Stable and robust nanotubes stretched from polymersomes

Joseph E. Reiner, Jeffrey M. Wells*, Rani B. Kishore, Candace Pfefferkorn†, and Kristian Helmerson‡

Physics Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899-8424

Communicated by William D. Phillips, National Institute of Standards and Technology, Gaithersburg, MD, December 14, 2005 (received for review August 15, 2005) We create long polymer nanotubes by directly pulling on the Phospholipid nanotubes, however, are transient and tend to membrane of polymersomes using either optical tweezers or a collapse unless steps are taken to make them more stable. micropipette. The polymersomes are composed of amphiphilic Attempts to cross-link self-assembled systems composed of diblock copolymers, and the nanotubes formed have an aqueous (or other low-molecular-weight amphiphiles) have met with core connected to the aqueous interior of the polymersome. We limited success (9, 15). Conversely, supramolecular structures stabilize the pulled nanotubes by subsequent chemical cross- from the self-assembly of higher-molecular-weight amphiphiles, linking. The cross-linked nanotubes are extremely robust and can such as block copolymers, are more amenable to stabilization by be moved to another medium for use elsewhere. We demonstrate cross-linking. Polymersomes (polymer vesicles) (16) have been the ability to form networks of polymer nanotubes and polymer- chemically cross-linked to form structures that are stable indef- somes by optical manipulation. The aqueous core of the polymer initely (17). Worm-like micelles of diblock copolymers with nanotubes together with their robust character makes them diameters as small as 10 nm and lengths exceeding 1 ␮m were interesting candidates for nanofluidics and other applications in cross-linked without any disruption of the cylindrical morphol- . ogy (18). Recently, spontaneous formation and subsequent cross-linking of water-filled nanotubes from the self-assembly of cross-link ͉ optical tweezers ͉ nanofluidics ͉ vesicles amphiphilic triblock copolymers was reported (19).

Here, we report on the directed formation of water-filled BIOPHYSICS anotubes are among the most promising structures in nano- nanotubes by pulling, using either optical tweezers or a micropi- Ntechnology. Carbon nanotubes are already finding applications pette, on bilayer membranes of polymersomes composed of in composite materials requiring high strength-to-weight ratio, and amphiphilic diblock copolymers. Although the stretching of many more applications are expected in the near future (1). nanotubes from polymersomes using optical tweezers to pull on Nanotubes also can naturally occur in biology. Transport of genetic a microsphere, attached as a handle to the diblock copolymer material from phage viruses to bacteria can occur via protein membrane, has been reported (20),§ generating sufficient forces nanotubes (2), and phospholipid nanotubes between live cells, to form a nanotube by pulling directly on the polymer membrane purported to be a conduit for intercellular organelle transport, were is much more difficult. An alternative to using more force is to CHEMISTRY recently observed (3). The use of nanotubes for transport of modify the membrane such that it is easier to pull. It is well biological molecules is particularly exciting, with potentially signif- known that the elastic and viscoelastic properties of mem- icant applications in biotechnology. Gold nanotubules in polymer branes can be dramatically altered by the inclusion of surfactants membranes have been used to separate small biological molecules such as detergents or charged lipids. Charged lipids have been on the basis of molecular size (4) and DNA fragments according to used to increase the fluidity of lipid membranes such that optical base recognition. Experiments on the movement of DNA in tweezers can readily stretch nanotubes from lipid membranes nanofabricated structures (5, 6) showed that new transport mech- (12). Detergent-like surfactants have also been used to alter the anisms occur when spatial confinement in one or more dimensions membrane properties of polymersomes, to increase water per- is less than the radius of gyration. Similar effects also have been meability and susceptibility to lysis (21). By incorporating a observed with DNA fragments passing through multiwalled carbon detergent-like, triblock copolymer in the formation of the poly- nanotubes (7). All of these nanotubular structures have an interior mersomes, we make the membranes sufficiently fluid to form aqueous environment suitable for biological molecules, but with the nanotubes by pulling directly on the membranes with optical exception of ref. 6, the length of an