We thank Beate Nitschke for her help with protein preparation and Robin Ghosh, Michael Schweikert (Stuttgart), and Maximilan Ulbrich (Freiburg) for insightful discussions. This work was supported by the Stuttgart Research Center Systems Biology (SRCSB) and a grant from the Baden W&#252;rttemberg Foundation (BiofMO-6 to SN).

We declare no conflicts of interest.

The protocol presented here provides an introduction to the use of DIB membranes to study the interplay between lateral ion channel movement and channel function using single-molecule TIRF microscopy. To obtain the best possible data, the preparation of stable DIB membranes with as many well-separated channels as possible is crucial for obtaining time series of individual particles, which can be analyzed satisfactorily.
Critical parameters to be optimized include the choice of lipid, the lipid concentration in the oil phase, and the protein and detergent concentrations in the aqueous droplets. The lipids employed are unusual, in that they show no clear phase transition at low temperatures. DPhPC is a commonly used lipid to produce stable membrane systems40. In principle, any lipid which maintains its fluid environment at low temperatures may be suitable for this application. In addition, the lipid should not be sensitive to oxidation. The detergent concentration in the droplets should be as low as possible to avoid membrane rupture. Stable membranes and good protein incorporation rates are generally achieved with detergent concentrations below the critical micelle concentration (cmc), given that the membrane protein does not precipitate.
If the DIB membranes do not tolerate specific detergents21,41, or if the proteins do not integrate from low detergent solution into the DIB membranes, the protein channels can first be reconstituted into small unilamellar lipid vesicles (SUVs), which are then fused to the DIB membranes from the droplet side, as has been successfully shown for E. coli MscL42. Sometimes, DIB membranes do not form because the lipid concentration in the oil phase is too low. To prevent DIB membranes from bursting, one should also be aware that the osmotic pressure between the hydrogel and the droplet must be precisely balanced without affecting the Ca2+-flux from cis to trans excessively. Optimized agarose thickness and mesh size seem to be crucial to observe the diffusion of membrane proteins. Any drying of the agarose layer should be avoided. The thickness can be determined using atomic force microscopy17. By varying the agarose concentration, volume, and rotation speed during spin coating, the mesh size and thickness of the hydrogel can be optimized. Note, however, that hydrogel layer thickness affects image contrast. To capture membrane proteins in DIBs, the agarose hydrogel can be replaced by custom-synthesized, non-crosslinked, Ni-NTA-modified, low-melting agarose to trap them via a His-tag17. An excessively high fluorescence background is often caused by rupture of the DIB membranes. This is particularly a problem with multi-well chambers, as the Ca2+-sensitive dye diffuses into the hydrogel. In this case, adjacent wells should be avoided. Fluorescence bleaching of the Ca2+-sensitive dye above the membrane should not be a significant limiting factor, as it is exchanged by unexcited dyes in the bulk of the droplet (Figure 3A) outside the TIRF evanescent field. The localization precision for the protein is given by the accuracy of fitting the spots and the pixel size.
Weak fluorescence signals can be caused by low Ca2+-flux through the channel. Possible reasons include: (i) inaccurate TIRF settings (e.g., laser intensity), (ii) the osmotic Ca2+ pressure across the membrane, or (iii) the intrinsic Ca2+-permeability of the channels is too low. To cope with the first issue, laser intensity, TIRF angle, and camera gain need to be optimized. The latter two issues can be overcome by the application of an electrical potential across the membrane2,43. However, the application of external voltages can distort the result, as electrical effects can influence the channel opening of ligand-gated or mechanosensitive ion channels that are actually not voltage-controlled. Examples of such channels are the mitochondrial protein translocase TOM-CC27, and its channel-forming subunit Tom4026,44,45,46. Finally, it should be noted that, inserting membrane proteins into DIB membranes in a specific orientation to achieve a desired functionality is tricky, and quantitative studies are rare47,48. In some cases, the orientation of the integrated proteins is random. This is a serious problem for studying membrane proteins, because certain membrane proteins are activated on only one side of the membrane.
TIRF microscopy is a powerful method for addressing single-molecule events in planar supported membranes49. Examples include assembly and folding pathway elucidation of channel proteins such as &#945;-hemolysin50, perfringolysin O51, and OmpG52. These studies included FRET as an additional technique. In addition, activation of the mechanosensitive ion channel MscL has previously been studied by mechanical stimulation of supported DIB bilayers42 using current measurements. Based on this work, future studies could combine the platform described here with single-molecule FRET experiments to address mechanosensitive channels at the single-molecule level in an optical manner17. Injection of buffer into the droplet, stretching the inner DIB monolayer, or targeted binding of individual channels to the underlying hydrogel can be used to further study not only the physical mechanism of mechanically activated channels, which respond to membrane tension and/or curvature as shown for MscL and MscS, the two-pore domain K+-channels, TREK-1, TREK-2, and TRAAK, and PIEZO (for review, see53), but also local binding to the cellular cytoskeleton, as shown for the touch-sensitive ion channel NOMPC54,55.

