Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates

Investigating the cellular internalization pathways of single molecules or single nano-objects is important to understanding cell-matter interactions and to applications in drug delivery and discovery. Imaging and tracking the motion of single molecules on cell plasma membrane require high spatial resolution in three dimensions (3D). Fluorescence imaging along the axial dimension with nanometer resolution has been highly challenging but critical to revealing displacements in trans-membrane events. Here, utilizing a plasmonic ruler based on the sensitive distance dependence of near-infrared fluorescence enhancement (NIR-FE) of carbon nanotubes on a gold plasmonic substrate, we probe ~10 nm scale trans-membrane displacements through changes in nanotube fluorescence intensity, enabling observations of single nanotube endocytosis in 3D. Cellular uptake and trans-membrane displacements show clear dependences to temperature and clathrin assembly on cell membrane, suggesting that the cellular entry mechanism for a nanotube molecule is via clathrin-dependent endocytosis through the formation of clathrin-coated pits on cell membrane.


Introduction
The interactions of molecules and nanostructured materials with mammalian cells have aroused a great deal of scientific interest with implications to many biological and medical applications in drug discovery, nanomedicine and toxicology. 1 The uptake pathway and subsequent intracellular trafficking have been intensely studied and debated for a broad range of nanomaterials, including fullerene, 2 quantum dots (QDs), 3 magnetic nanoparticles 4 and carbon nanotubes (CNTs). [5][6][7] An interesting question is in regards to the cellular internalization pathways and the dependence of the pathways on the size of molecules or nanomaterials. It is 2 important to investigate the upper size limit for molecules simply inserting and diffusing through the cell plasma membrane, and the lower size limit for nanomaterials becoming too small for wrapping by a highly curved lipid bilayer to undergo endocytosis.
As an example, two distinct pathways for carbon nanotube cellular entry have been suggested, direct insertion or diffusion through the lipid bilayer, 5,[8][9][10] and clathrin-dependent endocytosis. 6,[11][12][13] In most cases, ensembles of CNTs (including bundles and aggregates of tubes) are investigated in cellular uptake experiments through imaging of fluorescent dye labels, 5,6 and have suggested endocytotic internalizations of CNTs 6,13,14 except in several reports proposing direct insertion. 5,8 At the individual nanotube level, imaging CNT-cell interactions through detecting the intrinsic near-infrared (NIR) photoluminescence 7,15 of CNTs has been performed.
However, thus far, there has been no direct three dimensional (3D) imaging of single nanotube molecules traversing cell membrane to directly observe either insertion or endocytosis pathway.
In a theoretical study, Gao et al. suggested high curvature of cargo particles such as a bare single-walled CNT (diameter down to 1 nm) could cause elevated elastic energy associated with lipid-bilayer wrapping involved in endocytosis, and an optimum radius of curvature of ~14 nm exists for endosome formation around a cylindrical particle. 16 The question of whether an individual CNT (rather than bundles or aggregates of tubes) can undergo endocytosis remains an interesting open problem.
Imaging and tracking single events of specific molecules on cell membrane can offer new insights into various mechanisms of interest to biological systems. 17 Imaging single molecule trans-membrane motion requires nanometer spatial resolution along the axial dimension. Recent progresses in 3D single particle tracking have led to various new techniques to resolve the location of a single nanoparticle with high precision and elucidate interactions between the tracked nanoparticle with its surroundings. 18 For instance, by confining illumination to a ~ 100 nm optical section with evanescent waves travelling at the cover slip-cell interface, total internal reflection fluorescence microscopy (TIRFM) has been developed to image cell membrane, nearby cytoplasm and membrane-related events. 19 Further, combined with two-dimensional (2D) super-resolution techniques, 20 high axial resolution can also be achieved by imposing zdependent asymmetry into two orthogonal axes x and y, 21,22 or using two stimulated emission depletion (STED) beams to generate a central zero in three dimensions. 23 Single-molecule axial tracking has also been realized by feedback tracking, including focusing two circularly scanning 3 laser beams at different z-depths. 24 Still, it remains highly challenging to image trans-membrane motion of a single molecule (such as a single nanotube) with sensitivity on the order of the thickness of the plasma membrane (~10 nm), thus probing the pathway and kinetics of singlemolecule transportation across the plasma membrane.