individual channel is only on the tweezers. We subsequently cross-link the to form order of 1 ␮m. stable and robust nanotubes that can be removed for use Extremely long, water-filled nanotubes can be formed from elsewhere. We also demonstrate the formation of polymer self-assembling amphiphilic molecules. For example, phospholipids vesicle–nanotube networks using optical tweezers. can spontaneously form nanotubes under appropriate conditions (8). Vesicle membranes composed of self-assembled amphiphilic Results and Discussion molecules can exhibit both elastic and viscoelastic properties, which, Optical Pulling and Cross-Linking of Polymer Nanotubes. Fig.1isa under the application of a localized force, can result in the forma- sequence of images that shows a polymer nanotube being pulled tion of a nanotube. Pioneering work by Evans et al. (9) demon-

strated the formation of phospholipid nanotubes by using a mi- Conflict of interest statement: No conflicts declared. cropipette to pull on phospholipid vesicle () membranes. Freely available online through the PNAS open access option. More recently, Orwar and coworkers (10) adopted the technique of Abbreviation: TEM, transmission EM. Evans to create elaborate networks of interconnected by phospholipid nanotubes and demonstrated transport through these *Present address: NASA Langley Research Center, Hampton, VA 23681-2199. networks. Similarly, optical tweezers have been used to create †Present address: Gettysburg College, Gettysburg, PA 17325. phospholipid nanotubes by pulling directly on membranes of lipo- ‡To whom correspondences should be addressed. E-mail: [email protected]. somes (11, 12) or by pulling on a microsphere attached to a lipid §The experiment of ref. 20 found that the intermonolayer friction of a polymersome membrane (13). Alternatively, simultaneous hydration and suction- membrane of a particular formulation was Ϸ1 order of magnitude higher than that of a typical phospholipid membrane. That is, the force required to pull a nanotube from their driven flow has been used to create extremely long phospholipid polymer membrane is Ϸ1 order of magnitude higher than the force required to pull a nanotubes in microfluidic devices (11, 14). nanotube from a typical phospholipid membrane.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510803103 PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1173–1177 Downloaded by guest on September 28, 2021 Fig. 2. Video fluorescence microscopy images of a pulled nanotube after Fig. 1. Video images of the fluorescence from the membrane dye DiO-C16 cross-linking showing attachment to the parent polymersome (a), severing showing the creation of a nanotube from a polymersome by directly pulling from the parent polymersome by the application of a short, high-energy UV on the polymer membrane with optical tweezers. The nanotube was stretched laser pulse (b), and deflection (in the direction indicated by the arrow) of the from the membrane at a rate of Ϸ10 ␮m͞s. (Scale bar: 10 ␮m.) rigid, cross-linked polymer nanotube by optical tweezers (c). The optical tweezers is not on in a and b, whereas in c the location of the optical tweezers is indicated by X. from the membrane of a giant polymersome¶ using optical tweezers. We have been able to pull nanotubes up to Ϸ1cmin length and were limited by the travel of our microscope stage. nanotubes can be moved from the chamber in which they are Typically, the nanotube retracts back to the parent polymersome formed to another container with a different buffer. when the optical trap is switched off. The images of Fig. 1 were The rigidity of the nanotube suggests that the entire nanotube taken by using video fluorescence microscopy of the dye DiO- is cross-linked; however, the use of the pluronic F-127 may limit C16 in the polymer membrane. The apparent diameter of the the maximum extent of cross-linking relative to a pure copoly- nanotube is Ϸ250 nm, which is the diffraction limit of the optical mer system. In a systematic study of cross-linking within worm- microscope. The retraction of the nanotube to the parent vesicle, like micelles formed from a blend of cross-linkable and non- when the end of the nanotube is released, is indicative of the cross-linkable copolymers (22), percolation behavior (indicating transient nature of pulled structures from self-assembled mem- complete cross-linking) was observed for mol fractions of cross- branes; the system wants to relax to the lower-energy configu- linkable copolymers Ͼ0.15. Because we only use 8 wt % (Ϸ1.7% ration. This behavior is readily observed for nanotubes pulled mol fraction) pluronic F-127 in the formation of our polymer- from membranes (9). Although such nanotubes can somes, it seems