SB, SM and DM was supported by institutional funding from the CNRS, Inserm and Aix-Marseille University and program grants from the French National Research Agency (ANR-17-CE15-0032-01 and ANR-18-CE15-0021-02) and the French &#34;Investissement d&#39;Avenir&#34; (ANR-10-INBS-04 France-BioImaging, ANR-11-LABX-054 labex INFORM). KW acknowledges &#34;BioTechNan&#34;, a program of interdisciplinary environmental doctoral studies KNOW in the field of Biotechnology and Nanotechnology. EB acknowledges the financial support of the National Science Centre of Poland (NCN) under project no. 2016/21/D/NZ1/00285, as well as the French Government and the Embassy of France in Poland. M&#321; acknowledges the financial support from the Polish Ministry of Development (CBR POIR.02.01.00-00-0159/15-00/19) and the National Center for Research and Development (Innochem POIR.01.02.00-00-0064/17). TT acknowledges financial support from the National Science Center of Poland (NCN) under project no. 2016/21/B/NZ3/00343 and from the Wroclaw Biotechnology Center (KNOW).

1. Setting specification for assembling a custom-made svFCS setup
NOTE: The simplicity of the proposed svFCS setup allows easy installation, operation, and maintenance at a low cost while ensuring efficiency in photon recovery. For more details, see Supplementary material.
<ol>
	<li>Experimental room and safety
	<ol>
		<li>Install the system in a room stabilized at around 21 &#176;C.</li>
		<li>Avoid direct airflow on the passive (or active) optical table and follow the laser safety rules for optical alignment.</li>
	</ol>
	</li>
	<li>Hardware and software 
	NOTE: Supplementary material details the installation steps depicted in Figure 2.
	<ol>
		<li>Write the main acquisition and control software in LabVIEW using a state machine and event structure architecture where a multifunction acquisition board drives most of the controllers. 
		NOTE: The correlator, laser, and power meter are controlled or monitored by their own software.</li>
		<li>Adapt the hardware and software installation procedures according to the hardware used.</li>
	</ol>
	</li>
	<li>Optical setup 
	NOTE: Figure 3 illustrates the optical bench modules used in the following sections to control the quality of the optical alignments. All of the optical element specifications are listed in Table of materials. The procedure to build the setup is widely detailed in Supplementary material. This system comprises a continuous wave laser, a motorized inverted microscope equipped with an immersion water objective, an avalanche photodiode detector coupled to a single photon counting module, and a hardware correlator. A microscope incubation chamber with vibration-free heaters has been specially designed to control the temperature for experiments on living cells. By convention, the XY axis corresponds to the optical path&#39;s perpendicular plane, and the Z-axis corresponds to the optical path.</li>
</ol>
2. Daily checkpoint before running the experiment
<ol>
	<li>Control the excitation path (Figure 3, <img alt="Equation 3" src="/files/ftp_upload/61823/61823equ03v2.jpg" /> &#38; <img alt="Equation 4" src="/files/ftp_upload/61823/61823equ04v2.jpg" />).
	<ol>
		<li>Open all the iris diaphragms.</li>
		<li>Measure the laser power with the power meter, keeping the first iris fully open.</li>
		<li>Turn the half-wave plate (HWP) to find the maximum power.</li>
		<li>Check the alignment using the irises if the laser power is lower than usual, and move L1 and M1 alternately, if necessary.</li>
		<li>Note the power value in the experiment laboratory notebook.</li>
	</ol>
	</li>
	<li>Control the detection path (Figure 3, <img alt="Equation 5" src="/files/ftp_upload/61823/61823equ05v2.jpg" /> &#38; <img alt="Equation 6" src="/files/ftp_upload/61823/61823equ06v2.jpg" />).
	<ol>
		<li>Place the water, a coverslip, and a droplet of a 2 nM rhodamine 6G (Rh6G) solution on the objective.</li>
		<li>If the fluorescence signal (count number on the APD, recorded with the LabVIEW software) is lower than usual, remake the Rh6G solution, check the positioning and the coverslip&#39;s number on the objective lens, or eliminate bubbles, if any.
		<ol>
			<li>If the fluorescence signal is still lower than usual, place the power meter inside the optical path to block the beam.</li>
			<li>Turn off the APD (hereafter, APD refers to the APD and the single photon counting module).</li>
			<li>Remove the sample.</li>
			<li>Clean and replace the objective lens with a reflective target.</li>
			<li>Check the laser beam on the reflective target by removing the power meter from the light path. Make sure that the target&#39;s beam is centered, and the back reflection reaches the first iris on the line <img alt="Equation 4" src="/files/ftp_upload/61823/61823equ04v2.jpg" /> (Figure 3).</li>
			<li>If not, adjust the center positioning with M2 or the back reflection with the dichroic mirror.</li>
			<li>If microscope coupling is correct, push the objective lens back, add a drop of water, a coverslip, and a droplet of a more concentrated Rh6G solution (i.e., 200 nM), and set a lower laser power than for the classical measurements (few &#181;W).</li>
			<li>Turn on the APD and optimize APD and pinhole alignment, alternately, with their respective XYZ adjustment screws while monitoring the intensity signal (LabVIEW software).</li>
			<li>Change the coverslip and add a lower concentration of Rh6G (2 nM). Move the pinhole along the Z-axis to find a position where the molecular brightness ratio increases, and the waist is minimum.</li>
			<li>Close the iris until the signal drops down: the laser beam size reaches the back aperture size of the objective (i.e., the minimal waist size, see Supplementary material).</li>
			<li>Launch the correlator software and record data (see section 7 for data recording).</li>
			<li>Check the ACF, which should display a low amount of noise, give a small waist size, and a high count-rate per molecule per second (see section 7 for data analysis and waist size evaluation).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. General considerations for svFCS data recording and analysis
<ol>
	<li>Record and analyze the fluorescence data following this general scheme (see sections 7, 8, and 9): (1) fluorescence recording and ACF generation (correlator software), (2) unexpected discarding of data, an average of retained data, fitting with the appropriate model (with homemade Igor Pro software), (3) diffusion law plot (homemade MATLAB software 1), and (4) optional diffusion law comparison (homemade MATLAB software 2). The different software programs are available upon request. 