Here we report the use of an NIR fluorescence enhancement (NIR-FE) phenomenon on plasmonic gold substrates [25][26][27][28] to track cellular internalization of individual single-walled carbon nanotubes (SWCNTs) in 3D, with an axial resolution on the order of ~ 10 nm owing to the highly sensitive dependence of fluorescence enhancement to the distance between SWCNT and gold. 25 SWCNTs exhibit intrinsic band-gap fluorescence in the 0.9-1.4 m NIR II region upon excitation in the visible or NIR I (600-900 nm). [29][30][31][32][33][34][35][36][37] Recently, we observed fluorescence enhancement of SWCNTs by > 10 fold on a solution-phase grown gold films (called Au/Au films) containing patchworks of nanostructured Au islands. The fluorescence enhancement rapidly decreases as the distance between SWCNT and Au increases, with an exponential decay distance (1/e decay distance) of a mere ~6 nm. 25,27 By taking advantage of this interesting effect, we demonstrate single molecule trans-membrane imaging with high sensitivity to axial motion and elucidate the cellular internalization pathway for individual nanotubes.

Results
Plasmonic ruler based on fluorescence enhancement. We used water-soluble high-pressure CO conversion (HiPCO) SWCNTs (diameter ~0.7-1 nm) in our cell entry imaging experiments.
The nanotubes were stably suspended by mixed surfactants of 75% C18-PMH-mPEG (5kD for each PEG chain, 90kD in total) and 25% DSPE-PEG(5kD)-NH 2 (see Methods) with amine groups covalently conjugated with RGD peptide ligands capable of selectively binding to α v β 3integrin over-expressed on U87-MG glioblastoma cells. 38 Since the radius of gyration of a 5kD PEG chain is ~3.5 nm in aqueous solution, 39 a water-soluble SWCNT is wrapped by a ~7 nm thick polymer coating radially to form a cylinder with ~15 nm diameter (inset of Figure 1b), greater than the diameter of ~1 nm of bare nanotubes. Atomic force microscope (AFM) imaging of SWCNTs on glass substrate after calcination showed most nanotubes lying horizontally with an average length of ~1m (Supplementary Figure S1). We found that the length distribution did not affect the major results of our experiments (Supplementary Figure S2), suggesting a general nanoscopic 'ruler' applicable to different lengths of SWCNTs.
4 Figure 1a shows a scanning electron microscope (SEM) image of an NIR fluorescence enhancing Au/Au film comprised of gold nano-islands with abundant gaps in between. The UV-Vis-NIR extinction spectrum of the same Au/Au film (Figure 1a inset) shows a plasmon resonance peak located at ~800 nm, facilitating fluorescence enhancement in the NIR region. 40 To reveal the distance dependence of fluorescence enhancement, we coated Au/Au films with progressively thicker Al 2 O 3 by atomic layer deposition (ALD), and deposited nanotubes on the substrates by drop-drying an aqueous nanotube suspension. The surface density of nanotubes within the drop-dried spot can be seen from the AFM image in Supplementary Figure S1.
Comparing the nanotube fluorescence intensity on Au/Au films with different spacer thicknesses to that on bare glass, we found a decreasing trend of enhancement factor, from ~8-fold enhancement to almost no enhancement, as the spacer increases in thickness ( Figure 1b). The exponential fit showed a surprisingly short 1/e decay distance of ~6 nm considering the average length of our SWCNTs is much greater, ~ 1m. Since the nanotubes are long and may not lie perfectly flat on the substrate, the separation of any part of a long tube from the gold surface is greater than or equal to the thickness of Al 2 O 3 spacer. For this reason, we suggest that the measured enhancement vs. distance data corresponds to the minimum distance between Au and the length of a nanotube in the case when the nanotube is non-parallel to the Au surface.
3D tracking of single nanotubes at 37 °C. We exploited the ultra-sensitive fluorescence enhancement of SWCNTs to gold-nanotube distance for probing motion of nanotubes in the direction normal to the gold surface. We stained trypsinized U87-MG cells at 4 º C by highly diluted SWCNTs (~20 pM) with PEG and RGD functionalization and placed the cells on an Au/Au substrate kept at 4 º C. Imaging with an InGaAs 2D detector (excitation ~658 nm) in the 1.1-1.7 m emission range revealed a brightly fluorescent spot overlaying with a single cell (Figure 1c inset), attributed to a single nanotube sandwiched between Au/Au and cell membrane on the proximal side to the substrate (see Supplementary Figure S3 for further evidence of bright nanotubes sandwiched between cell and Au), exhibiting strong NIR fluorescence enhancement due to proximity to the gold surface. 27 Due to trypsinization and staining at 4 º C to prevent unwanted endocytosis before tracking started, most cells lost extracellular matrix and turned into round shape, but they still remained viability and could internalize nanomaterials from the outside once temperature allowed. 27 We identified the single nanotube by one single peak in the 5 emission spectrum [~1150 nm in Fig.1c, corresponding to (7,6) chirality] and the sinusoidal dependence 41 of fluorescence on the polarization of laser excitation (Figure 1d).