The present protocol aims to describe how to study the correlation between membrane mobility and ion channel permeability of membrane proteins in polymer-supported droplet-interface bilayer (DIB) membranes1,2,3.
The present technique complements an impressive array of advanced optical and surface analytical tools, such as single particle tracking4,5, fluorescence correlation spectroscopy6,7, and high-speed atomic force microscopy8,9,10. These provide valuable insights into the dynamic composition and structure of membranes that influence membrane-based reactions11,12,13. While the movement and lateral diffusion of proteins depends on the local density of proteins in the membrane, individual protein molecules can also be trapped in lipid rafts14 and by protein-protein interactions15,16. Depending on the protein domains protruding from the membrane into the extracellular environment or cytosol, protein mobility can vary from highly mobile to completely immobile. However, due to the complexity of the membrane and its peripheral structures, it is often difficult to decipher the interplay between the nature of lateral mobility and protein function17.
DIB membranes have proven to be an efficient platform for biophysical single-molecule analyses of membrane proteins18,19,20,21,22. They are formed by lipid self-assembly through the contact of aqueous droplets with hydrogel-supported substrates in a lipid/oil phase. Similar to the commonly used supported lipid bilayers (SLBs)1,23,24,25, DIBs allow local tuning of the lateral mobility by temporary or permanent binding of proteins to the polymer matrix when functionalized with suitable ligands17. The latter can serve as a model system for biochemical processes in cell membranes with a heterogeneous protein distribution10.
The experimental approach described here relies on the fluorescence of Ca2+-sensitive dyes to measure the Ca2+ ion flux through individual channels in close proximity to the membrane2,22 using TIRF microscopy. This optical approach limits the illumination of the sample to a distance close to the membrane, which, due to the physical properties of the evanescent excitation light, leads to a significant reduction of the fluorescence background. The latter is a prerequisite if high spatial and temporal resolution is required for the detection of single molecules. In contrast to classical electrophysiological methods26,27, no membrane voltages are required to study ion flux through individual channels. Furthermore, the method does not require labeling with fluorescent dyes or molecules that could interfere with the lateral movement of the channels in the membrane.
The method is particularly useful for studying protein channels embedded in the membrane at the single molecule level without using classical electrophysiology. Using mitochondrial protein-conducting channel TOM-CC from Neurospora crassa28,29,30 and OmpF, which supports the diffusion of small hydrophilic molecules across the outer membrane of Escherichia coli17,31, we illustrate how the membrane mobilities and channel activities of the two proteins can be studied and correlated. We suggest that this approach, although optimized for TOM-CC and OmpF, can be readily applied to other protein channels.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-diphytanoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850356C</td>
			<td></td>
		</tr>
		<tr>
			<td>100x Oil objective Apochromat N.A. 1.49</td>
			<td>Nikon</td>
			<td>MRD01991</td>
			<td>TIRF microscope&#160;</td>
		</tr>
		<tr>
			<td>10x Hoffmann modulation contrast objective NA 0.25&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope to assess DIB membrane formation&#160;&#160;</td>
		</tr>
		<tr>
			<td>10x objective N.A. 0.25</td>
			<td>Nikon</td>
			<td>MRL00102</td>
			<td>TIRF microscope&#160;</td>
		</tr>
		<tr>
			<td>40x Hoffmann modulation contrast objective NA 0.55</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope to assess DIB membrane formation&#160;&#160;</td>
		</tr>
		<tr>
			<td>488 nm laser, 100 mW&#160;&#160;</td>
			<td>Visitron</td>
			<td></td>
			<td>TIRF microscope&#160;</td>
		</tr>
		<tr>
			<td>Adhesive tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;kta pure&#160;&#160;</td>
			<td>Cytiva</td>
			<td></td>
			<td>Protein purification system</td>
		</tr>
		<tr>
			<td>Bradford assay kit, Pierce</td>
			<td>Thermo Fisher&#160;</td>
			<td>23236</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Roth</td>
			<td>5239.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chelax 100 resin</td>
			<td>Biorad</td>
			<td>143-2832</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MC1024452500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis cassettes Slide-A-Lyzer 20 k MWCO</td>
			<td>Thermo Fisher&#160;</td>
			<td>87735</td>
			<td></td>
		</tr>
		<tr>
			<td>DIB chamber&#160;</td>
			<td>Custom made</td>
			<td></td>
			<td>PMMA chamber for DIB membranes</td>
		</tr>
		<tr>