	NOTE: The hardware correlator has a minimum sampling time of 12.5 ns (i.e., a sampling frequency of 80 MHz). It provides a temporal resolution that is at least 1,000 lower than the typical resident time of freely diffusing small molecule in solution and 106 smaller than the diffusion time of membrane proteins within a confocal observation volume.</li>
</ol>
4. Cell culture and transfection
<ol>
	<li>Seed the Cos7 cells in 8-well chambered coverglass with #1.0 borosilicate glass bottom at a density of 10,000 cells/well using complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 U/mL), and L-glutamine (1 mM).</li>
	<li>Culture the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for 24 h.</li>
	<li>Remove the medium, add 300 &#181;L of the fresh complete culture medium per well, and preincubate the cells for 30 min at 37 &#176;C.</li>
	<li>Dilute 0.5 &#181;g of the plasmid DNA encoding Thy-1 protein fused with eGFP25 in 50 &#181;L of serum-free DMEM. Vortex briefly to mix.</li>
	<li>Dilute 1.5 &#181;L of the DNA transfection reagent in 50 &#181;L of serum-free DMEM, and mix the solution well.</li>
	<li>Add the diluted transfection reagent directly into the prepared DNA solution, and mix the compounds immediately.</li>
	<li>Incubate the prepared mixture for 10 to 15 min at room temperature.</li>
	<li>Add 10 &#181;L of the combined DNA/transfection reagent complexes dropwise onto the medium in each well, and homogenize by gently swirling the plate.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 3 h.</li>
	<li>After the incubation, replace the medium containing DNA/transfection reagent complexes with 400 &#181;L of fresh complete DMEM, and culture the cells for 16 h before the svFCS experiment.</li>
</ol>
5. Preparation of cells for svFCS measurements
<ol>
	<li>Remove the culture medium.</li>
	<li>Wash the cells gently two to three times with serum-free Hank&#8217;s balanced salt solution (HBSS) buffer containing Ca2+ and Mg2+ supplemented with 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), pH 7.4 (HBSS/HEPES).</li>
	<li>Maintain the cells in HBSS/HEPES buffer during all svFCS acquisitions.</li>
</ol>
6. Pharmacological treatment
<ol>
	<li>Remove the culture medium, and wash the cells two to three times with serum-free HBSS supplemented with 10 mM HEPES, pH 7.4 (HBSS/HEPES).</li>
	<li>Incubate the cells with 1 U/mL of cholesterol oxidase (COase) solution in HBSS/HEPES buffer for 1 h at 37 &#176;C.</li>
	<li>Remove the solution, and maintain the cells in the presence of 0.1 U/mL of COase in HBSS/HEPES buffer while performing the svFCS measurements.</li>
</ol>
7. Spot size calibration
<ol>
	<li>Prewarm the microscope chamber at 37 &#176;C.</li>
	<li>Prepare a standard 2 nM solution of Rh6G by serial dilution.</li>
	<li>Drop 200 &#181;L of 2 nM Rh6G solution on a glass coverslip placed on the water-immersion objective.</li>
	<li>Start all the hardware and software.</li>
	<li>Measure and adjust the 488 nm laser beam power to 300 &#181;W.&#160;Depending on the brightness and the photo-stability of the fluorescent probe used, adapt this power according to (1) the fluorescence intensity (on the LabVIEW software), which should be stable, (2) the ACF shape (on the correlator software), which should have a constant shape over the time, and (3) the fitting parameters giving a small waist size and a high count rate per molecule (photons per molecule per second, typically few tens to hundreds of photons per molecule per second). 
	NOTE: The amplitude of the ACF (called G(0)) is inversely proportional to the number of the molecule (i.e., the concentration of the fluorescent probe). For the waist size calibration, this is a good quality control candidate parameter. Therefore, G(0) should be similar for the same concentration from day to day as it links the waist size and concentration. For cell measurements, as FCS is more accurate for low concentration, G(0) should be high for the proper parameter fitting extraction.</li>
	<li>Set the svFCS illumination/detection microscope port with the LabVIEW software.</li>
	<li>Turn on the APD.</li>
	<li>Close the iris until the signal drops down to obtain the minimal waist size, or close it for bigger waist size.</li>
	<li>Record several ACFs of selected duration (namely a run) to improve statistical reproducibility, typically 10 runs lasting for 20 s each with the correlator software.</li>
	<li>Turn off the APD.</li>
	<li>Use the Igor Pro software to check and discard the runs with strong fluctuations due to molecular aggregates. Perform this step manually&#8212; it should be user-independent after users have been trained.</li>
	<li>Fit the average of the retained ACFs with a 3D diffusion model.</li>
	<li>Extract from the fitting parameters the average diffusion time <img alt="Equation 2" src="/files/ftp_upload/61823/61823equ02.jpg" />&#160;and save it into a &#8220;.txt&#8221; file (the file format is dictated by the Igor Pro software).</li>
	<li>Check the count-rate per molecule per second (a good performance indicator) by dividing the average intensity (extracted from the fluorescence trace) by the number of molecules (extracted from the ACF). 
	NOTE: Make sure that this value is high and stable from day to day for the same acquisition parameters.</li>
	<li>Knowing the diffusion coefficient of Rh6G in aqueous solution at 37 &#176;C (D) and <img alt="Equation 2" src="/files/ftp_upload/61823/61823equ02.jpg" /> (see 7.13), calculate the experimental waist size &#969; according to: <img alt="Equation 1" src="/files/ftp_upload/61823/61823equ01.jpg" />.</li>
	<li>Apply the procedure for every waist size modification required to plot the FCS diffusion law and before any new experimental series of svFCS data acquisition.</li>
</ol>
8. svFCS data acquisitions on cells
<ol>
	<li>Measure and adjust the 488 nm beam power between 2 and 4 &#181;W. Depending on the brightness and the photostability of the fluorescent probe used, adapt this power to allow a high count rate per molecule (typically several thousand of photons per molecule per second), while keeping the photobleaching low (i.e., a stable intensity trace on the LabVIEW software).</li>
	<li>Equilibrate samples for 10 min at 37 &#176;C before starting the measurements.</li>
	<li>Set the epi-fluorescence illumination microscope with the LabVIEW software.</li>
	<li>Choose a cell with an appropriate fluorescent probe location and (low) fluorescence signal intensity. 
	NOTE: The lower the fluorescence is, the better the FCS measurements are (see step 8.1).</li>
	<li>Set the svFCS illumination/detection microscope port with the LabVIEW software.</li>
	<li>Turn on the APD.</li>
	<li>Perform an xy-scan of the selected cell with the LabVIEW software.</li>
	<li>Perform a z-scan and locate the confocal spot at the maximal fluorescence intensity by choosing the plasma membrane at the top and start the data acquisition. To maximize the separation between the two membranes, preferably perform the scan in the nuclear area of the cell.</li>
	<li>Record one series of 20 runs lasting for 5 s, each with the correlator software. 