Once an individual nanotube was identified, we increased the incubation temperature from 4 º C to 37 º C in situ and tracked the fluorescence of the SWCNT over time at a frame rate of 0.3 frame/sec after the temperature stabilized at 37 º C. The (7,6) tube in Fig.1c  SWCNTs have been reported to rotate freely in water. 43 However, in our case the nanotubes showed small rotations during endocytosis due to confinement and interactions with the membrane and the long 3s integration time that had averaged out the rotational effects.
It is also possible that some nanotubes were bound to the cell membrane perpendicular to the Au-cell interface initially and endocytosed vertically as suggested recently for large multiwalled nanotubes. 44 The laser excitation used in our experiments was not able to excite and 6 resolve these tubes efficiently. Also noticeable is that we had not observed a brightly fluorescent nanotube evolving into a dim state in a time scale of several seconds (the estimated rotation time if assuming free rotation in a viscous medium corresponding to the slow translational diffusivity measured), suggesting none of the SWNTs imaged had changed orientation from in the x-y plane to pointing to the z-direction during endocytosis.
Given the control experiments ruling out other possibilities of fluorescence decay, the observed fluorescence decrease of single nanotubes on cells at 37 º C hinted axial motion of nanotubes away from the Au/Au substrate due to ultra-sensitive Au-SWCNT distance dependence of nanotube fluorescence intensity. Based on the nanoscopic ruler effect of fluorescence enhancement, we rationalized that during 4 º C staining, RGD functionalized SWCNTs selectively attached to the α v β 3 -integrin receptors on the cell membrane ( Figure 2f) without entering the cytoplasm due to blocked endocytosis at 4 º C. 6 At 37 º C, endocytosis was activated with the formation of a vesicle wrapping around the surfactant-coated nanotube via clathrin-associated invagination of the plasma membrane (Figure 2g), followed by vesicle pinching-off ( Figure 2h) and clathrin uncoating to undergo the endocytotic pathway. 45 In the first ~ 20 s at 37 º C, the nanotube was sandwiched between the plasma membrane and the underlying Au/Au film (Figure 2f), exhibiting high fluorescence due to close proximity to the fluorescence enhancing surface (Figure 2b). The distance from the membrane to substrate could reach 4~8 nm 46 in less than 5 min 47 according to the Derjaguin-Landau-Verwey- Overbeek (DLVO) model. 48 Over time (from Figure 2b to 2c), clathrin assembled on the inner surface of plasma membrane and the membrane bent inwards to form clathrin-coated pits and wrapped around the nanotube. Due to invagination, the nanotube was displaced away from the Au substrate (Figure 2g), leading to reduced fluorescence enhancement (Figure 2c). At later times (from Figure 2c to 2d), the clathrin-coated pit continued to grow and finally pinched off to form a complete vesicle enclosing the nanotube. At this point the nanotube was >20 nm away from the Au substrate due to the two lipid bilayers in between (Figure 2h). This large spatial separation led to very little enhancement from the plasmonic substrate ( Figure 2d). A complete sequence of such regulated events including clathrin assembly, pits formation, budding and clathrin uncoating usually takes tens of seconds to a few minutes at 37º C, depending on the size of the cargo molecule. 45 In the case of this particular SWCNT, the time required to complete 7 fluorescence decrease was ~ 250 s (Figure 2e), within the reported time range to complete clathrin cluster assembly 49 but on the higher side, similar to the time needed to internalize relatively large cargo molecules (~400 s) such as reovirus particles (~85 nm in diameter). 45

3D tracking of single nanotubes at various temperatures.
To support that trans-membrane motion of single nanotubes at 37 º C was indeed the cause of fluorescence decrease, we imaged single nanotubes on U87-MG cells at several other temperatures, including 4 º C, 25 º C and 42 º C.