reasonable that we should observe rigid nano- be kept from retracting by attaching them to surfaces or other tubes after cross-linking. vesicles (10), the resulting structure is quite fragile and typically stable for only a few hours. For many applications, the nanotubes Nanotube Formation Using a Micropipette. Not surprisingly, we also must be highly stable such that they can be moved from container can pull polymer nanotubes using micropipettes, similar to the to container. Although there have been attempts to stabilize technique demonstrated with lipid membranes (9, 10), and pulled lipid nanotubes by imbedding, in the lipid bilayer, mac- subsequently cross-link them as described earlier. Alternatively, romolecules that contain cross-linkable monomers (9), this we generate flow by expelling buffer containing the appropriate strategy has not been successful for small (Ͻ200 nm) diameter cross-linking reagents from a micropipette tip into a chamber lipid nanotubes. Conversely, polymers can be readily cross- containing a fairly high density of polymersomes in buffer. This linked to form stable structures. flow causes the polymersomes to move past the micropipette tip, We stabilize our pulled polymer nanotubes by free radical and occasionally one of the polymersome membranes sticks to polymerization of the hydrophobic butadiene tails (17). We the end of the tip. The flow continues to push the polymersome introduce the cross-linking reagents using a flow- device. The downstream pulling a nanotube out of the membrane, with the nanotubes became cross-linked and stable within 1 min of adding other end attached to the micropipette tip. Simultaneously, the the reagents. Fig. 2a is a fluorescence image of a cross-linked cross-linking reagent flowing from the tip causes the resulting polymer nanotube attached to the parent polymersome. The structure to become rigid over time. The process continues nanotube is curved because of the fluid flow as the cross-linking repeatedly as the polymersomes flow past the micropipette tip, reagents were introduced. (Straighter, cross-linked nanotubes generating many rigid and straight polymer nanotubes dangling are produced by reducing the flow velocity.) A pulsed UV laser from the end of the micropipette tip. As a final step, additional (optical scalpel) is used to cut the nanotube free from the cross-linking reagents are added to the chamber to further stabilize all of the structures produced. similarly cross-linked, parent polymersome. Fig. 2b is a fluores- cence image of the polymer nanotube after application of the Nanotube Morphology and Diameter by Transmission Electron Mi- UV pulse. The rigidity of the polymer nanotube is now evident. croscopy (TEM) Imaging. To obtain a better measurement of the In contrast to the situation before cross-linking where the diameters and morphologies of our stable polymer nanotubes, nanotube retracts to the parent polymersome (at a rate of Ϸ10 we imaged them by TEM. Fig. 3 contains TEM images of a ␮m͞s) after release of the end of the nanotube from the optical sample of cross-linked, polymer nanotubes. Fig. 3 Left shows a trap, the cross-linked nanotube does not change shape after its relatively uniform nanotube and one that is corrugated. A connection to the polymersome is severed. Further evidence of number (Ϸ80%) of cross-linked nanotubes imaged in this sample the rigidity of the nanotube is seen in Fig. 2c, in which pulling on appeared corrugated. The corrugation appears only after cross- the end of the severed nanotube with optical tweezers (in the linking and depends on whether the nanotube is under some direction indicated by the arrow) displaces the nanotube without ʈ tension during the cross-linking. This result suggests that the changing its shape. The cross-linked, polymer nanotubes are corrugation is due to the cross-linking process and not due to extremely robust, maintaining their shape even after several polymer vesicles fusing together linearly (23), like a string of weeks of storage at room temperature. In contrast to lipid nanotubes pulled from liposomes, our cross-linked polymer ʈThe corrugation also can be seen under fluorescence microscopy with nanotubes contain- ing the membrane dye DiO-C16 and appears only after the addition of the cross-linking ¶We typically pulled nanotubes from giant polymersomes with diameters of Ͼ5 ␮m, reagents. When care is taken to ensure that both ends of the nanotube are held securely because these polymersomes adhered more readily to the coverslip surface and therefore and the nanotube is under some tension, the fraction of nanotubes that are corrugated, did not move as we pulled on the membrane. as measured with fluorescence microscopy, can be Ͻ10%.