	NOTE: Make sure that the duration of each run is long enough to obtain ACFs with reduced noise. Long acquisitions are susceptible to photobleaching or unexpected substantial variations (e.g., aggregates). Adapt the number of runs, their duration, and the number of series to the samples, but make sure that they remain constant within the same bulk of experiments for reproducibility.</li>
	<li>Turn off the APD.</li>
	<li>Discard unexpected runs with the Igor Pro software.</li>
	<li>Fit the average ACF with a 2-species 2D diffusion model. Adapt this model to the type of diffusion behavior of the target molecule.</li>
	<li>Save the fitting parameters into the previous file (see step 7.13).</li>
	<li>Perform 10 to 15 series of recordings on at least 10 different cells, and reproduce steps 8.3 to 8.13. Check that the single file obtained contains the waist size information and the fitting parameters of the 10&#8211;15 recordings.</li>
	<li>To establish a single diffusion law, analyze at least four waist sizes varying between 200 and 400 nm. This range is defined by the diffraction optical limit, but is objective- (numerical aperture) and laser (wavelength)-dependent. 
	NOTE: As the waist size calibration is not absolute and has some degree of uncertainty, a dedicated MATLAB software28 accounting for the x and y error (namely &#969;2 and td) was built to fit the diffusion law.</li>
	<li>Start the MATLAB software 1 and select a folder containing all the &#8220;.txt&#8221; files corresponding to at least four waist size experiments.</li>
	<li>Plot &#60;td&#62; versus &#60;&#969;2&#62;, namely the diffusion law. Two major parameters can be extracted: the y-axis intercept (t0) and the effective diffusion coefficient (Deff, inversely proportional to the slope).</li>
</ol>
9. Diffusion laws of different experimental condition comparison
NOTE: If necessary, reproduce sections 7 and 8 for different experimental conditions. A dedicated software (MATLAB software 2) was developed to determine whether these diffusion laws are similar or not according to the t0 and Deff values28. It tests two hypotheses: the two values are different, or the two values are not different at a threshold set above a probability of false alarm (PFA). An arbitrary PFA value of 5% (T = 3.8) is considered the upper limit of significance between two parameters (t0 or Deff), indicating that there is only 5% chance that the two values are identical.
<ol>
	<li>Create an &#8220;.xls&#8221; file containing the characteristic diffusion law values of each condition to compare (i.e., a file containing the t0, t0 error, Deff and Deff error for the non-treated (NT) and treated (COase) conditions as a table).
	<ol>
		<li>Start the MATLAB software 2.</li>
		<li>Select the &#8220;.xls&#8221; file.</li>
		<li>Analyze the generated color-coded 2D plot, where the t0 and Deff statistical tests are to be plotted on the x- and y-axes, respectively (Figure 4). The higher T is, the greater is the difference between the compared values.</li>
	</ol>
	</li>
</ol>
10. Cholesterol concentration measurements
<ol>
	<li>Cell treatment and lysis
	<ol>
		<li>Seed the Cos7 cells in triplicate in 6-well plates at 4 &#215; 105 cells/well and incubate in 2 mL of complete DMEM at 37 &#176;C with 5% CO2 overnight to allow the cells to attach to the plate.</li>
		<li>Remove the culture medium and wash the cells three times with phosphate-buffered saline (PBS).</li>
		<li>Add 1 mL of HBSS/HEPES buffer containing (or not, for controls) 1 U/mL of Coase, and incubate for 1 h at 37 &#176;C with 5% CO2.</li>
		<li>Replace the medium with 1 mL of HBSS/HEPES containing 0.1 U/mL of Coase, and incubate for 1 h at 37 &#176;C with 5% CO2.</li>
		<li>Remove the solution and harvest the cells.</li>
		<li>Wash the cells three times with PBS, and centrifuge at 400 &#215; g for 5 min at room temperature.</li>
		<li>Lyse the cells with radioimmunoprecipitation assay buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP40, 10 mM, MgCl2, 1 mM ethylenediamine tetraacetic acid, 2% glycerol, protease and phosphatase inhibitor cocktail) for 30 min on ice.</li>
		<li>Centrifuge the lysates at 10000 &#215; g for 10 min at 4 &#176;C and collect the supernatant.</li>
	</ol>
	</li>
	<li>Quantify total protein concentration for each sample by modified Bradford&#39;s protein assay using the working solution according to the manufacturer&#39;s recommendations.</li>
	<li>Cholesterol concentration measurement
	<ol>
		<li>To determine total cellular cholesterol level enzymatically, use the appropriate kit (e.g., Amplex Red Cholesterol Assay Kit) according to the manufacturer&#39;s recommendations.</li>
		<li>For each reaction, mix the sample containing 5 &#181;g of protein with Amplex Red reagent/horseradish peroxidase/cholesterol oxidase/cholesterol esterase working solution, and incubate for 30 min at 37 &#176;C in the dark.</li>
		<li>Measure the fluorescence using excitation of 520 nm, and detect the emission at 560&#8211;590 nm using a microplate reader.</li>
		<li>Subtract the background from the final value, and determine cholesterol concentration using a standard curve.</li>
		<li>Calculate the final cholesterol content in ng of cholesterol per &#181;g of protein.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Here, we have described the implementation of the svFCS module on a standard fluorescent microscope, a powerful experimental approach to decipher the dynamics of the plasma membrane organization in living cells thanks to the FCS diffusion law analysis. Conceptually, the svFCS is based on a simple principle: correlation measurements of fluorescence in the time domain while varying the size of the illumination area23. This strategy has been instrumental in deducing nanoscopic information from microscopic measurements, which helps decipher the main physicochemical elements contributing to the plasma membrane organization in steady state25 and physiological processes30,31,32,33. Altogether, these svFCS analyses unambiguously demonstrate the existence of lipid-dependent nanodomains in various cell types and their direct implication in tuning different signaling events.
Within this framework, there are some optical aspects that need to be considered while building the svFCS setup to optimize the photon budget and minimize optical aberrations. Thus, we recommend using a microscope from which the tube lens can be removed when the svFCS measurement is performed. Moreover, a single iris plays a key role in the svFCS setup: it changes the beam size at the back aperture of the objective, thus directly varying the effective waist size (i.e., the effective excitation volume). The beam diameter should fit the objective back pupil to obtain the smallest waist size34. This option, which helps tune the waist size, ensures optimization of the photon budget and is easy to implement. Finally, a minimal number of optical parts are used along the light path; the less complex the system, the fewer the photons that are lost. All of these options significantly improve the robustness of svFCS experiments.
