to achieve the imaging speed and localization 
 precision required to fully characterize the transient mobilities 
 of membrane constituents , we optimized interferometric scattering 
 microscopy for high - speed imaging .
the apparatus 
 used here for high - speed iscat imaging is remarkably simple : a linearly 
 polarized illumination beam passes through a polarizing beamsplitter 
 and a quarter waveplate before entering a microscope objective .
light 
 scattered by the sample interferes with the weak reflection at the 
 membrane  water interface , and this light is collected by the 
 same objective before being imaged onto a cmos camera ( figure 1a ) . in previous applications of iscat ,
imaging speeds 
 of up to 1 khz were achieved by scanning the incident light over a 
 large sample area ( tens of m ) using acousto - optic 
 deflectors . here , by simply underfilling the objective to create a 
 field of view of a few m
, we are able to record 
 trajectories containing tens of thousands of data points with a 50-fold 
 increase in temporal resolution . in this experimental arrangement
, 
 20 nm aunps appear as a clear drop of 9% in the reflected 
 beam intensity relative to that of the incident light ( figure 1b ) .
pbs , polarizing beamsplitter ; 
 qwp , quarter waveplate ; o , oil - immersion microscope objective with 
 a numerical aperture of 1.42 .
( b ) representative interferometric scattering 
 image of a single 20 nm gold nanoparticle after subtraction of a median 
 background .
( c ) time 
 traces of the positions of two immobilized 20 nm gold nanoparticles 
 acquired with 10 s exposure time at 50 khz frame rate ( black 
 and red , upper panel ) .
subtracting these two traces removes the nanometer 
 motion due to vibrations and drift to yield the true localization 
 error of the measurement ( blue , lower panel ) .
( d ) histogram of the 
 interparticle distance from the time traces in c ( n = 5  10 ) .
the single - particle localization precision 
 equates to /2 = 1.9 nm , where  is the 
 full width at half - maximum .
in contrast to fluorescence imaging , where the localization 
 precision 
 scales with the number of photons detected from the emitter , the precision of iscat is governed by the ratio 
 of the shot - noise - induced fluctuations in the background light intensity 
 to the size of the iscat signal and can 
 be tuned by adjusting the incident power and magnification of the 
 imaging system . to avoid difficulties arising from nonstandard point 
 spread functions common to interferometric imaging techniques with 
 coherent light sources , we determined 
 the localization precision by recording the relative motion of two 
 20 nm particles immobilized on a cover glass ( figure 1c ) . assuming that the particles are completely immobile
, the 
 fluctuations in the interparticle distance return a single - particle 
 localization precision of 1.9 nm at an exposure time of only 10 s 
 with heating of the particle surface of less than 1.5 k at the incident 
 power density used . while state - of - the - art 
 fluorescence microscopy can also achieve 1 nm precision , this comes 
 at the expense of acquisition speed due to the limited achievable 
 photon flux .
likewise , dark - field microscopy has been used to track 
 light scattered from gold nanoparticle labels at 40 khz frame rate 
 ( 25 s exposure time ) , although with a localization precision 
 of only 17 nm . thus , iscat achieves an 
 unprecedented combination of localization precision and speed with 
 nanoscopic probes .
we first examined the diffusion of 20 nm 
 aunp / ctxb bound to gm1 
 in a slb made from dopc doped with 0.03 mol % gm1 on a plasma - cleaned 
 glass substrate .
streptavidin 
 linker , and we estimate that a 20 nm aunp has approximately 25 bound 
 ctxb .
although a 20 nm aunp is much larger ( 50 mda ) than the 
 single - molecule fluorescent probes more typically used in tracking 
 measurements , such as gfp ( 27 kda ) , its size does not significantly 
 affect receptor diffusion as the viscous forces of the membrane dominate 
 the movement of the aunp / ctxb complex .
smaller nanoparticle labels 
 on the order of 10 nm or below could also be imaged and tracked , although in this case we used larger particles 
 to ensure confident differentiation from residual vesicles that are 
 sometimes encountered on the bilayer surface .