1174 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510803103 Reiner et al. Downloaded by guest on September 28, 2021 Fig. 4. Images illustrating the creation of polymer nanotube–vesicle net- Fig. 3. Low-resolution (Left) and high-resolution (Right) TEM images of works. (Left) Sequence of images illustrating the pulling of a nanotube from cross-linked polymer nanotubes. The nanotubes, which were relatively the membrane of a polymersome using optical tweezers (shown in red) and straight just after cross-linking, were washed, dried, and stained with uranyl the attachment of the nanotube to another polymersome. (Right) Composite acetate before placing them in the TEM. This process of preparing the nano- image from video fluorescence microscopy of a network of polymer nano- tubes for the TEM most likely caused them to bend and also bundle together. tubes and polymersomes, containing the membrane dye DiO-C16, assembled We observed that the majority of nanotubes in this sample appeared to be using optical tweezers. (Scale bar: 10 ␮m.) (Inset) Scanning confocal micros- corrugated, which we later discovered occurred as a result of cross-linking. The copy image of a nanotube pulled from a polymersome encapsulating sulfor- wall of the nanotube is visible in the high-resolution image as the region along hodamine B dye in buffer. Variations in the intensity are due to movement of the edge of the nanotube that is slightly darker. A collapsed cross-linked the nanotube during the scan. (Scale bar: 10 ␮m.) polymersome is apparent in the upper portion of the low-resolution image. BIOPHYSICS giant worm-like micelles have been observed to form from the beads. Corrugation of tubular lipid vesicles have been explained diblock copolymer PBd-PEO (18). To observe the water-filled as a Rayleigh-type instability arising during front propagation core, we pulled nanotubes from polymersomes encapsulating because of a sudden increase in membrane tension (24). Cor- buffer containing the water-soluble, fluorescent dye sulforho- rugated patterns observed in cylindrical polymer gels (25) have damine B. Because the permeability of the polymer membrane been attributed to stress associated with the spatial changes in to water is Ͼ1 order of magnitude less than the corresponding the polymer density across the thickness of the polymer bound- permeability for lipid membranes (21), it is a reasonable as- ary, which limits the diffusion of water. Similarly, periodic, sumption that any water in the core of the pulled polymer CHEMISTRY corrugated patterns are observed in stiff polymer films sup- nanotube comes from the water enclosed by the parent poly- ported on relatively soft, elastic media due to a strain-induced mersome. Fig. 4 Right Inset is a fluorescence image, taken with elastic buckling instability (26). Because we flow in chemicals to a laser scanning confocal microscope, of a cross-linked, pulled perform the free radical polymerization reaction, the polymers polymer nanotube and the parent polymersome. The fluores- in the outer layer of the bilayer membrane structure will, most cence from the excited sulforhodamine B dye evident in the likely, cross-link before the molecules in the inner layer, possibly parent polymersome also can be seen in the pulled nanotube, giving rise to stresses that may result in corrugation of the indicating that buffer containing dye is in the core of the nanotube. nanotube. Also visible in Fig. 3 Left is a cross-linked polymersome that appears to have collapsed, possibly during dehydration of the Polymer Nanotube-Vesicle Network. The polymer membranes can sample. The rigidity of the cross-linked polymer membrane is be manipulated, with either optical tweezers or micropipettes, to apparent by the folds in the collapsed polymersome. Fig. 3 Right form nanotube network structures such as Y-junctions, similar to is a magnified view of another pair of nanotubes. Again, one of those demonstrated with lipid membranes (9, 10). Fig. 4 Right the nanotubes has a relatively uniform profile, whereas the other contains a composite image of a network composed of polymer is highly