			<td>Digital power meter and energy console</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Laser power meter&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Roth&#160;</td>
			<td>4720.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Double distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse TS 100 Hoffmann modulation contrast microscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope to assess DIB membrane formation&#160;&#160;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8042.2</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera iXon Ultra 897</td>
			<td>Andor</td>
			<td></td>
			<td>TIRF microscope&#160;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>32205-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed angle rotor Ti70</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-8, CalciFluorTM</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-362561</td>
			<td>Ca2+-sensitive dye</td>
		</tr>
		<tr>
			<td>French press cell disruption homogenizer</td>
			<td>Igneus</td>
			<td>Igneus 40000 psi</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP filter</td>
			<td>AHF</td>
			<td></td>
			<td>Filter seeting used for excitation of DIB-membranes by epifluorescence with white light source&#160;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>4878</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 40 mm x 24 mm x 0.13 mm</td>
			<td>Roth</td>
			<td>1870.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 10 mL</td>
			<td>Roth</td>
			<td>X033.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 100 mL</td>
			<td>Roth</td>
			<td>X049.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 500 mL</td>
			<td>Roth</td>
			<td>EY49.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block&#160;</td>
			<td>Eppendorf</td>
			<td>Thermomixer comfort</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Minitube&#160;</td>
			<td>HT200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Roth&#160;</td>
			<td>9205.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadcane</td>
			<td>Sigma-Aldrich</td>
			<td>296317</td>
			<td></td>
		</tr>
		<tr>
			<td>His Trap HP 1 mL</td>
			<td>Cytiva</td>
			<td>29051021</td>
			<td>Ni-NTA column</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>1.04716.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Honeywell</td>
			<td>10314243</td>
			<td></td>
		</tr>
		<tr>
			<td>KLM spin coater&#160;</td>
			<td>Schaefer Tec</td>
			<td>SCV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>List medical L/M-3P-A vertical pipette puller</td>
			<td>Artisan Technology Group</td>
			<td>57761-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9414</td>
			<td></td>
		</tr>
		<tr>
			<td>M8 Stereomicroscope</td>
			<td>Wild</td>
			<td></td>
			<td>Stereomicrosope&#160;</td>
		</tr>
		<tr>
			<td>Matlab&#160;</td>
			<td>MathWorks</td>
			<td>R2022a</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroFil pipette tips</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 gas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>3957.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoliter 2010 injector&#160;</td>
			<td>World Precision Instruments</td>
			<td>Nanoliter 2010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecyl-b-D-maltoside</td>
			<td>Glycon Biochemicals</td>
			<td>D97002-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA agarose, non-crosslinked</td>
			<td>Cube Biotech</td>
			<td>124115393</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>NIS-Elements AR software</td>
			<td>Nikon</td>
			<td>MQS31100/MQS42560/MQS42580/MQS42780/MQS41930</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>n-octyl-polyoxyethylene</td>
			<td>Sigma-Aldrich</td>
			<td>40530</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 gas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>Roth&#160;</td>
			<td>6367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode sensor&#160; Si, 400 - 1100 nm, 500 mW</td>
			<td>Thorlabs</td>
			<td>S130C</td>
			<td>Sensor for laser power meter&#160;</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Diener Electronics</td>
			<td>Zepto</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparative ultracentrifuge Optima</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-band TIRF-filter 446/523/600/677 HC</td>
			<td>AHF</td>
			<td></td>
			<td>Filter setting used for excitation of DIB-membranes with 488 nm laser&#160;</td>
		</tr>
		<tr>
			<td>Resource Q 1 mL</td>
			<td>Cytiva</td>
			<td>17117701</td>
			<td>Anion exchange column</td>
		</tr>
		<tr>
			<td>Silicon oil AR 20</td>
			<td>Sigma-Aldrich</td>
			<td>10836</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Roth</td>
			<td>2326.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Super LoLux camera</td>
			<td>JVC</td>
			<td></td>
			<td>Stereomicrosope&#160;</td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>Gerhardt</td>
			<td>THL 500/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-E Fluorescence microscope&#160;&#160;</td>