tracking the aunp 
 at 50 khz with 10 s exposure time , we 
 acquired trajectories that reveal circular nanoscopic regions of confinement , 
 suggesting anomalous behavior ( figure 2a ) . 
 to examine the time dependence of particle mobility quantitatively , 
 we calculated the mean - square displacement ( msd ) as a function of 
 the time interval . using the relationship 
 msd  t , we can classify the motion 
 of the particle based on the value of the anomalous diffusion coefficient 
  , with  = 1 indicating brownian diffusion and  
  1 suggesting anomalous diffusion .
a logarithmic plot of the 
 diffusion coefficient versus time has the slope (   
 1 ) and thus provides a convenient representation of the nature of 
 the motion : a slope of zero indicates brownian diffusion , while negative 
 and positive slopes represent sub- and superdiffusion , respectively . 

according to this analysis , ctxb bound to gm1 in a slb formed on plasma - cleaned 
 glass exhibits subdiffusive lateral motion on the 20 s to 10 
 ms time scale and brownian diffusion at longer times ( figure 2a ) .
in contrast to previous reports where such data 
 showing the transition from anomalous to brownian diffusion had to 
 be generated from separate measurements at different acquisition speeds , these plots were constructed from single events 
 using a maximum time lag equal to 20% of the trajectory length , thus 
 spanning almost 5 orders of magnitude in time .
the circle markers 
 show the mean time - dependent diffusion coefficient of 35 trajectories , 
 while the red lines indicate the range of observed behaviors ( one 
 standard deviation ) .
this diffusion analysis demonstrates that ctxb - bound 
 gm1 diffusion is confined on short ( < 10 ms ) time scales .
the macroscopic 
 diffusion coefficient ( 0.09 m / s ) for time lags 
 longer than 10 ms agrees well with independently recorded results 
 for fluorescently labeled ctxb on an identical slb with fluorescence 
 correlation spectroscopy ( 0.14  0.01 m / s ) .
iscat experiments with 40 nm particles resulted 
 in similar trajectories and time - dependent mobilities with minor differences 
 for sub - ms time lags ( figure 2b ) .
sample trajectories and time - dependent mobilities of ( a ) 
 20 nm aunp / ctxb / gm1 on glass and ( b ) 40 nm aunp / ctxb / gm1 on glass . 

the open circles represent the mean behavior of all acquired trajectories , 
 and the red lines the standard deviation resulting from inhomogeneous 
 broadening .
total number of frames used for the 
 log  log plots : ( a ) 1.75  10 and ( b ) 2.05 
  10 .
number of data points per trajectory : 5  
 10 . in the experiments described 
 above
, the membrane supports were 
 cover glass subjected to oxygen plasma cleaning , a process known to 
 functionalize the surface with hydroxyl groups that are capable of 
 immobilizing the hydroxyl - containing gm1 head groups in the lower 
 leaflet . to investigate the potential origin of the 
 observed anomalous behavior , we modified the membrane  substrate 
 interaction and the degree of potential interleaflet coupling while 
 keeping modifications to the assay to a minimum .
first , we repeated 
 our gm1 tracking experiments using chemically inert mica as a substrate . 
 because of difficulties with removing all membrane - proximal vesicles 
 on mica , and because lipid vesicles and 20 nm aunps have similar signal 
 contrasts in iscat , we used 40 nm labels to ensure that all tracked 
 objects corresponded to ctxb / gm1 . during the acquisition of more than 
 5  10 frames we observed no confinement events and 
 exclusively brownian motion for time delays > 100 s ( figure 3a ) . for delays < 100 s , we observed superdiffusive 
 behavior , which we attribute to dynamic error in our measurements 
 at early times due to the sub - nanometer localization precision achieved 
 for 40 nm particles .