Regarding the protocol itself, a few critical steps have to be considered. The most important is an appropriate alignment of the optical paths that is crucial for successful svFCS measurements (protocol, section 2). This is easy to check by analyzing the fluorescence signal from a 2 nM Rh6G solution, which should be ~200 kHz under 300 &#181;W laser illumination. All irises should be opened, and the ACFs should have an important amplitude (typically G0~1.5&#8211;2.0). Another critical point concerns the cells and their preparation for svFCS analysis (protocol, sections 4&#8211;8). Their density has to be adapted so that isolated cells to be observed are available for analysis. Non-adherent cells have to be immobilized on a chambered coverglass by using poly-L-lysine solution. The fluorescence signal from cell labeling should not be too strong, or it will result in very flat ACFs that are difficult to fit, and the fit parameters are burdened with an important error. Additionally, nonhomogeneous labeling and fluorescence aggregates in cells make the svFCS measurements extremely difficult to interpret. Finally, cholesterol oxidase treatment affects cell viability, and the svFCS analysis should not exceed one hour after the treatment. It is also better to record the fluorescence fluctuations from the upper plasma membrane as it is not attached to the support, and there is no risk of hindered diffusion of molecules due to the physical interactions with the support.
There have been enough advances in the svFCS technique for its use in different approaches owing to the diversity of modalities for adjusting the detection volume, making it possible to study various biological processes in living cells. An alternative to adjusting the size of the excitation volume is to use a variable beam expander35. It is also possible to simply modulate the size of the illumination area by recording the fluorescent signal from the intercept of the plasma membrane along the z direction36. This can be done on a standard confocal microscope for which a theoretical framework has been developed to derive the diffusion law37,38.
Although the svFCS method offers spatio-temporal resolution, which is necessary for the characterization of the inhomogeneous lateral organization of the plasma membrane, the geometrical modes of confinement are not mutually exclusive. A deviation of t0 in one direction or the other exclusively reveals a dominant mode of confinement25. Moreover, another important limitation of the present svFCS method results from the classical optical diffraction limit (~200 nm). This is unquestionably greater than the domains confining the molecules within the cell plasma membrane. Therefore, the analysis of the confinement is inferred from the t0 value, extrapolated from the diffusion law.
This drawback has been overcome by implementing alternative methods. Initially, using metallic films drilled with nanoapertures offered the possibility of illuminating a very small membrane area (i.e., below the optical diffraction limit of single nanometric apertures of radii varying between 75 and 250 nm)39. The transition regime predicted from the theoretical diffusion law for isolated domain organization was thus reported, and it allowed a refinement of the characteristic size of the nanometric membrane heterogeneities and a quantitative estimate of the surface area occupied by lipid-dependent nanodomains39. Alternatively, nanometric illumination has also been developed using near-field scanning optical microscopy40 or planar optical nanoantennas41. More recently, combining stimulated emission depletion (STED) and FCS has provided a powerful and sensitive tool to document the diffusion law with very high spatial resolution. This STED-FCS gives access to molecular diffusion characteristics on a nanoscale occurring within a short period of time, allowing the study of the dynamic organization of lipid probes at the plasma membrane42,43. However, the incomplete suppression of fluorescence in the STED process challenges the analysis of the auto-correlation curves in FCS.
A new fitting model has been developed to overcome this difficulty, improving the accuracy of the diffusion times and average molecule numbers measurements44. Finally, for slow molecular diffusion at the plasma membrane, the svFCS principle can be applied to data recorded by image correlation spectroscopy45. Recently, it has been demonstrated that combining atomic force microscopy (AFM) with imaging total internal reflection-FCS (ITIR-FCS) contributes to the refinement of the nature of the mechanism hindering molecular diffusion at the plasma membrane, especially near the percolation threshold membrane configuration because of a high density of nanodomains46.
In conclusion, establishing diffusion law by svFCS has provided the experimental evidence to infer local heterogeneity created by dynamic collective lipids and membrane proteins&#8217; associations. As stated by Wohland and co-workers46, &#8220;the FCS diffusion law analysis remains a valuable tool to infer structural and organizational features below the resolution limit from dynamic information&#8221;. Still, we need to develop new models to refine the interpretation of the diffusion law that should allow for a better understanding of the dynamics of the molecular events occurring at the plasma membrane.

The complexity of plasma membrane organization 
The current understanding of cell membrane organization has to take into account several aspects1. First, a complex lipid composition varies not only between cell types, but also within a single cell (membrane organelles/plasma membrane). Besides, associated or intrinsic membrane proteins are mostly organized in dynamic multimeric complexes, with large domains extending outside of the membrane, accounting for a significantly larger area than that of the transmembrane domains alone. Moreover, membrane-associated proteins exhibit specific lipid-binding or lipid-interacting capacities that play roles in regulating protein function. These depend directly on the local composition and accessibility of the lipids2.
Finally, a significant level of asymmetry is observed between two membrane leaflets due to the intrinsic asymmetric structure of membrane proteins and the distribution of lipids. Indeed, a lipid metabolic balance between synthesis and hydrolysis, combined with lipid flip-flop between the leaflets, generates such asymmetric distribution. As any transport across the bilayer is constrained by the free energy required to move the polar head group through the hydrophobic interior of the membranes, it is usually assisted by selective transporters. For each cell type, asymmetry tends to be firmly maintained. Altogether, these factors contribute to lateral inhomogeneity or compartmentalization of the plasma membrane3,4.
We enrich this representation of the plasma membrane by taking into account the intrinsic molecular diffusion within and across the bilayer, which contributes to the dynamic lateral heterogeneity on a scale of tenths to hundreds of nanometers and microseconds to seconds. For instance, lipid-dependent membrane nanodomains&#8212;the so-called lipid rafts, defined as cholesterol, and sphingolipid-rich signaling platforms&#8212;contribute to the compartmentalization of the plasma membrane5,6. However, the current view of membrane organization is not restricted to lipid rafts alone. Membrane nanodomains are more complex and heterogeneous in composition, origin, and function. Still, their presence at the plasma membrane has to be tightly coordinated, and dynamic interactions between proteins and lipids seem to be important in the spatial distribution and chemical modification of membrane nanodomains1,3,7,8.
