events. We can observe antibodies interacting with their membrane epitopes in a cellular environment, providing insight relevant to therapeutic antibodies.The antibody concentration, non-illumination interval, and photo bleaching steps are critical to optimize. Suboptimal parameters will compromise the results. Single-antibody labeling can be extended to simultaneously evaluate membrane protein binding by multiple antibodies within the same cellular environment.

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Research

JoVE Journal

Bioengineering

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Learn single-antibody labeling to visualize nanoscale antibody-epitope interactions in T cell membranes using single-molecule localization microscopy. This protocol enables direct observation of therapeutic antibody binding in native cellular environments.

Single-Molecule Localization Microscopy of Membrane Proteins using Single-Antibody Labeling

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