when superimposed 
 on a subdiffusive slope , this effect results in a leveling off that 
 resembles brownian diffusion ( figure 2b ) . for 
 20 nm particles , where the localization precision was lower ( 2 nm ) , 

this effect was not observed ( figure 2a ) . to 
 investigate the reason for anomalous diffusion further , we modified 
 the degree of interleaflet coupling between gm1 hydrocarbon chains 
 by replacing the native gm1 ( nt - gm1 ) with an analogue 
 ( do - gm1 ) that has the unsaturated glycerophospholipid 
 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine ( dope ) 
 in place of the naturally occurring sphingosine base .
tracking the 
 motion of do - gm1 on plasma - cleaned glass substrates 
 using 40 nm aunps revealed purely brownian diffusion at delays > 100 
 s as well . upon examination of individual trajectories for do - gm1 on glass , we found only extremely rare and short - lived 
 binding events that under a macroscopic diffusion analysis were representative 
 of brownian motion ( figure 3b ) .
this switch 
 from anomalous to brownian diffusion was observed although the interaction 
 between the headgroup and the plasma - cleaned glass substrate remained 
 unchanged from the nt - gm1 measurements .
the comparison 
 of nt - gm1 and do - gm1 on plasma - cleaned 
 glass demonstrates that surface roughness is unlikely to be the cause 
 of anomalous diffusion , which is further supported by afm measurements 
 of the plasma - treated glass surface that show the rms roughness to 
 be only 0.6 nm . brownian motion of gm1 in supported lipid bilayers on 
 mica substrates 
 and with modified lipid tail groups .
sample trajectories and time - dependent 
 mobilities of ( a ) 40 nm aunp / ctxb / nt - gm1 on mica 
 and ( b ) 40 nm aunp / ctxb / do - gm1 on glass .
the change 
 in lipid tail structure upon modification of nt - gm1 
 is indicated by the molecular structures .
total number 
 of data points used for constructing log  log plots : ( a ) 3.1 
  10 , ( b ) 1.07  10 .
we next examined the dependence of confinement 
 on gm1 concentration 
 by determining the fraction of time the particle was confined relative 
 to the total length of each trajectory .
for nt - gm1 
 on plasma - treated glass , the mean confined fraction for all gm1 concentrations 
 measured ( 0.031 mol % ) was 0.72  0.03 with zero representing 
 no confinement and one completely immobilized ( figure 4a ) .
ctxb was considered to be confined when the label traveled 
 less than 50 nm in 10 ms , as inferred from d = 1 
 m / s of free diffusion , that is , an average travel 
 of 200 nm in 10 ms .
in contrast , all control experiments exhibited 
 negligible confinement events and zero slope at t > 100 s in the corresponding log  log plots of diffusion 
 coefficient versus time . the control experiments included the following : 
 tracking biotinylated 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine 
 ( dppe ) in dopc bilayers with streptavidin - functionalized aunps on 
 plasma - cleaned glass substrates in the absence and presence of nt - gm1 ( 1.0% dppe , 1.0% gm1 ) ; do - gm1/ctxb 
 at various receptor concentrations on plasma - cleaned glass ( 0.03 and 
 1% do - gm1 ) ; and nt - gm1/ctxb on freshly 
 cleaved mica ( 0.03% gm1 ) ( figure 4a ) . from 
 this
we follow that interleaflet coupling between gm1 in the lower 
 and upper leaflets , combined with membrane  substrate interactions , 
 is the reason for transient molecular confinement and thus anomalous 
 diffusion . as mentioned , the hydroxyl - containing headgroups of gm1 
 lipids in the lower leaflet couple efficiently with hydroxyl groups 
 on the plasma - cleaned surface .
while dppe has saturated chains that 
 could potentially lead to interleaflet interactions , its headgroup 
 does not contain hydroxyl groups for direct substrate interaction .
do - gm1 can interact with the substrate through its hydroxyl - containing 
 headgroup , but its unsaturated tails do not exhibit strong interleaflet 
 coupling .