The svFCS principle and its application to probe the organization of the plasma membrane 
Although much progress has been made in the analysis of membrane domains, mainly through biophysical techniques, the determinants that dictate the local organization of the plasma membrane need to be refined with appropriate spatial and temporal resolution. Determinants based on tracking individual molecules provide excellent spatial precision and allow the characterization of different modes of motion9,10,11,12, but have a limited temporal resolution with classical low camera frame rates and require more experimental effort to record a significant number of trajectories. Alternatively, the diffusion coefficient of membrane components can be evaluated by Fluorescence Recovery After Photobleaching (FRAP)13 or Fluorescence Correlation Spectroscopy (FCS)14. The latter has received more attention, mainly because of its high sensitivity and selectivity, microscopic detection volume, low invasiveness, and wide dynamic range15.
The conceptual basis of FCS was introduced by Magde and colleagues about 50 years ago16,17. It is based on recording the fluctuation of fluorescence emission with a high temporal resolution (from &#181;s to s)18. In its modern version, measurements in living cells are performed by a small confocal excitation volume (~0.3 femtoliters) positioned within a region of interest (e.g., at the plasma membrane); the fluorescence signal generated by diffusing fluorescent molecules going in and out of the observation volume is collected with very high temporal resolution (i.e., the time of arrival of each photon on the detector). Then, the signal is computed to generate the autocorrelation function (ACF), from which the average time td (diffusion time) for which a molecule stays within the focal volume is extracted, together with the mean number of particles, (N), present in the observation volume, which is inversely proportional to the amplitude of the ACF. This last parameter might be useful information on the molecule concentration within the observation volume.
Since then, a growing number of FCS modalities have been implemented thanks to rapidly developing instrumentation in biophotonics, allowing the description of dynamic phenomena occurring in living systems. Still, a molecular species would experience a more overlapping distribution of the diffusion coefficient values, which is usually reflected by an anomalous diffusion characteristic, in which molecules diffuse with a nonlinear relationship in time19, and difficulty in identifying the biological meaning of this anomalous subdiffusion. In the past, this difficulty has been somewhat overcome by recording the molecular diffusion by FRAP from areas of various sizes, rather than from just one area, thereby providing additional spatial information. This enabled, for instance, the conceptualization of membrane microdomains20,21,22.
A translation of this strategy to FCS measurements (i.e., the so-called spot variation Fluorescence Correlation Spectroscopy (svFCS)) was established by varying the size of the focal volume of observation, allowing the fluctuation in fluorescence to be recorded on different spatial scales23. Thus, the svFCS approach provides indirect spatial information allowing for the identification and determination of molecular diffusion modes and type of membrane partitioning (isolated versus contiguous domains24) of studied molecules. By plotting the diffusion time td as a function of the various spatial scales defined by the waist (&#969;) value, which corresponds to the detection beam radius size in this case23,25, one can characterize the diffusion law of a given molecule in a given physiological condition. The svFCS is, therefore, a perfect analog to single-particle tracking in the time domain26. Under the Brownian diffusion constraint, one should expect a strictly linear relationship between the diffusion time td and the waist &#969; (Figure 1)23,25. The origin of the deviation of the diffusion law from this scheme can be attributed to nonexclusive reasons, such as cytoskeleton meshwork, molecular crowding, dynamic partitioning in nanodomains, or any combination of these and other effects (Figure 1), and needs to be tested experimentally25.
Here we provide all necessary control checkpoints for the daily use of a custom-made svFCS optical system built from scratch, which complements our previous protocol reviews27,28 on that experimental approach. Further, as a proof of concept, we give guidelines regarding the calibration of the setup, the preparation of cells, data acquisition, and analysis for the establishment of svFCS diffusion law (DL) for Thy1-GFP, a plasma membrane glycosylphosphatidylinositol-anchored protein, which is known to be localized in lipid-raft nanodomains29. Finally, we demonstrate how the partial destabilization of lipid-raft nanodomains by cholesterol oxidase treatment impacts the diffusion properties of Thy1-GFP. Additionally, a detailed description of building a svFCS setup from scratch is provided in Supplementary material.

<table>
	<tbody>
		<tr>
			<td>Aligment tool</td>
			<td>Spanner Wrench for SM1-Threaded Retaining Rings</td>
			<td>Thorlabs</td>
			<td>SPW602</td>
		</tr>
		<tr>
			<td>Avalanche Photodiode and Single Photon Counting Module (SPCM)</td>
			<td>Single-Photon Counting Module, Avalanche Photodiode</td>
			<td>Excelitas</td>
			<td>SPCM-AQRH-15</td>
		</tr>
		<tr>
			<td></td>
			<td>BNC 50 &#937; plug to 50 &#937; plug lead 2 m</td>
			<td>RS Components</td>
			<td>742-4315</td>
		</tr>
		<tr>
			<td></td>
			<td>Coaxial cable 415 Cinch Connectors, RG-316, 50 &#937; With connector, 1.22 m, RoHS2</td>
			<td>RS Components</td>
			<td>885-8172</td>
		</tr>
		<tr>
			<td></td>
			<td>Tee 50&#937; RF Adapter BNC Plug to BNC Socket 0 &#8594; 1GHz</td>
			<td>RS Components</td>
			<td>546-4948</td>
		</tr>
		<tr>
			<td></td>
			<td>Brennenstuhl 2.5 m, 8 Socket Type E &#8211; French Extension Lead, 230 V</td>
