Name: Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules (Video) | JoVE Journal
Uploaded: 2009-11-02T16:05:00.000+00:00
Duration: 10 min 57 s
Description: Learn single molecule FRET measurements using an automated scanning confocal microscope with surface-immobilized DNA. This method enables real-time study of nucleic acid dynamics and enzyme interactions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Chandran Sabanayagam for his help in developing the system, Joshua Edel for advice on designing the microfluidic device and the staff at the Rowland Institute at Harvard University for machining parts of the setup. We acknowledge useful discussions with members of Meller&#x2019;s group at the Rowland Institute and at Boston University, and members of T. Ha&#x2019;s group at UIUC for helpful information. Finally, we appreciate the financial support from the National Science Foundation (PHY-0701207) and the National Institute of Health (GM075893).

1 - Assembling the microfluidic cell
The microfluidic cell is made by fusing a patterned, 2 mm thick, polydimethylsiloxane (PDMS) disk to a freshly cleaned quartz cover slip. The PDMS disk contains four 30 μl rectangular chambers that are accessed by inlet and outlet flexible capillaries connected to a buffer reservoir and a syringe pump, respectively, for the controlled flow of buffer. The ﬂow cell is supported on its back side by a glass window and placed on a custom-made cell holder containing a water cooled thermoelectric element to regulate temperature.
<ol>
 <li>To make the patterned PDMS disk, mix 10 parts silicone elastomer base with 1 part curing agent, mix well and leave degassing for 1 hour in vacuum.</li>
 <li>Gently pour the elastomer mix onto the microfluidic cell mold. In our case, this is a 1" silicon wafer with four strips (1x104 x 1x103 x 300 μm) made from negative photoresist. Leave this to cure on a hot plate at 70° C for two hours.</li>
 <li>Carefully peel the cured PDMS disk from the mold using a razor blade. Fuse the disk onto a 1'' glass window (containing 8 x 2 mm-diameter pre-drilled holes at both ends of the channels) by plasma oxidizing both surfaces for 30 seconds. Fuse the glass windows onto the flat side of the PDMS disk (the side not containing the channels).</li>
 <li>Insert a 2-inch long flexible fused silica capillary tubing through each glass window's hole until it reaches the channel. Inserting a needle first and then following with the capillary may facilitate this process. Seal the glass window holes using a fast curing silicone casting compound such as Kwik-Cast.</li>
 <li>Rinse the channels with ethanol and water, and plasma-activate the cell together with a freshly cleaned, 1-inch circular quartz cover slip for 30 seconds. We suggest cleaning this cover slip using piranha*. Fuse the cover slip to the side of the cell containing the channels, sealing the chambers.</li>
 <li>Leave the assembled cell to cure overnight at room temperature.</li>
</ol>
* Piranha is a solution of H2O2 and H2SO4 in 1:2.5 proportions. To clean the cover slips, immerse them in piranha at 90° C for 20 min.
2 - Activating the surface for molecule immobilization
For nucleic acid studies, the simplest surface immobilization scheme consists of first coating a glass or quartz cover slip with biotinylated bovine serum albumin (BSA) and then with streptavidin. This allows the biotinylated sample to be immobilized with high specificity for days at room temperature. 
<ol>
 <li>Connect flexible tubing to the capillaries for easy buffer injection using a syringe and needle.</li>
 <li>Inject a solution of 0.1 mg/ml of biotinylated BSA in buffer A (10 mM TRIS-HCl, pH 8.0, 50 mM NaCl, filtered using 0.02 μm filters) into the channel and incubate for at least 30 min.</li>
 <li>Flow 300-500 μl of buffer A to remove any free biotinylated BSA from the channel, being careful not to trap any air bubbles inside the chamber.</li>
 <li>Inject a solution of 0.1 mg/ml of streptavidin in buffer A and incubate for 15 min. Then repeat Step 3.</li>
 <li>Flow through a 3-5 pM solution of the biotinylated sample. Perform a surface scan to check the coverage of fluorescent molecules. If necessary, increase the sample concentration to obtain better coverage. Incubate for 10 min and repeat Step 3.</li>
</ol>
The microfluidic cell is mounted on a motorized x-y stage allowing coarse (up to 25 mm) motion using optically encoded DC motors, and ﬁne (1 nm) motion using two closed-loop piezo actuators (Physik Instrumente model M-014 or similar). This can be done either before or after activating the surface for sample immobilization.
3 - Preparing the Imaging Buffer (IB)
The IB consists of an enzymatic oxygen scavenging system and a triplet quencher, such as Trolox. Since the IB is flushed through constantly overnight, at least 10 ml should be prepared in advance.
<ol>
 <li>Prepare 10 ml of 0.8% w/v D-glucose, at least 35 mM TRIS-HCl pH 8.0 and 50 mM NaCl in deionized water. The higher concentration of buffer is important for two reasons: dissolving Trolox will acidify the solution, and the enzymatic reaction will also lower the pH of the solution over time. Since this solution has a long shelf life it can be stored as a stock solution.</li>
 <li>Dissolve 5 mg of Trolox in the buffer prepared in Step 1 by vortexing for a couple of minutes and then shaking for &gt;10 min. Trolox is not very soluble in water at pH &gt; 7, so do not rush this step. Filter the solution using a 0.2 μm filter and leave degassing in vacuum for 10 minutes.</li>