corrugated. By measuring the outer diameter of the nanotubes and polymersomes. As illustrated in Fig. 4 Left, nanotube in this image and using a (unilamellar) membrane optical tweezers are used to grab onto the polymersome mem- thickness of 8 nm,** we obtain an average inner diameter of Ϸ70 brane and stretch out a polymer nanotube. The end of the nm for the uniform nanotube and Ϸ25 nm at the constrictions nanotube, held by the optical trap, is pulled through the mem- of the corrugated nanotube. Similar diameters were obtained for brane of another polymersome and then released. Once re- nanotubes from other magnified TEM images. leased, the nanotube retracts until it reaches the polymersome membrane through which it was inserted, thus forming a struc- Evidence for Aqueous Core. For applications of nanotubes as ture composed of two polymersomes joined by a polymer conduits for transporting biological material, it is necessary that nanotube. The two Y-junctions apparent in Fig. 4 Right were the core of the nanotube contain water. Although our nanotubes formed by pulling an additional nanotube from a polymersome are formed from amphiphilic copolymers, the question remains that already had a nanotube attached to it. If the additional whether the core of the nanotube is aqueous or micellar, because nanotube was pulled from the membrane at a location suffi- ciently close to the attachment point of the first nanotube, the attachment point of the additional nanotube would move along **We obtain a membrane thickness of 8 nm for our polymers using the curve in figure 3 of the polymersome membrane toward the attachment point of the ref. 27. This value is consistent with measurements based on our TEM images (circled first nanotube and then along the length of the first nanotube, region of figure 3), which appear to give a membrane thickness of Ϸ12 nm but systematically overestimate the actual value because the images are not true cross- thus forming a nanotube Y-junction. (The location of the sectional views. attachment point of the additional nanotube to the first nano-

Reiner et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1175 Downloaded by guest on September 28, 2021 tube is such that all nanotubes emanating from the Y-junction experience only tension and no sheer or bending force.) This process is the same mechanism of forming Y-junctions originally demonstrated with nanotubes pulled from liposome membranes using micropipettes (9, 10). We also have demonstrated the formation of a Y-junction by directly pulling on the wall of a nanotube to form an additional nanotube. Fig. 5. Illustration of the flow-cell device used for cross-linking polymer nanotubes. The sample chamber consists of a microscope slide with an Ϸ1cm Conclusions and Outlook diameter hole drilled through the center. The bottom of the chamber is a glass coverslip (not shown) sealed to the microscope slide by silicone grease. The We demonstrate the controlled formation of polymer nanotubes channel was formed by using two pieces of double-sided tape (each piece is Ϸ2 by directly pulling on a membrane composed of amphiphilic cm in length with a thickness of Ϸ70 ␮m) spaced by 2 mm between the upper diblock copolymers using optical tweezers. We subsequently surface of the microscope slide and an additional glass coverslip. This coverslip make them stable and robust by chemical cross-linking. The only covered the channel region up to the edge of the sample chamber. The ability to pull directly with optical tweezers provides additional sample chamber was left uncovered to allow for evaporation to pull fluid to capabilities to form supramolecular structures of interest. For be pulled through the channel. example, we have observed that the optical trap can be used to pull a nanotube directly from the wall of an existing nanotube to porating Ͻ10 wt % F-127 stably form vesicles (29). Fifty form a junction and that we can readily insert the end of a microliters of this polymer mixture was applied to Ϸ1-cm length polymer nanotube through the membrane of a polymersome. We of two parallel platinum wires (1 mm diameter, spaced