			<td>RS Components</td>
			<td>768-5500</td>
		</tr>
		<tr>
			<td></td>
			<td>Mascot, 6W Plug In Power Supply 5V dc, 1.2A, 1 Output Switched Mode Power Supply, Type C</td>
			<td>RS Components</td>
			<td>452-8394</td>
		</tr>
		<tr>
			<td></td>
			<td>Crystek CCSMACL-MC-24 Reference Oscillator Power Cable RF Adapter</td>
			<td>RS Components</td>
			<td>792-4079</td>
		</tr>
		<tr>
			<td>Fluorescence filtering</td>
			<td>535/70 ET Bandpass, AOI 0&#176; Chroma Diameter 25 mm</td>
			<td>AHF filter</td>
			<td>F47-539</td>
		</tr>
		<tr>
			<td></td>
			<td>Laser Beamsplitter zt488 RDC, AOI 45&#176; Chroma 25.5 x 36 x 1 mm</td>
			<td>AHF filter</td>
			<td>F43-088</td>
		</tr>
		<tr>
			<td></td>
			<td>496/LP BrightLine HC Longpass Filter, AOI 0&#176; Chroma Diameter 25</td>
			<td>AHF filter</td>
			<td>F37-496</td>
		</tr>
		<tr>
			<td>Hardware correlator</td>
			<td>80 MHz Digital Correlator</td>
			<td>Correlator.com</td>
			<td>Flex02-12D</td>
		</tr>
		<tr>
			<td>Laser</td>
			<td>LASER LASOS LDM-XT fiber coupled, 488 nm, 65 mW</td>
			<td>Lasos</td>
			<td>BLD-XT 488100</td>
		</tr>
		<tr>
			<td>Laser safety</td>
			<td>High-Performance Black Masking Tape, 1&#34; x 180&#39; (25 mm x 55 m) Roll</td>
			<td>Thorlabs</td>
			<td>T743-2.0</td>
		</tr>
		<tr>
			<td></td>
			<td>Lens Tissues, 25 Sheets per Booklet, 5 Booklets</td>
			<td>Thorlabs</td>
			<td>MC-5</td>
		</tr>
		<tr>
			<td></td>
			<td>Laser Safety Glasses, Light Orange Lenses, 48% Visible Light Transmission</td>
			<td>Thorlabs</td>
			<td>LG3B</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss Axiovert 200M Motorized Inverted Fluorescence Microscope 
			Fine and coarse focusing, reflector turret rotation, objective nosepiece rotation, switching camera ports, and internal light shutters</td>
			<td>Carl Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>C-Apochromat 40x/1,2 W Korr.selected for FCS 
			(D=0.14-0.19 mm) (WD=0.28 mm at D=0.17 mm), UV-VIS-IR</td>
			<td>Carl Zeiss</td>
			<td>421767-9971-711</td>
		</tr>
		<tr>
			<td></td>
			<td>Adapter W0.8 / M27x0.75 H &#34;5&#34;</td>
			<td>Carl Zeiss</td>
			<td>000000-1698-345</td>
		</tr>
		<tr>
			<td></td>
			<td>Middle ring W0.8 - W0.8 H &#34;5&#34;</td>
			<td>Carl Zeiss</td>
			<td>000000-1698-347</td>
		</tr>
		<tr>
			<td>Optical path</td>
			<td>D25.4mm Mirror, Protected Silver</td>
			<td>Thorlabs</td>
			<td>PF10-03-P01</td>
		</tr>
		<tr>
			<td></td>
			<td>D25.4mm, F=60.0.mm, Visible Achromat</td>
			<td>Thorlabs</td>
			<td>AC254-060-A</td>
		</tr>
		<tr>
			<td></td>
			<td>D25.4mm, F=35.0.mm, Visible Achromat</td>
			<td>Thorlabs</td>
			<td>AC254-035-A</td>
		</tr>
		<tr>
			<td></td>
			<td>25 &#181;m mounted pinhole</td>
			<td>Thorlabs</td>
			<td>P25S I</td>
		</tr>
		<tr>
			<td></td>
			<td>25.4mm Mounted Zero, Order 1/2 Waveplate 488 nm</td>
			<td>Thorlabs</td>
			<td>WPH10M-488 (HWP)</td>
		</tr>
		<tr>
			<td></td>
			<td>20mm Polarizing Beamsplitter Cube 420-680 nm</td>
			<td>Thorlabs</td>
			<td>PBS201</td>
		</tr>
		<tr>
			<td></td>
			<td>Rotation Stage 56 mm x 26 mm Threaded ID</td>
			<td>Thorlabs</td>
			<td>RSP1/M</td>
		</tr>
		<tr>
			<td></td>
			<td>52 mm x 52 mm Kinematic Platform Mount</td>
			<td>Thorlabs</td>
			<td>KM100B/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Adjustable Prism Clamp</td>
			<td>Thorlabs</td>
			<td>PM3/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Beam block - active area 19 mm x 38 mm</td>
			<td>Thorlabs</td>
			<td>LB1/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Iris Diaphragm 1 mm to 25 mm Aperture</td>
			<td>Thorlabs</td>
			<td>ID25/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Left-Handed Kinematic Cylindrical Lens Mount</td>
			<td>Thorlabs</td>
			<td>KM100CL</td>
		</tr>
		<tr>
			<td></td>
			<td>1&#34; Optic Holder, M4 Tap</td>
			<td>Thorlabs</td>
			<td>MFF101/M</td>
		</tr>
		<tr>
			<td></td>
			<td>1&#34; Stackable Lens Tube</td>
			<td>Thorlabs</td>
			<td>SM1L03</td>
		</tr>
		<tr>
			<td></td>
			<td>Stackable Lens Mount for 1&#34; optic-usable depth &#189;</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
		</tr>
		<tr>
			<td></td>
			<td>Stackable Lens Mount For 1&#34;Optic-usable Depth 2&#34;</td>
			<td>Thorlabs</td>
			<td>SM1L20</td>
		</tr>
		<tr>
			<td></td>
			<td>Small Optical Rails 600mm, metric</td>
			<td>Thorlabs</td>
			<td>RLA600/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Small Optical Rails 75mm, metric</td>
			<td>Thorlabs</td>
			<td>RLA075/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Small Optical Rails 150mm, metric</td>
			<td>Thorlabs</td>
			<td>RLA150/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Rail Carrier, Counterbored Hole 1&#34;x 1&#34;</td>
			<td>Thorlabs</td>
			<td>RC1</td>
		</tr>
		<tr>
			<td></td>
			<td>Rail Carrier, Perpendicular Dovetail</td>
			<td>Thorlabs</td>
			<td>RC3</td>
		</tr>
		<tr>
			<td></td>
			<td>High Precision Translating Lens Mount for 1 inch</td>
			<td>Thorlabs</td>
			<td>LM1XY/M</td>
		</tr>
		<tr>
			<td></td>
			<td>&#189; &#34; (12mm) Dovetail Translation Stage</td>
			<td>Thorlabs</td>
			<td>DT12/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Rail Clamps</td>
			<td>Thorlabs</td>
			<td>CL6</td>
		</tr>
		<tr>
			<td></td>
			<td>Metric XYZ Translation Stage (Includes PT102)</td>
			<td>Thorlabs</td>
			<td>PT3/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Black Rubberized Fabric</td>
			<td>Thorlabs</td>
			<td>BK5</td>
		</tr>
		<tr>
			<td></td>
			<td>Ball Driver kit/ 6 tools</td>
			<td>Thorlabs</td>
			<td>BD-KIT/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Adapter with External M6 x 1.0 Threads and External M4 x 0.7 Threads</td>
			<td>Thorlabs</td>
			<td>AP6M4M</td>
		</tr>
		<tr>
			<td></td>
			<td>Mounting Base, 25 mm x 58 mm x 10 mm, 5 Pack</td>
			<td>Thorlabs</td>
			<td>BA1S/M-P5</td>
		</tr>
		<tr>
			<td></td>
			<td>Lens Mount for 25.4mm optic</td>
			<td>Thorlabs</td>