nt - gm1 has saturated tails and a hydroxyl - containing 
 headgroup but can not interact directly with the chemically inert mica 
 surface .
on the basis of the weak dependence of confinement on gm1 
 concentration , we conclude that although both substrate interactions 
 and interleaflet coupling are required for anomalous diffusion , the 
 number of confinement events is essentially independent of gm1 concentration 
 and likely determined by the density of hydroxyl pinning sites on 
 the surface .
this conclusion is further supported by experiments showing 
 that transient binding of nt - gm1-bound ctxb can be 
 induced on mica by plasma cleaning the surface ( figure 4a , pc - mica ) , indeed suggesting that surface functionalization 
 plays a critical role .
( a ) confined 
 fraction 
 for different nt - gm1 concentrations in dopc slbs 
 on glass , synthetically modified gm1 ( do - gm1 ) on 
 glass , dppe with a biotin / streptavidin linker in the presence of nt - gm1 on glass , and nt - gm1 on freshly 
 cleaved and plasma - cleaned mica .
the minimum usable concentration 
 ( 0.03% ) was defined as the lowest where specific binding of ctxb - labeled 
 nanoparticles was still observed .
( b ) gaussian - like ( upper panel ) 
 and ring - like ( lower panel ) spatial distributions of confinement events 
 at 0.03% gm1 on glass obtained by averaging 228 and 93 confinement 
 events , respectively . for classification , each confinement site was 
 fit to a rayleigh distribution and the coefficient of determination , r , was calculated . all confinement events with r > 0.95
as the sites with low r values 
 contain both ring - like confinement events and other non - gaussian shapes , 
 the ring - like sites were selected by thresholding the standard deviation 
 of distances from the confinement center of mass over the overall 
 confinement to be smaller than 0.45 .
the optimum ratio was determined 
 visually by inspecting the resulting averaged spatial distributions . 

( c ) individual ring - like confinement events exhibiting 
 35 distinct binding sites .
the black lines represent trajectories 
 after averaging 25 consecutive frames , equivalent to 0.5 ms time averaging , 
 and help visualize the stepping motion between gm1 receptor sites . 

( d , e ) radial probability density plots and corresponding 
 fits to a rayleigh distribution for all confinement events recorded 
 at 0.03% and 1.0% nt - gm1 concentrations .
the distribution 
 histograms were computed from 2  10 data points 
 in each case .
( f ) residuals from the fits of d and e as well as similar 
 plots for 0.1 and 0.3% nt - gm1 concentration . (
g ) 
 change in the fwhm of gaussian - like events as a function of nt - gm1 concentration .
error bars are the standard deviation 
 of the mean confinement size . to examine the transient immobilization events in more detail 
 we 
 superimposed all confinement events for 0.03% gm1 on plasma - cleaned 
 glass .
we identified two different populations consisting of gaussian - like 
 and ring - like spatial distributions ( figure 4b ) with confinement sizes < 20 nm .
individual ring - like confinement 
 events often exhibited two to five distinct binding sites that were 
 revisited frequently ( figure 4c ) and showed 
 little variation with label size ( 20 and 40 nm aunp ) . to quantify 
 any dependence on nt - gm1 concentration , we combined 
 all confinement events and computed radial probability density plots 
 as a function of nt - gm1 molar concentration ranging 
 from 0.03 to 1.0% gm1 . at 0.03% concentration ( figure 4d ) ,
the histogram obtained from a total of 2  10 data points was well described by a rayleigh distribution , 
 indicating that the confinement distributions were mostly gaussian - like . 
 as the gm1 concentration increased 33-fold to 1.0% , the fit to the 
 rayleigh distribution deteriorated ( figure 4e ) , a trend that scaled with nt - gm1 concentration 
 ( figure 4f ) .
in addition , for gaussian - like 
 events , although the number of confinement events did not change , 
 the confinement size increased linearly with nt - gm1 
 concentration ( figure 4 g ) .