 <li>Prepare the "gloxy" enzymatic solution by mixing 60 μl water, 20 μl of 5X buffer A, 20 μl of catalase, 1 mg glucose oxidase and 100 μl of 10 mg/ml BSA. Filter the solution using a 0.22 μm centrifuge filter.</li>
 <li>Gently mix the buffer from Step 2 with the "gloxy" solution from Step 3 avoiding the formation of air bubbles.</li>
 <li>Flow the IB through the channel at a constant rate of 5 μl/min using a mechanical syringe pump to control the flow rate. Bubble argon gas into the IB reservoir to prevent atmospheric oxygen from re-dissolving into the buffer.</li>
</ol>
The surface scanning, molecule localization and acquisition of single molecule intensity traces is controlled by a LabView program that permits automated data collection1. The program scans 20x20 μm2 areas at 0.2 μm resolution, with a rate of 0.1 μm/ms. Data is immediately processed to yield intensity-weighted locations of all pixels above the background counts. Once the immobilized molecules are found, they are moved one by one into the confocal volume and the intensities of the donor and acceptor fluorophore are recorded as a function of time until both fluorophores photobleach. The stage is then programmed to move to a new origin 100 μm away, and the scanning process is repeated.
4 - Representative Results
Below are some images showing the assembled microfluidic cell before surface activation and after being mounted on the microscope ready for molecule immobilization. If the channel is activated successfully, surface scans should be similar to the scans depicted below showing emission of the donor dye (green) and acceptor (red) after direct donor excitation. These molecules are moved one by one into the probing volume and the fluorescence is recorded over time until both dyes photobleach, as shown in the single molecule traces. From traces like these one can obtain the FRET efficiency which informs on the instantaneous donor-acceptor separation, which in turn is used to explore the structure and dynamics of molecules of biological interest.
<img src="/files/ftp_upload/1542/1542fig1tmb.jpg" alt="Microfluidic device diagram, chromatography method, showcasing fluid channels and capillary action." border="0" data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1: Microfluidic cell with four channels, each with inlet/outlet capillaries. Please <a href="https://www.jove.com/files/ftp_upload/1542/1542fig1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1542/1542fig2tmb.jpg" alt="Optical excitation setup with spectroscopy components, diagram for emission analysis and spectral fitting." border="0" data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2: Cell mounted on the microscope ready for measurements. Please <a href="https://www.jove.com/files/ftp_upload/1542/1542fig2.jpg">click here</a> to see a larger version of figure 2.
<img src="/files/ftp_upload/1542/1542fig3tmb.jpg" alt="Transient absorption spectroscopy setup; optical excitation and emission measurement system." border="0" data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3: Setup for sm-FRET measurements. Please <a href="https://www.jove.com/files/ftp_upload/1542/1542fig3.jpg">click here</a> to see a larger version of figure 3.
<img src="/files/ftp_upload/1542/1542fig4tmb.jpg" alt="Fluorescence microscopy image, cell distribution, intensity heatmap, cellular biology experiment." border="0" data-altupdatedat="1787630408000" data-alt="Figure 4"><img src="/files/ftp_upload/1542/1542fig5tmb.jpg" alt="Fluorescence microscopy image; protein distribution analysis in cellular matrix; intensity scale." border="0" data-altupdatedat="1787630408000" data-alt="Figure 5"> Figures 4 and 5: Red and green channel of a surface scan of immobilized FRET molecules excited with green laser. Please click to see a larger version of <a href="https://www.jove.com/files/ftp_upload/1542/1542fig4.jpg">figure 4</a>, or <a href="https://www.jove.com/files/ftp_upload/1542/1542fig5.jpg">figure 5</a>.
<img src="/files/ftp_upload/1542/1542fig6tmb.jpg" alt="Photon emission time graph; red-green trace in optical measurement; data fluctuation analysis." border="0" data-altupdatedat="1787630408000" data-alt="Figure 6"><img src="/files/ftp_upload/1542/1542fig7tmb.jpg" alt="Photon detection graph; photons per ms vs. time, key for optical excitation analysis." border="0" data-altupdatedat="1787630408000" data-alt="Figure 7"> <img src="/files/ftp_upload/1542/1542fig8tmb.jpg" alt="Photon emission analysis graph showing intensity vs time, illustrating transient absorption changes." border="0" data-altupdatedat="1787630408000" data-alt="Figure 8"><img src="/files/ftp_upload/1542/1542fig9tmb.jpg" alt="Photon count graph, time vs photons per ms, fluorescence emission, data analysis." border="0" data-altupdatedat="1787630408000" data-alt="Figure 9"> Figures 6, 7, 8, and 9: Intensity trajectories of donor (green) and acceptor (red) fluorophores labeled to a DNA molecule 8, 10, 16 and 18 basepairs apart. Please click to see a larger version of 
<a href="https://www.jove.com/files/ftp_upload/1542/1542fig6.jpg">figure 6</a>, <a href="https://www.jove.com/files/ftp_upload/1542/1542fig7.jpg">figure 7</a>,<a href="https://www.jove.com/files/ftp_upload/1542/1542fig8.jpg">figure 8</a>, or <a href="https://www.jove.com/files/ftp_upload/1542/1542fig9.jpg">figure 9</a>.