It is known that at temperatures lower than 37 º C, cell functions such as active uptake are impaired, and endocytosis is completely blocked at 4 º C. 6 On the other hand, cell functions are more active at elevated temperatures until excessive heating begins to cause damages. 50 In a typical experiment at 4 º C, we observed a single nanotube (evidenced by polarization dependence in Figure 3a where the average length of our SWCNTs was 2a = 1 m, and they were coated with long PEG chains with an overall radius of b ~7.5 nm. The as-calculated effective radius lied in the vesicle wrapping region for nanoparticles but was on the high side, as suggested by the model of Gao et al. 16 An optimum size for the cellular entry of both Au nanoparticles and SWCNTs has been reported 15,55 to be ~25 nm in effective radius, smaller than the effective radius of the SWCNTs used in the current work. Therefore, although endocytosis mediated by membrane wrapping was more favorable than direct insertion in our case, the large effective capture radius slowed down this process with a relatively high apparent activation barrier.

Block of endocytosis. Previous work has found that cellular internalization of ensembles of
CNTs involves the clathrin-dependent endocytosis pathway. 6 Potassium depletion, as well as hypertonic medium incubation, are the two methods known to perturb endocytosis by removing membrane-associated clathrin lattice. 59

Discussion
This work exploited the sensitive distance dependence of NIR fluorescence enhancement of single carbon nanotube molecules on a gold plasmonic substrate to probe ~10 nm scale transmembrane displacements through changes in nanotube fluorescence intensity, presented the first 3D tracking of individual SWCNTs and established the nanotube entry pathway to be clathrindependent endocytosis. Compared to other existing 3D single particle imaging and tracking techniques that either require sophisticated implementation or are limited with insufficient axial and/or temporal resolution, 18 the sensitive distance dependence of fluorescence enhancement based on the plasmonic effect presents a facile, inexpensive and sensitive probing of sub-10 nm distance changes in biological systems. Notably, spatial sensitivity and range of distance 11 measurements are difficult to optimize simultaneously. Our current method allows for probing subtle distance changes of <10 nm along the z-axis, but tracking displacements beyond ~20 nm becomes difficult due to the short plasmonic ruler length. It is necessary to resort to other fluorophores with different plasmonic ruler lengths, such as synthetic dyes, fluorescent nanoparticles or fluorescent proteins to extend the measurable range along the z-axis. Our preliminary results have indeed identified that the plasmonic fluorescence enhancement vs. distance profile is characteristic to each fluorophore and also depends on the type of plasmonic substrates. Suitable combinations of fluorophore-substrate could lead to a library of 'nanoscopic rulers' spanning various ranges of distances to probe molecular motions at the nanoscale.
We envisage that the reverse process of endocytosis, exocytosis 7,60 of single nanotubes or molecules could also be investigated by utilizing fluorescence enhancement phenomena on plasmonic substrates. Further, similar to trans-membrane processes, imaging with suitable plasmonic rulers may also offer a platform to decipher important biological pathways involving translocation motions of molecules inside cells. Distance information and protein conformational changes inside the cytoplasm could also be revealed by introducing plasmonic metal nanoparticles and fluorophores inside live cells.

Methods
Preparation of Au/Au films. The solution-phase Au/Au film synthesis can be found in detail in another publication of our group. 26 Briefly, a glass slide was immersed in a 25 mL solution of 3 mM chloroauric acid, to which 400 L of ammonia was added under vigorous agitation. The substrate was then allowed to sit in the seeding solution with gentle shaking for 1 min, after which it was rinsed with water. Then the substrate was submerged into a 25 mL solution of 1 mM sodium borohydride on an orbital shaker at 100 rpm for 5 min. Following a second rinsing step, the seeded substrate was soaked in a 25 mL solution of 1 mM chloroauric acid and 1 mM hydroxylamine under agitation for 15 minutes. It was then rinsed with water and soaked in 1 mM cysteamine ethanol solution for 1 h to render it hydrophilic and biocompatible. UV-Vis-NIR absorbance measurements. UV-Vis-NIR absorbance curve of the as-made Au/Au film on glass substrate was measured by a Cary 6000i UV-Vis-NIR spectrophotometer, background-corrected for any glass contribution. The measured range was 400-1200 nm. Scanning electron microscopy (SEM) imaging. Au/Au film grown on glass was imaged via SEM. Image was acquired on an FEI XL30 Sirion SEM with FEG source at 5 kV acceleration voltage.