the unique ability of iscat to measure the 
 motion of individual membrane components at their intrinsic time ( s ) 
 and length ( nm ) scales makes it an ideal method for investigating 
 molecular membrane mobility .
while other imaging approaches have contributed 
 a great deal to our understanding of the nanoscale structure and organization 
 of cell membranes , potential experimental artifacts and poor spatiotemporal 
 precision have impeded their ability to fully capture the behavior 
 of membrane constituents .
because iscat induces minimal perturbations 
 to the system being studied while providing high spatial and temporal 
 resolution , it is able to capture nanoscopic behavior that is beyond 
 the reach of other imaging approaches . given the possibility 
 of defects in slbs , it is important to emphasize that our results 
 rule out bilayer imperfections as a cause for the observed behavior 
 for a number of reasons . if defects were responsible , we would expect 
 the same diffusive properties for do - gm1 and nt - gm1 in contrast to experimental observations ( figures 2 and 3 ) . even in the event 
 that differences in do - gm1 and nt - gm1 lipid properties could cause a different propensity for the 
 formation of defects at such low gm1 concentrations , such defects 
 would also be present when tracking dppe in an nt - gm1-doped slb , an environment where we observed no anomalous diffusion 
 ( figure 4a ) .
dppe - tracking in gm1-doped slbs 
 also shows that time - dependent mobilities induced by particle cross - linking 
 or label - membrane interactions are unlikely .
if the labels caused 
 transient immobilization , anomalous subdiffusion would have been observed 
 in the control experiments ( figure 4a ) .
we 
 also rule out nonspecific binding of the gold nanoparticle to the 
 membrane , as particles not functionalized with ctxb did not interact 
 with gm1-containing slbs but were easily washed away .
comparison of 
 the results obtained using inert and plasma - cleaned mica substrates 
 excludes phase separation and nanoscopic aggregation due to free ctxb 
 in solution as a possible cause for the transient immobilization . 

our results therefore suggest that transient immobilization of gm1 
 in one leaflet can affect the diffusion of gm1-bound ctxb in the other 
 leaflet through interactions between the lipid tails .
when we 
 compare the mobility of nt - gm1 and do - gm1 on cover glass with nt - gm1 on mica , 
 we find that only the combination of nt - gm1 with 
 a plasma - treated surface leads to time - dependent mobility .
our experiments 
 on an effectively single component system also make effects due to 
 lipid composition or substrate - induced asymmetry of the lipid distribution 
 unlikely .
a recent study of the distribution of gm1 in dopc slbs on 
 uv / ozone - treated silica showed that 85% of the total gm1 is present 
 in the upper leaflet with a linear relationship for 05% gm1 
 content . because the reported effect 
 is a consequence of the lipid headgroup charge , nt - gm1 and do - gm1 on glass
would be expected to exhibit 
 the same distribution between the two leaflets given that both experiments 
 were performed on identical , plasma - cleaned glass substrates . taken 
 together
, our results suggest that two conditions need to be met for 
 anomalous diffusion : ( a ) gm1 molecules in the lower leaflet must be 
 immobilized through interactions with the surface and ( b ) the immobilized 
 molecules must interact with ctxb - cross - linked gm1 molecules in the 
 upper leaflet through the hydrophobic core of the bilayer via long , 
 straight aliphatic chains .
how many cross - linked gm1 molecules are 
 required for this to occur remains to be addressed in future work .
our ability to super - resolve the spatiotemporal dynamics of the 
 label during confinement events provides important clues to the nanoscopic 
 origin of the observed anomalous diffusion . to illustrate this
, it 
 is helpful to consider the molecular details of the tracking assay . 