<ol><li>Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Molecule+Techniques%2C+a+Laboratory+Manual%5BTitle%5D)+AND+%22CSHL+Press%22%5BJournal%5D)">Single-Molecule Techniques, a Laboratory Manual.</a> CSHL Press. , (2008).</li>
<li>Sabanayagam, C. R., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+scanning+confocal+microscope+for+single+molecule+analysis%5BTitle%5D)+AND+%22Applied+Pys.+Letters%22%5BJournal%5D)">High-throughput scanning confocal microscope for single molecule analysis.</a> Applied Pys. Letters. , 84-87 (2004).</li>
<li>Sabanayagam, C. R., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+Fluorescence+resonance+energy+transfer+to+measure+distances+along+individual+DNA+molecules%3A+Corrections+due+to+nonideal+transfer%5BTitle%5D)+AND+%22J.+Chem.+Phys%22%5BJournal%5D)">Using Fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer.</a> J. Chem. Phys. 122, 61103-61105 (2004).</li>
<li>Echeverra, A., Aitken, C., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Oxygen+Scavenging+System+for+Improvement+of+Dye+Stability+in+Single-Molecule+Fluorescence+Experiments%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments.</a> Biophysical Journal. 94, 1826-1835 (2008).</li>
<li>Rasnik, I., McKinney, S. A., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonblinking+and+long-lasting+single+molecule+fluorescence+imaging%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Nonblinking and long-lasting single molecule fluorescence imaging.</a> Nature Methods. 3 (11), 891-893 (2006).</li>
<li>Boukobza, E., Sonnenfeld, A., Haran, G., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immobilization+in+Surface-Tethered+Lipid+Vesicles+as+a+New+Tool+for+Single+Biomolecule+Spectroscopy%5BTitle%5D)+AND+%22J.+Phys.+Chem%22%5BJournal%5D)">Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy.</a> J. Phys. Chem. 105, 12165-12170 (2001).</li>
<li>Di Fiori, N., Meller, A. Article in preparation. , Forthcoming.</li>
</ol>