Atomic layer deposition (ALD) process. Low temperature ALD process was used to coat the as-made Au/Au film with desirable thicknesses. Deposition was carried out on amine group functionalized hydrophilic Au/Au substrate at 100 ℃ in ~300 mTorr pure nitrogen environment where trimethylaluminum (TMA) and water vapor were used as precursors. For each cycle of ALD, water pulse was 0.5 s in duration, followed by a 40 s purging time, a 0.5 s TMA pulse and a 30 s purging time. To deposit the Al 2 O 3 layers of 5nm, 10 nm, 15 nm and 20 nm, 50, 100, 150 and 200 cycles were used, respectively. Preparation of water soluble SWCNT-PEG-RGD bioconjugate. The preparation of water soluble SWCNT fluorophores can be found in detail in another publication of our group with some modification. 34 In general, raw HiPCO SWCNTs (Unidym) were suspended in 1 wt% sodium deoxycholate aqueous solution by 1 hour of bath sonication. This suspension was ultracentrifuged at 300,000 g to remove the bundles and other large aggregates. To further remove any remaining bundles and keep only bright single nanotubes, a gradient separation was used to purify the as-made SWCNTs. The supernatant was first concentrated and then layered to the top of a 10 wt%/20 wt%/30 wt%/40 wt% sucrose step gradient, followed by ultracentrifugation at 300,000 g for 1 hour. Only the top 1 mL was retained by careful fractionation and 0.75 mg/mL of C18-PMH-mPEG (5 kD for each PEG chain, 90 kD in total) (poly(maleic anhydride-alt-1-octadecene)-methoxy(polyethyleneglycol, 5000) MW = 90,000 in total), synthesized by our group) along with 0.25 mg/mL of DSPE-PEG(5 kD)-NH 2 (1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol, 5000)], Laysan Bio) was added. The resulting suspension was sonicated briefly for 5 min and then dialyzed at pH 7.4 in a 3500 Da membrane (Fisher) with a minimum of six water changes and a minimum of two hours between water changes. To remove aggregates, the suspension was ultracentrifuged again for 1 hour at 300,000 g. This surfactant-exchanged SWCNT sample has lengths ranging from 100 nm up to 3.0 μm, with the average length of ~1 μm as shown in Supplementary Figure S1. These amino-functionalized SWCNTs were further conjugated with RGD peptide according to the protocol that has been used in our group. Briefly, an SWCNT solution with amine functionality at 300 nM after removal of excess surfactant, was mixed with 1 mM sulfo-SMCC at pH 7.4 for 2 h in PBS at pH 7.4. After removing excess sulfo-SMCC by filtration through 100-kDa filters (Amicon), RGD-SH (cyclo-RGDFC, Peptides International) was added together with tris(2-carboxyethyl)phosphine (TCEP) at pH 7.4. The final concentrations of SWCNT, RGD-SH and TCEP were 300 nM, 0.1 mM and 1 mM, respectively. The reaction was allowed to proceed for 2 days at 4 º C before purification to remove excess RGD and TCEP by filtration through 100-kDa filters. Atomic force microscopy (AFM) imaging. AFM image of the as-made SWCNT conjugate was acquired with a Nanoscope IIIa multimode instrument in the tapping mode. The sample for imaging was a drop-dried sample on glass, the same one for calibration curve measurement of the distance dependence of fluorescence enhancement, prepared by drop-drying 0.5 L watersoluble PEGylated and functionalized SWCNTs (0.45 nM) solution containing 0.05 wt% Triton X-100 on a bare glass substrate and calcination at 350 ºC for 15 min.

Distance-dependence of plasmonic fluorescence enhancement.