when drawn to scale , it is clear that for a nanoparticle label with 
 a single ctxb subunit immobilized on the membrane , some flexibility 
 remains due to the distance between gm1 and the center of mass of 
 the particle ( figure 5a ) .
simple geometric 
 arguments suggest that nanoscopic rocking of the label can induce 
 variations in the center of mass on the order of 15 nm for both 20 
 and 40 nm aunps ( figure 5b ) . when observed 
 over tens of milliseconds , such fluctuations result in a 2d gaussian 
 spatial distribution around the center of mass if the position of 
 the label can be determined on a time scale shorter than that of the 
 rocking motion ( figure 4b ) . a second surface - bound 
 ctxb on the nanoparticle may transiently bind to another , possibly 
 diffusing gm1 , and although the gm1/ctxb dissociation constant is 
 very low ( kd  10 ) , we frequently observed this behavior . on average , these multiple - bound 
 particles yield trajectories centered around an immobilized central 
 ctxb that resembles a ring - like structure on the nanoscale with a 
 radius comparable to the maximum motion of the center of mass of the 
 label ( figure 4c ) .
( a ) schematic of the tracking assay 
 during a transient binding event drawn to scale for a 20 nm aunp . 

the curved arrow indicates the achievable nanoscopic rocking motion 
 of the label and the dashed lines the achievable fluctuations in the 
 center of mass of the label even for a completely immobilized ctxb . 

( b ) comparison of nanoscopic rocking motion for 20 and 40 nm labels . 

due largely to the ratio between the radius of curvature of the particle 
 and the separation between adjacent ctxb subunits , the confinement 
 sizes are expected to be similar for both labels .
as multiple binding becomes more likely with increasing gm1 
 concentration , 
 the radial probability density plot representative of all confinement 
 events is expected to further deviate from a rayleigh distribution . 
 at low gm1 concentration ( 0.03% ) , there is 1 receptor in an 
 area of 40 nm , meaning that other ctxb subunits on the 
 aunp will rarely encounter any gm1 receptors upon approaching the 
 membrane during nanoscopic rocking . as a result , we obtain an excellent 
 fit to a rayleigh distribution ( r > 
 0.95 ) 
 ( figure 4d , e ) . upon increasing the receptor 
 concentration 30-fold
, however , the immediate presence of gm1 becomes 
 more likely and transient binding to individual receptors occurs more 
 often . as a consequence , the deviation between the radial probability 
 density histogram and the rayleigh fit increases with gm1 concentration . 

even for gaussian - like confinement events , an increase in nearby gm1 
 concentration should lead to an increase in the apparent confined 
 area because nanoscopic rocking will result in the particle on average 
 spending more time near the membrane surface due to transient binding 
 with additional gm1 ( figure 4 g ) , even if it 
 does not lead to cross - linking and long - term multiple binding . the tight lateral confinement on the < 20 nm scale and the observation 
 of ring - like confinement events
are both consistent with transient 
 immobilization of a single membrane - bound ctxb subunit on the nanometer 
 scale . given that transient binding requires both native gm1 lipid 
 tail domains and a plasma - cleaned substrate as evidenced by the studies 
 on mica and with do - gm1 , we propose that the most 
 likely origin is clustering of gm1 on the lower leaflet by hydrogen 
 bonding to nanoscopic patches of surface hydroxyl groups from the 
 plasma cleaning process .
although in principle one could envision 
 that a single pinned gm1 molecule is responsible for immobilization 
 of five ctxb - bound gm1 molecules in the upper leaflet , such a prospect 
 appears energetically unlikely .
we conclude that the lower leaflet 
 is likely scattered with numerous < 10 nm patches of high gm1 density 
 with higher viscosity that communicate with the upper leaflet through 
 transbilayer interactions , leading to transient ( < 10 ms ) nanoscopic 
 confinement .
this interpretation is further supported by the 
 weak dependence 
 of the observed confinement events on label size .
both 20 and 40 nm 
 labels produce similarly sized confinement events in line with expectations 
 based on the schematic in figure 5b , a consequence 
 largely of the ratio between the radius of curvature and the separation 
 between adjacent ctxb subunits .