Although the above protocol has been optimized for our particular system and for a specific choice of dyes (TMR and ATTO647N), it is by no means the only proved method. Recently, an alternative oxygen scavenging system consisting of protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) was shown to increase the photostability of certain dyes4. Likewise, other triplet-state quenchers such as &beta;-mercaptoethanol (BME) can be used instead of Trolox, although these might not suppress long-lasting blinking of some dyes, in particular Cy55.
Although immobilization via biotin-streptavidin-biotinylated bovine serum albumin (BSA) is the preferred choice for nucleic acids studies, a polyethylene glycol (PEG)-coated surface is better suited for studies of proteins since it prevents non-specific adhesion1. Additionally, sub-diffraction-limited size vesicles can be used to encapsulate the biomolecule of interest in cases where it can be affected by surface interactions6.
In this article we have used a confocal setup equipped with silicone avalanche photodiodes to acquire the fluorescence intensity traces. This protocol works equally well with Total Internal Reflection Fluorescence microscopes (TIRF) equipped with CCD cameras. Regardless of the microscopy used, sm-FRET can inform on spatial resolutions approaching the angstrom when the donor and acceptor signal are properly corrected7.

<table><tbody><tr><td>Fused silica capillary tubing</td><td>Polymicro Technologies</td><td>TSP150375</td><td></td></tr><tr><td>Fused silica cover slip</td><td>Esco Products</td><td>R425000</td><td></td></tr><tr><td>Tubing</td><td>Diba Industries</td><td></td><td></td></tr><tr><td>Biotinylated BSA</td><td>Sigma-Aldrich</td><td>A8549</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9085</td><td></td></tr><tr><td>Streptavidin</td><td>Thermo Fisher Scientific, Inc.</td><td>0021122</td><td></td></tr><tr><td>Sylgard 184 Elastomer Kit</td><td>Dow Corning</td><td>3097366-1004</td><td>Silicone Elastomer Kit</td></tr><tr><td>Trolox</td><td>Aldrich</td><td>238813</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C3155</td><td></td></tr><tr><td>Glucose Oxidase</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Kwik-Cast Sealant</td><td>World Precision Instruments, Inc.</td><td>Kwik-Cast</td><td>Silicone Casting Compound</td></tr></tbody></table>

automated system for single molecule fluorescence measurements of surface-immobilized biomolecules