			<td>Nikon</td>
			<td>MEA53100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>9090.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Roth</td>
			<td>8952.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath&#160;</td>
			<td>Bandelin Sonorex</td>
			<td>RK 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum pump</td>
			<td>Vacuubrand</td>
			<td>MD 4C NT</td>
			<td></td>
		</tr>
		<tr>
			<td>White light source for epifluorescence illumination (100 W)</td>
			<td>Nikon</td>
			<td>MBF72655</td>
			<td>TIRF microscope&#160;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Roth</td>
			<td>2363.2</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-molecule imaging of lateral mobility and ion channel activity in lipid bilayers using total internal reflection fluorescence (tirf) microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Real-time electrode-free optical single-channel recording reveals the interplay between lateral protein movement and the function of individual ion channels in DIB membranes. Reconstitution of the mitochondrial TOM core complex (Figure 1A) into a DIB membrane (Figure 4D) illustrates a strong temporal correlation between lateral mobility and ion permeability (Figure 5A). The TOM-CC gating appears to be sensitive to the mode of lateral movement17. Moving channels show Ca2+ flux through their pores and high fluorescence point intensities. Trapped, non-moving molecules show low and medium fluorescence intensities. For the general protein import pore of mitochondria TOM-CC, this single molecule approach revealed a strong temporal correlation between lateral mobility and ion permeability, suggesting that the TOM-CC channel has mechanosensitive properties17. A lateral stop of freely moving TOM-CC molecules is accompanied by a partial or complete closure of the TOM-CC channel. Imaging DIB membranes with OmpF (Figure 1E and Figure 4F), which is almost fully embedded in the membrane, shows no stop-and-go effects (Figure 5B). Random stops of OmpF are not associated with a change in intensity and, thus, with the closing of its pores. Based on the fluorescence signals38, the positional accuracy of the individual channels can be estimated in the range between 5 and 10 nm. However, it should be noted that this accuracy cannot be achieved if the channels wobble slightly due to mobile anchoring with the agarose hydrogel, as shown, for example, for the TOM-CC molecules in an intermediate state with a root mean displacement of 120 nm (Figure 5A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64970/64970fig01.jpg" /> 
Figure 1: Isolation of TOM-CC. (A) Cryo EM structure of N. crassa TOM-CC30,39. Mitochondria from a N. crassa strain containing a Tom22 with a 6xHis tag were solubilized in DDM and subjected to Ni-NTA affinity chromatography (B) and anion-exchange chromatography (C). (D) SDS-PAGE of isolated TOM-CC. (E) Crystal structure (PDB, 1OPF) and (F) SDS-PAGE of purified E. coli OmpF. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64970/64970fig02.jpg" /> 
Figure 2: Flow chart of PMMA chamber assembly. Step 1: A glass coverslip is spin-coated with an agarose hydrogel. Step 2: The spin-coated coverslip is mounted in a custom-made PMMA microscopy chamber. Step 3: Additional low melt agarose is added into the inlet port of the PMMA chamber on a 35 &#176;C hot plate. Step 4: Lipid monolayers are formed around aqueous droplets at a buffer/oil interface (left) and on the agarose hydrogel/oil interface (right). Step 5: Individual aqueous droplets are pipetted into the wells of the PMMA chamber to form a lipid bilayer upon contact of the two lipid monolayers. Step 6: Formation of the DIB membranes is validated by Hofmann modulation contrast microscopy. Step 7: Images of selected areas of the DIB membranes with inserted ion channels are acquired by TIRF microscopy. Green: 0.75% agarose; yellow: 2.5% agarose containing Ca2+ ions; magenta: lipid/oil phase; dark blue: aqueous droplet buffer containing Ca2+-sensitive dye and protein. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64970/64970fig03.jpg" /> 
Figure 3: Experimental setup. (A) Schematic representation of a DIB membrane in a PMMA well. The bilayer rests on an ultrathin 0.75% agarose film to allow TIRF imaging of Ca2+-flux through an ion channel over time using a Ca2+-sensitive fluorescence dye (Fluo-8) in trans. (B) The Ca2+-flux is controlled exclusively by the osmotic pressure from cis to trans. This allows both the determination of the position in the membrane and the open-closed state of the channel. The channel shown here is the protein-conducting channel TOM-CC of N. crassa mitochondria30. (C) Trajectory of a single TOM-CC channel. Green: moving channel; yellow: non-moving channel. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64970/64970fig04.jpg" /> 