To determine the calibration curve, on the bare glass substrate and each Au/Au substrate with a certain thickness of Al 2 O 3 coating, 0.5 L water-soluble, PEGylated and functionalized SWCNTs (0.45 nM) solution containing 0.05 wt% Triton X-100 was drop-dried to form a uniform spot with diameter of ~2 mm. All spots were imaged in epifluorescence setup with a 658-nm laser diode (100 mW, Hitachi) focused to a 750 μm diameter spot by focusing the laser near the back focal length of a ×10 objective lens (Bausch & Lomb). The resulting NIR photoluminescence (PL) was collected using a liquid-nitrogen-cooled, 320 × 256 pixel, two-dimensional InGaAs camera (Princeton Instruments) with a sensitivity ranging from 800 to 1,700 nm. The excitation light was filtered out using an 1,100 nm long-pass filter (Omega) so that the intensity of each pixel represented light in the 1,100 -1,700 nm range. The exposure time was 100 ms. Images were taken in a 2D scanning mode, flat-field-corrected to account for non-uniform laser excitation, and then stitched automatically using LabVIEW to recover the original shape of the spot. Integral intensity for each stitched spot was done using the roipolyarray function in MATLAB software. Cell incubation and staining. The U87-MG cell medium containing 1 g· L -1 D-glucose, 110 mg· L -1 sodium pyruvate, 10% fetal bovine serum, 100 IU· mL -1 penicillin, 100 μg·mL -1 streptomycin and L-glutamine was used. Cells were maintained in a 37 ℃ incubator with 5% CO 2 . To stain cells with SWCNT-PEG-RGD, trypsinized U87-MG cells were mixed with SWCNT-PEG-RGD at a nanotube concentration of 1 nM (for imaging on glass; this higher concentration helped to find the brighter single nanotubes from a distribution including many dim tubes) or 20 pM (for imaging on Au/Au which helped to visualize the otherwise dim tubes by enhancement) at 4 º C for 1 h, followed by washing the cells with 1x PBS to remove all free conjugates in the suspension. For the hypertonic treatment, the original cell medium was completely removed and the cells were incubated in 1x PBS supplemented with 0.45 M sucrose at 37 º C for 0.5 h before trypsinized and stained. For the potassium depletion treatment, the original cell medium was removed and the cells were incubated in a potassium-free buffer containing 0.1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1.4 M NaCl and 25 mM CaCl 2 at 37 º C for 0.5 h before trypsinized and stained. Note that for the K + -depletion treatment, the potassium-free buffer was used to replace 1x PBS wherever 1x PBS was needed throughout the entire procedure. High-magnification NIR photoluminescence imaging. 5 L of stained U87-MG cell suspension was transferred to 200 L of 1x PBS or potassium-free buffer (for potassium depletion treatment), placed into an 8-well chamber slide (Lab-Tek™ Chambered Coverglass, 1.0 Borosilicate). An Au/Au coated glass substrate or a bare glass chip was then placed on top. Capillary force formed a very thin layer of liquid between the substrate and coverglass, allowing a monolayer of cells residing in between. The chamber slide was kept in a temperature controlled chamber (BC-260W, 20/20 Technology, Inc.) for epifluorescence imaging. Temperature was always kept at 4 º C at the beginning for at least 5 min to ensure the cell membrane in close contact with the hydrophilic gold surface. The temperature of the imaging cell was controlled by heat exchanger (HEC-400, 20/20 Technology, Inc.), and the CO 2 gas flow was kept at 1 L/min by a gas purging system (GP-502, 20/20 Technology, Inc.). Single nanotube imaging and tracking were done using a 658-nm laser diode excitation with an 80 μm diameter spot focused by a ×100 objective lens (Olympus). The resulting NIR photoluminescence was collected using a liquid-nitrogen-cooled, 320 × 256 pixel, two-dimensional InGaAs camera (Princeton Instruments) with a sensitivity ranging from 800 to 1,700 nm. The excitation light was filtered out using an 1100 nm long-pass filter (Thorlabs) so that the intensity of each pixel represented light in the 1,100 -1,700 nm range. To initiate the active uptake of single nanotubes, the chamber temperature was increased from the initial temperature of 4 º C and stabilized at the set temperature (usually took 2 min). The camera took images to record endocytotic process continuously with an exposure time of 3 s. Matlab 7 was used to process the images for any necessary flat-field correction and extract trajectories and time courses of PL changes from the video.       Figure S5. Average fluorescence intensity of SWCNTs immobilized on Au/Au substrate at different pH at 37 º C. All intensities were normalized based upon the maximum intensity at pH 7.4. SWCNTs were wrapped in PEG and conjugated with SPDP ligand (N-Succinimidyl 3-[2-pyridyldithio]-propionate) with activatable SH terminus and anchored onto the gold surface via the thiol-gold chemistry. We soaked the Au/Au substrate into SWCNT-PEG-SPDP solution to have enough thiolated SWCNTs adsorbed on the gold surface for ensemble measurement, removed the unbound nanotubes, placed the substrate in 1x PBS at 37 º C to mimic the cell imaging condition and imaged the nanotubes through ×100 objective when pH was adjusted between 5 and 9 by adding 0.1 M HCl or 0.1 M NaOH (as the pH of endosomes and lysosomes has been reported as around 5) 42 . Error bars were obtained by taking the standard deviation of all 319×256 pixels in the field of view (69 m by 86 m).