the proposal of nanoscopic aggregates 
 of gm1 on plasma - cleaned glass also agrees with afm measurements of 
 comparable assays , although these do not provide direct information 
 regarding whether the aggregation occurs in the lower or upper leaflet 
 of the bilayer .
our general mechanism 
 agrees with early observations of interleaflet coupling , although these required the presence of macroscopic 
 domains .
taken together , our work suggests that transient binding 
 can occur for nanoscopic domains and change the mobility of single 
 proteins bound to multiple receptors across a bilayer membrane .
the compounds 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine - n-(cap biotinyl ) 
 ( dppe ) , and gm1 bovine brain ganglioside ( gm1 ) were purchased from 
 avanti polar lipids ( alabaster , al ) . do - gm1 was prepared 
 as described before .
biotin - labeled cholera 
 toxin b ( ctxb ) subunits from vibrio cholera were 
 purchased from sigma - aldrich ( milwaukee , wi ) and reconstituted with 
 water to give a solution containing 0.05 m tris buffer , ph 7.5 , 0.2 
 m nacl , 3 mm nan3 , and 1 mm sodium edta .
gold nanoparticles 
 ( aunps ) functionalized with streptavidin were purchased from british 
 biocell international ( cardiff , u.k . ) , diluted to a concentration 
 of 9  10 particles / ml and incubated with a 10-fold 
 excess of biotin - ctxb at room temperature for 1 h. excess ctxb was 
 removed by centrifuging the aunp / ctxb sample for 2 min at 14000 g and resuspending the pellet in bilayer buffer ( 10 mm hepes , 
 ph 6.8 , 200 mm nacl and 2 mm cacl2 ) . on the basis of a 
 streptavidin contact area of 25 nm , and assuming that 
 streptavidin covers 50% of the surface area of an aunp , we estimate 
 that there are 25 ctxb per 20 nm aunp and 100 ctxb per 40 nm aunp . 
 for dppe tracking experiments , streptavidin - functionalized aunps were 
 added directly to the bilayer .
lipids in organic solvent 
 were mixed in a glass vial and the solvent evaporated first under 
 a gentle stream of nitrogen for 5 min and then under vacuum for 30 
 min .
the dried lipid film was resuspended to a lipid concentration 
 of 1 mg / ml in bilayer buffer .
lipid suspensions were vortexed for 
 1 min , hydrated at room temperature for 30 min and then passed 21 
 times through a 100 nm polycarbonate membrane using a mini extruder 
 ( avanti polar lipids ) , resulting in clear suspensions of suvs .
1.5 borosilicate cover glass 
 ( menzel - glser , braunschweig , germany ) was etched in 2:1 h2o2:hcl for 10 min , followed by thorough rinsing 
 with ultrapure water ( merck millipore , billerica , ma ) .
the clean substrates 
 were dried with a gentle stream of nitrogen and etched with oxygen 
 plasma for 8 min at 50 w power immediately prior to vesicle deposition 
 ( diener electronic , plasma system femto ) .
mica substrates were prepared 
 by bonding a 22 mm square sheet of mica ( agar scientific , essex , u.k . ) 
 to a clean cover glass using optical adhesive ( norland optical adhesive 
 61 ) . immediately prior to vesicle deposition ,
the mica was cleaved 
 leaving a thin , optically transparent layer adhered to the cover glass .
sample chambers were 
 assembled by placing a culturewell silicon gasket ( grace bio - laboratories , 
 bend , or ) onto the glass or mica substrate .
slbs were formed by adding 
 20 l bilayer buffer followed by 10 l of the 1 mg / ml 
 suv suspension to the 30 l sample well and incubating for 5 
 min .
excess suvs were rinsed away with 3 ml bilayer buffer and then 
 2.5 l of aunp / ctxb or aunp / streptavidin solution was deposited 
 and allowed to incubate for 5 min .
a collimated beam from a solid - state 
 diode laser (  = 532 nm ) was focused through an under - filled 
 60 , 1.42 na oil immersion microscope objective ( olympus ) onto 
 a small region of the sample .
a portion of the light scattered from 
 the aunp together with the incident light reflected at the membrane / water 
 interface was collected by the objective and focused onto a fast cmos 
 camera ( photon focus mv - d1024 - 160-cl-8 , lachen , switzerland ) for imaging 
 with 100 magnification for an effective pixel size of 106 nm . 