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

To study the dynamics of individual molecules over an extended period of time, molecules must first be localized in space. We accomplish this by immobilizing them on the surface of a microfluidic cell. The procedure starts with the assembly of the cell, which contains four chambers where the biomolecules will be fixed.The chambers are connected to the exterior through inlet and outlet capillaries. The cell is made by fusing a patterned PDMS disc to a quartz cover slip. The assembled cell is then mounted on the fluorescence microscope and flexible tubings are connected to the capillaries to allow the easy exchange of solutions through the chambers to activate the surface for molecule immobilization first flow, a solution of biotinylated BSA through one of the channels.BSA will absorb to the chamber. Surfaces then flow a solution of streptavidin, which will in turn bind to the biotin. Finally, flow your biotinylated oligonucleotide, which will bind to the streptavidin, thus anchoring the molecule of interest to the surface of the quartz cover slip.An imaging buffer designed to maximize the lifetime stability and intensity of the fluoro fours is flowed through the chamber at a constant rate throughout the entire acquisition period. The automated setup scans different sections of the surface for molecules having both donor and acceptor flora fours, and then moves them one by one into the probing volume obtaining for each a fluorescence intensity time trace until both Fluor fours photo bleach. Hi, I'm Nicholas Dfi from the laboratory of Professor Miller in the biomedical engineering department at Boston University.Today we will show you a procedure to obtain single molecule F traces from immobilized nucleic acids using a confocal setup. We use this procedure in our laboratory to study nucleic acid dynamics and nucleic acid and enzyme interactions. So let's get started.The Microfluid Excel is made by fusing a patterned two millimeter thick poly dimethyl silicone or PDMS disc to a freshly cleaned quartz cover slip. To make the patterned PDMS disc combined 10 parts of silicone elastomer base with one part curing agent mix well and Degas for one hour in a vacuum. After the elastomer base has been Degas, pour it gently into the microfluidic cell mold.Leave the elastomer mix to cure on a hot plate at 70 degrees Celsius for two hours. When cured, carefully peel the PDMS disc off from the mold using a razor blade. The PDMS disc contains four 30 microliter rectangular chambers.Fuse the flat side of the PDMS disc, the side not containing the channels, onto a one inch glass window by plasma oxidizing both surfaces for 30 seconds. The glass window contains eight to two millimeter holes at both ends of the channels which were previously drilled. Next, a two inch long flexible fused silica capillary tubing is inserted through each glass window hole.To do this first insert a needle and then insert the capillary tubing until it reaches the channel. Seal the glass window holes using a fast curing silicone casting compound such as quick cast. Rinse the channels with ethanol and water after plasma activating the cell and a freshly piranha cleaned one inch circular quartz cover slip for 30 seconds.Fuse the cover slip to the side of the cell containing the channels. The chambers are now sealed. Leave the assembled cell curing overnight at room temperature.After the assembled microfluid Excel has cured overnight, it is placed in a custom made holder and mounted on the stage of the confocal microscope to begin activating the surface of the cell. For molecule immobilization, connect flexible tubing to the capillaries of the cell to facilitate buffer injection using a syringe and a needle. Now inject a solution of 0.1 milligrams per milliliter biotinylated BSA in buffer A into the channel and incubate for at least 30 minutes.After the 30 minute incubation flow. 300 to 500 microliters of buffer A To remove any unabsorbed biotinylated, BSA from the channel, be careful not to trap any air bubbles in the chamber. Next, inject a solution of 0.1 milligrams per milliliter STR din in buffer A and incubate for 15 minutes.After the 15 minute incubation flow, 300 to 500 microliters of buffer A to remove any unabsorbed strep din from the channel. As before, avoid trapping any air bubbles in the chamber. Finally, flow a solution of three to five picomolar of the biotinylated sample.To check the coverage of fluorescent molecules, a surface scan is performed. Incubate the biotinylated sample for 10 minutes and repeat the flow of buffer. A As shown previously, the imaging buffer or IB consists of an enzymatic oxygen scavenging system and a triplet quencher such as trol locks to make the IB first dissolve.Five milligrams of trol locks into 10 milliliters of glucose buffer by vortexing for a couple of minutes and shaking for at least 10 minutes. Filter the solution and leave it to gassing in a vacuum for 10 minutes. Gently mix the TRO lock solution with the Glocky enzymatic solution while avoiding the formation of air bubbles.Fill the reservoir with the IB solution. Then using a mechanical syringe pump control the flow rate, flow the IB through the channel at a constant rate of five microliters per minute, bubble argon gas into the IB reservoir to prevent atmospheric oxygen from dissolving into the buffer. The surface scanning, molecule localization and acquisition of single molecule intensity traces is controlled by the lab view program, which scans 20 by 20 micrometer squared areas at 0.2 micrometer resolution with a rate of 0.1 micrometer per millisecond.The data from the surface scanning is immediately processed to yield the intensity weighted locations of all pixels above the background counts. Once the immobilized molecules are found, they are moved one by one into the confocal volume and the intensities of the donor and acceptor fluorophore are recorded as a function of time until both flora fours photo bleach. The stage is then programmed to move to a new origin, 100 millimeters away and the scanning process is repeated.The assembled MICROFLUID Excel is shown here. These are surface scans showing single molecules in the green donor and red acceptor channels. Some typical single molecule traces showing the acceptor intensity in red and donor in green are shown.We've just shown you how to obtain single molecule traces from immobilized biomolecules using a confocal setup. When doing this procedure, it's important to remember to do every step carefully and take your time since this is a challenging experiment. So that's it.Thanks for watching and good luck with your experiments.

automated-system-for-single-molecule-fluorescence-measurements

Research

JoVE Journal

Biology

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

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

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

This article presents a method for spatiotemporal analysis of mobile, single-molecule Förster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

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

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

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.

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

Learn single molecule FRET measurements using an automated scanning confocal microscope with surface-immobilized DNA. This method enables real-time study of nucleic acid dynamics and enzyme interactions.

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Explore More Videos