			<td>LMR1/M</td>
		</tr>
		<tr>
			<td></td>
			<td>SM1 FC/APC Adapter</td>
			<td>Thorlabs</td>
			<td>SM1FCA</td>
		</tr>
		<tr>
			<td></td>
			<td>Kinematic Mirror Mount For 1 inch Optics</td>
			<td>Thorlabs</td>
			<td>KM100</td>
		</tr>
		<tr>
			<td></td>
			<td>Silicon Power Head, 400-1100nm, 50mW</td>
			<td>Thorlabs</td>
			<td>S120C</td>
		</tr>
		<tr>
			<td></td>
			<td>12.7 mm Post Holders, Spring-Loaded Hex-Locking Thumbscrew, 
			L=50 mm, 5 Pack</td>
			<td>Thorlabs</td>
			<td>PH50/M-P5</td>
		</tr>
		<tr>
			<td></td>
			<td>Post Holder with Spring-Loaded Hex-Locking Thumbscrew, L=20 mm</td>
			<td>Thorlabs</td>
			<td>PH20/M</td>
		</tr>
		<tr>
			<td></td>
			<td>12.7 mm x 50 mm Stainless Steel Optical Post, M4 Stud, 
			M6-Tapped Hole, 5 Pack</td>
			<td>Thorlabs</td>
			<td>TR50/M-P5</td>
		</tr>
		<tr>
			<td></td>
			<td>12.7 mm x 75 mm Stainless Steel Optical Post, M4 Stud, 
			M6-Tapped Hole, 5 Pack</td>
			<td>Thorlabs</td>
			<td>TR75/M-P5</td>
		</tr>
		<tr>
			<td></td>
			<td>USB Power and Energy Meter Interface</td>
			<td>Thorlabs</td>
			<td>PM100USB</td>
		</tr>
		<tr>
			<td></td>
			<td>12.7 mm x 30 mm Stainless Steel Optical Post, M4 Stud, 
			M6-Tapped Hole</td>
			<td>Thorlabs</td>
			<td>TR30/M</td>
		</tr>
		<tr>
			<td></td>
			<td>12.7 mm x 20 mm Stainless Steel Optical Post, M4 Stud, 
			M6-Tapped Hole</td>
			<td>Thorlabs</td>
			<td>TR20/M</td>
		</tr>
		<tr>
			<td></td>
			<td>750 mm long Structural Rail (detection box)</td>
			<td>Thorlabs</td>
			<td>XE25L750/M</td>
		</tr>
		<tr>
			<td></td>
			<td>350 mm long Structural Rail (detection box)</td>
			<td>Thorlabs</td>
			<td>XE25L350/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Quick Corner Cube for 25 mm Rails</td>
			<td>Thorlabs</td>
			<td>XE25W3</td>
		</tr>
		<tr>
			<td></td>
			<td>Right-Angle Bracket for 25 mm Rails</td>
			<td>Thorlabs</td>
			<td>XE25A90</td>
		</tr>
		<tr>
			<td></td>
			<td>Black posterboard 20&#34; x 30&#34; (508 mm x 762 mm), 1/16&#34; (1.6 mm) Thick, 5 Sheets</td>
			<td>Thorlabs</td>
			<td>TB5</td>
		</tr>
		<tr>
			<td></td>
			<td>Hinge for 25 mm Rail Enclosures</td>
			<td>Thorlabs</td>
			<td>XE25H</td>
		</tr>
		<tr>
			<td></td>
			<td>Lid Stop for 25 mm Rail Enclosures</td>
			<td>Thorlabs</td>
			<td>XE25LS</td>
		</tr>
		<tr>
			<td></td>
			<td>M4 Cap Screw Kit</td>
			<td>Thorlabs</td>
			<td>HW-KIT1/M</td>
		</tr>
		<tr>
			<td></td>
			<td>M6 Cap Screw Kit and Hardware kit</td>
			<td>Thorlabs</td>
			<td>HW-KIT2/M</td>
		</tr>
		<tr>
			<td></td>
			<td>Table Clamp, L-Shape, 5 Pack</td>
			<td>Thorlabs</td>
			<td>CL5-P5</td>
		</tr>
		<tr>
			<td></td>
			<td>SM1 Ring-Actuated Iris Diaphragm (&#216;0.8 - &#216;12 mm)</td>
			<td>Thorlabs</td>
			<td>SM1D12D</td>
		</tr>
		<tr>
			<td></td>
			<td>&#216;1&#34; SM1-Mounted Frosted Glass Alignment Disk w/&#216;1 mm Hole</td>
			<td>Thorlabs</td>
			<td>DG10-1500-H1-MD</td>
		</tr>
		<tr>
			<td></td>
			<td>Honeycomb Optical Table Top, Standa</td>
			<td>Standa</td>
			<td>1HB10-15-12</td>
		</tr>
		<tr>
			<td></td>
			<td>Optical Table support, Standa</td>
			<td>Standa</td>
			<td>1TS05-12-06-AR</td>
		</tr>
		<tr>
			<td>Sample nano-positionning</td>
			<td>Precision XYZ Nanopositioning</td>
			<td>Physik Instrumente</td>
			<td>PI P527-3.CD</td>
		</tr>
		<tr>
			<td></td>
			<td>Digital Multi-Channel Piezo Co, 3 Channels, -30 to 130 V 
			Sub- D Connector(s), Capacitive Sensors,</td>
			<td>Physik Instrumente</td>
			<td>PI E727-3.CD</td>
		</tr>
		<tr>
			<td>Temperature chamber</td>
			<td>Zeiss 200M Inverted Microscope Incubator System MATT BLK</td>
			<td>Digital Pixel</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Dual Channel Microprocessor Temperature Controller</td>
			<td>Digital Pixel</td>
			<td>DP_MTC_2000_DUO</td>
		</tr>
		<tr>
			<td></td>
			<td>Two Vibration Free Heater Modules</td>
			<td>Digital Pixel</td>
			<td>DP_150_VF</td>
		</tr>
		<tr>
			<td></td>
			<td>PT100 Temperature Sensor</td>
			<td>Digital Pixel</td>
			<td>DP_P100_TS</td>
		</tr>
		<tr>
			<td>Biological Reagents and Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture and transfection</td>
			<td>Cos7 cells</td>
			<td>ATCC&#174;</td>
			<td>CRL-1651&#8482;</td>
		</tr>
		<tr>
			<td></td>
			<td>8- well Lab-Tek chambers</td>
			<td>Thermo Fisher Scientific</td>
			<td>155411PK</td>
		</tr>
		<tr>
			<td></td>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
		</tr>
		<tr>
			<td></td>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
		</tr>
		<tr>
			<td></td>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td></td>
			<td>PBS buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
		</tr>
		<tr>
			<td></td>
			<td>PenStrep</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
		</tr>
		<tr>
			<td></td>
			<td>PolyJet Transfection Reagent</td>
			<td>SignaGen Laboratories</td>
			<td>SL100688</td>
		</tr>
		<tr>
			<td>Cholesterol content measurement</td>
			<td>Amplex Red Cholesterol Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12216</td>
		</tr>
		<tr>
			<td></td>
			<td>Protease Inhibitor Cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>87786</td>
		</tr>
		<tr>
			<td></td>
			<td>Phosphatase Inhibitor Cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>78420</td>
		</tr>
		<tr>
			<td></td>
			<td>ROTI Nanoquant Working Solution</td>
			<td>Roth</td>
			<td>K880</td>
		</tr>
		<tr>
			<td></td>
			<td>GloMax Discover Microplate Reader</td>
			<td>Promega</td>
			<td>GM3000</td>
		</tr>
		<tr>
			<td>svFCS measurements</td>
			<td>HBSS buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025092</td>
		</tr>
		<tr>
			<td></td>
			<td>Hepes buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
		</tr>
		<tr>
			<td></td>
			<td>Cholesterol oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>C8868</td>
		</tr>
		<tr>
			<td></td>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>83697-1G</td>
		</tr>
	</tbody>
</table>