the incident power density was between 20 and 30 kw / cm , which corresponds to focusing approximately 3 mw onto a 2  
 2 m spot size at the sample . for 
 a 20 nm aunp ,
this incident intensity at a wavelength of 532 nm leads 
 to a local heating effect that amounts to a < 1.5 k rise in temperature 
 at the particle surface .
we remark that 
 any trapping forces exerted by this beam are on the order of few femtonewtons , much too small to affect the diffusion on the 
 length scales studied in this work . for all trajectories , 

the two - dimensional msd for each time interval was calculated according 
 towhere t = 
 20 s 
 ( frame time ) ,   is the particle displacement during time 
 interval tn = nt , n is the total 
 number of frames in the trajectory , and n is a positive 
 integer that determines the time interval . the log  log plots 
 of the diffusion coefficient versus time ( figures 2 , 3 ) were generated using intervals 
 up to 20% of the trajectory length .
particle detection 
 was performed by combining the nonmaximum suppression algorithm with 
 a threshold based on the median absolute deviation .
briefly , the standard 
 deviation of the image in the absence of particles was estimated by 
 the median absolute deviation .
pixel values exceeding three times 
 this standard deviation were classified as candidate pixels for particles . 

the candidate pixels from the median absolute deviation threshold 
 were intersected with the candidate pixels from the nonmaximum suppression 
 algorithm to obtain to the nearest integer pixel value the position 
 of any candidate particle .
candidate particles were then segmented 
 into regions of interest corresponding to 954  954 nm , which for a magnification of 100 corresponded to 9  
 9 pixels ( effective pixel size of 106 nm ) .
a two - dimensional gaussian 
 function was then fit to each segmented region and candidate particles 
 satisfying the contrast upper and lower bounds were used as input 
 parameters for the consecutive frame .
particle trajectories were reconstructed 
 using a greedy algorithm that minimized the distance between detected 
 particle positions in consecutive frames . given the density of less 
 than one aunp / ctxb complex per square micrometer , any artifacts arising 
 due to this greedy algorithm were minimized .
 the 
 biological functions of the cell membrane are influenced by 
 the mobility of its constituents , which are thought to be strongly 
 affected by nanoscale structure and organization . 
 interactions with 
 the actin cytoskeleton have been proposed as a potential mechanism 
 with the control of mobility imparted through transmembrane  pickets  
 or gpi - anchored lipid nanodomains . 
 this hypothesis is based on observations 
 of molecular mobility using various methods , although many of these 
 lack the spatiotemporal resolution required to fully capture all the 
 details of the interaction dynamics . 
 in addition , the validity of 
 certain experimental approaches , particularly single - particle tracking , 
 has been questioned due to a number of potential experimental artifacts . 
 here , we use interferometric scattering microscopy to track molecules 
 labeled with 2040 nm scattering gold beads with simultaneous 
 < 2 nm spatial and 20 s temporal precision to investigate 
 the existence and mechanistic origin of anomalous diffusion in bilayer 
 membranes . 
 we use supported lipid bilayers as a model system and demonstrate 
 that the label does not influence time - dependent diffusion in the 
 small particle limit ( 40 nm ) . by tracking the motion of the 
 ganglioside lipid gm1 bound to the cholera toxin b subunit for different 
 substrates and lipid tail properties , we show that molecular pinning 
 and interleaflet coupling between lipid tail domains on a nanoscopic 
 scale suffice to induce transient immobilization and thereby anomalous 
 subdiffusion on the millisecond time scale . 
Results
Discussion
Materials
and Methods