Figure 4: Imaging TOM-CC and OmpF in DIB membranes. (A) DIB membrane imaged by Hoffmann modulation contrast microscopy showing the bilayer contact area between the hydrogel and the droplet. (B) Broken DIB membrane imaged as in (A). Arrow, edge of PMMA chamber. (C) DIB membrane imaged by TIRF microscopy without protein channels. (D) DIB membrane with reconstituted TOM-CC, imaged by TIRF microscopy. The white squares mark spots of high (SH), intermediate (SI), and low (SL) intensity. (E) Fitting the fluorescence intensity profile of the three spots marked in (A) to two-dimensional Gaussian functions reveals the position of individual TOM-CCs and the Ca2+ flux through the channel. (F) DIB membrane with reconstituted OmpF. (G) Gaussian fit of the fluorescent spot marked in (F). In contrast to the two-pore &#946;-barrel protein complex TOM-CC, the three-pore &#946;-barrel OmpF reveals only one permeability state. Pixel size, 0.16 &#956;m.&#160;<a href="https://www.jove.com/files/ftp_upload/64970/64970fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64970/64970fig05.jpg" /> 
Figure 5: Channel activity correlates with lateral mobility of TOM-CC. (A) The fluorescent amplitude trace (top) and corresponding trajectory (bottom) of TOM-CC indicate that the open-closed channel activity of TOM-CC correlates with the lateral membrane mobility of the complex. The trajectory displays three permeability states. Green: fully open state; yellow: intermediate permeability state; red: closed channel state; red star: TOM-CC in the intermediate state wobbles around its mean position by about &#177;60 nm. The positional accuracies37 based on the fluorescence signals in the fully open and intermediate states range between 5 nm and 10 nm. (B) Fluorescent amplitude trace (top) and corresponding trajectory (bottom) of OmpF. OmpF reveals only one intensity level, regardless of whether it is in motion or trapped. The trajectory segments corresponding to the time periods of trapped molecules are marked in grey. <a href="https://www.jove.com/files/ftp_upload/64970/64970fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer&#160;</td>
			<td colspan="3">Reagent concentrations</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>A1*</td>
			<td colspan="3">20 mM Tris-HCl pH 8.5, 0.1 % (w/v) n-dodecyl-&#946;-D-maltoside (DDM), 10% (v/v) glycerol, 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>A2*</td>
			<td colspan="3">20 mM Tris-HCl pH 8.5, 0.1% (w/v) DDM, 10% (v/v) glycerol, 1 M Imidazole and 1 mM PMSF</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>B1*</td>
			<td colspan="3">20&#8201;mM HEPES pH 7.2, 0.1% (w/v) DDM, 2% (v/v) dimethyl sulfoxide (DMSO)</td>
			<td>100 mL&#160;</td>
		</tr>
		<tr>
			<td>B2*</td>
			<td colspan="3">20&#8201;mM HEPES pH 7.2, 0.1% (w/v) DDM, 1 M KCl, 2% (v/v) dimethyl sulfoxide (DMSO)</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td colspan="5">* Degas and pass through a 0.22 &#956;m filter before use.</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer solutions for TOM-CC isolation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer&#160;</td>
			<td colspan="3">Reagent concentrations</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>LB*</td>
			<td colspan="3">1% (w/v) tryptone, 1% (w/v) NaCl and 0.5% (w/v) yeast extract&#160;</td>
			<td>1100 mL</td>
		</tr>
		<tr>
			<td>C1</td>
			<td colspan="3">2 mM MgCl2, and ~750 units DNAse and 50 mM Tris-HCl pH 7.5&#160;&#160;</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>C2</td>
			<td colspan="3">50 mM Tris-HCl, pH 7.5</td>
			<td>50 mL&#160;</td>
		</tr>
		<tr>
			<td>C3</td>
			<td colspan="3">4% (w/v) sodium dodecyl sulfate (SDS), 2 mM &#946;-mercaptoethanol and 50 mM Tris-HCl pH 7.5</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>C4</td>
			<td colspan="3">2% (w/v) SDS, 500 mM NaCl and 50 mM Tris-HCl pH 7.5</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>C5</td>
			<td colspan="3">0.5% (w/v) octyl polyoxyethylene (octyl POE), 1 mM EDTA and 20 mM Tris pH 8.5&#160;</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td colspan="5">* Sterilize before use.</td>
		</tr>
	</tbody>
</table>
Table 2: Buffer solutions for OmpF isolation.

Watch this Scientific Journal Video about Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy at JoVE.co

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

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

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

single-molecule-imaging-lateral-mobility-ion-channel-activity-lipid

Research

JoVE Journal

Biochemistry

2.9K Views.  University of Stuttgart. 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.

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

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

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

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

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.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Dual-Color End-Labeling and Vesicle Encapsulation of RNAs for smFRET-TIRF Studies

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.