spot variation fluorescence correlation spectroscopy for analysis of molecular diffusion at the plasma membrane of living cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

We generated a DL for Thy1-GFP expressed in Cos-7 cells (Figure 4, black squares). The diffusion law has a positive t0 value (19.47 ms &#177; 2 ms), indicating that Thy1-GFP is confined in nanodomain structures of the plasma membrane. The cholesterol oxidase treatment of the cells expressing Thy1-GFP resulted in the shift of the DL t0 value to 7.36 &#177; 1.34 ms (Figure 4, gray squares). This observation confirms that the nature of Thy1-GFP confinement depends on the cholesterol content and is associated with lipid raft nanodomains. These two diffusion laws are shown to be different according to the statistical test described above (see step 9.1.3) in terms of t0 and Deff values. In addition, we assessed the concentration of total cellular cholesterol in non-treated Cos-7 cells versus the cells treated with COase. A small, but significant, decrease in total cholesterol content is observed upon COase treatment (Figure 5). As this enzyme acts only on the cholesterol pool accessible at the outer leaflet of the plasma membrane, we assume that the observed decrease in cholesterol is associated only with the plasma membrane and results in the destabilization of lipid raft nanodomains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61823/61823fig01.jpg" /> 
Figure 1: Simulated fluorescence correlation spectroscopy (FCS) diffusion laws established by spot-variation FCS for different forms of membrane organization.&#160;(Upper panels) Schematic representation of membrane organization&#8212;(A) free diffusion, (B) meshwork barriers, and (C) trap/domain confinements&#8212;with the trajectory drawn for a single molecule (red). Blue circles denote the intersection of the membrane and laser beam of waist &#969;. (Lower panels) FCS diffusion laws represented by plotting the diffusion time td as a function of the squared radius &#969;2. Diffusion law projection (green dashed line) intercepts the time axis at (A) the origin (t0 = 0) in the case of free diffusion; (B) in negative axis (t0 &#60; 0) when there are meshwork barriers, or (C) in positive axis (t0 &#62; 0) when there are traps and domains (lipid rafts). D is the lateral diffusion coefficient for Brownian motion; Deff, the effective diffusion coefficient; Dmicro, the microscopic diffusion coefficient inside the meshwork traps; Din, the diffusion coefficient inside domains; Dout, the diffusion coefficient outside domains; L, the size of the side of a square domain; and rD, the radius of a circular domain. This figure has been modified from He and Marguet6. <a href="https://www.jove.com/files/ftp_upload/61823/61823fig01large.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/61823/61823fig2v2.jpg" /> 
Figure 2: Schematic view of svFCS hardware control.&#160;The computer controls all the devices through different communication protocols: serial (microscope, external shutter), USB (XYZ piezoelectric stage, correlator), and PCI (acquisition board). DAQ: data acquisition board, APD: avalanche photodiode, SPCM: single-photon counting module, DO: digital output. <a href="https://www.jove.com/files/ftp_upload/61823/61823fig2v2large.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/61823/61823fig03.jpg" /> 
Figure 3: Schematic view of excitation and emission optical paths of the svFCS setup.&#160;The svFCS setup contains four modules: (1) the output of a fibered 488 nm laser is collimated, (2) a combination of a half-wave plate and polarizing beam-splitter sets the optical power, (3) the laser beam focused on the sample after traveling through a tube-lens free motorized microscope, and (4) the fluorescence is detected through a confocal-like detection path onto an avalanche photodiode coupled to a single photon counting module, which delivers a signal to a hardware correlator. Simplicity gives the system its sensitivity, robustness, and ease of use (widely commented in Supplementary material). <a href="https://www.jove.com/files/ftp_upload/61823/61823fig03large.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/61823/61823fig04.jpg" /> 
Figure 4: The svFCS diffusion laws generated from diffusion analysis of Thy1-GFP expressed in Cos-7.&#160;svFCS diffusion laws of Cos-7 cells without treatment (NT, black squares) and after cholesterol oxidase treatment (COase, gray circles). The insert in the graph represents statistical testing of a significant difference between the two presented svFCS diffusion laws (according to Mailfert et al.28). The test value (T) should be above the threshold set at 3.8 when both diffusion laws are different. The higher it is, the greater is the difference between the diffusion laws. The value of T is color-coded. <a href="https://www.jove.com/files/ftp_upload/61823/61823fig04large.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/61823/61823fig05.jpg" /> 
Figure 5: Comparison of total cholesterol content in Cos-7 cells.&#160;Cos-7 cells were either non-treated (NT) or treated with 1 U/mL of cholesterol oxidase (COase) for 1 h. The data represent an example of one experiment in triplicate. A two-tailed, unpaired t-test was used to assess the statistical difference (&#945;=0.05). <a href="https://www.jove.com/files/ftp_upload/61823/61823fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table of materials: The list of optical elements required for the svFCS setup.
Supplementary material: This document describes the building of a svFCS setup from scratch. <a href="https://cloudflare.jove.com/files/ftp_upload/61823/61823_R1_Supp Mat_final.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells at JoVE.com

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

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Biochemistry

2.7K Views.  Aix Marseille Univ. This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Video: Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...