by a gap have been able to pull nanotubes up to Ϸ1 cm in length. We also of 3 mm), dehydrated under nitrogen gas, and then vacuum dried have pulled polymer nanotubes using micropipettes, similar to for 1 h. The resulting polymer film on the platinum wires then the technique demonstrated with lipid membranes (9, 10), and Ϸ subsequently cross-link them as described earlier. Alternatively, was hydrated in an electroformation chamber, containing 1ml we generate flow by expelling buffer containing the appropriate of 0.3 M sucrose solution, at 10 V and 10 Hz for 18 h. Five cross-linking reagents from a micropipette tip into a chamber hundred microliters of the polymersome suspension was trans- ferred to a centrifuge tube containing 10 ml of buffer (10 mM containing a fairly high density of polymersomes in buffer. This ͞ ϫ flow of reagents simultaneously directs the formation of nano- Mops 50 mM NaCl, pH 7.0) and centrifuged at 672 g for5min. tubes and cross-links them. A sample from the bottom of the centrifuge tube was used for The cross-linked nanotubes we form appear to be quite rigid, experiments. For some of the experiments, 1 mM sulforhodam- suggesting that the entire structure is cross-linked. Although the ine B (Sigma) dissolved in the 0.3 M sucrose solution was used, use of a pluronic to make the polymersome membrane more instead of the membrane dye DiO-C16. flexible may limit the maximum extent of cross-linking, it is not clear whether the pluronic is still incorporated in the polymer Flow Cell and Cross-Linking. The polymersomes and nanotubes membrane of the stretched nanotube or in the membrane, in were cross-linked by free radical polymerization (17). A mixture ␮ ͞ ␮ ͞ general, after cross-linking. Further studies, for example, using of 90 l of potassium persulfate (12.5 mg ml), 8 lof9mg ml ␮ ͞ ␮ fluorescently labeled pluronics, need to be performed to address sodium metabisulfite solution, and 8 l of 130 mg 300 l ferrous this issue. sulfate heptahydrate solution was applied to the polymer struc- Although we have demonstrated stabilization of nanotubes by tures by using a flow cell. The flow cell consisted of a channel chemically induced cross-linking of our polymers, photo-induced constructed using two strips of double-sided tape that was cross-linking also should be possible and may, in some cases, be sandwiched between a microscope slide, with a 1-cm-diameter a more convenient and flexible technique to use. Direct expo- hole drilled through the center, and a glass coverslip (Fig. 5). The sure of the polymersomes to UV did not induce cross-linking. 1-cm hole, sealed off on the lower side by another glass coverslip, We have observed that the chemical cross-linking process can using silicone high-vacuum grease (Dow-Corning), served as the produce corrugated nanotubes, and such structures may be sample chamber. The polymersomes, having a density greater useful for DNA sorting (5, 6). We also demonstrated the than water because of the encapsulated sucrose, eventually (we formation of polymer vesicle–nanotube networks that include typically wait 60 min) reside at the bottom of a chamber and stick Y-junctions. The ability to create stable, biologically compatible to the coverslip surface, after which they are readily accessible nanotubes of essentially arbitrary length in nontrivial geometries for optical manipulation and imaging on an inverted microscope. lends itself to many applications in biotechnology, such as The fluid channel, created by the double-sided tape and cover- nanofluidics, as well as opening up new opportunities for slip, is open to air at one end and empties into the top portion investigations of other systems, such as the behavior of long of the chamber at the other end. Cross-linking reagents are molecules effectively confined to one dimension. introduced at the open end of the channel and move toward and empty into the chamber region by capillary flow. Although the Materials and Methods flow velocity in the channel is quite high, the flow velocity in the Polymersome Preparation. Polymersomes were formed by using an chamber is substantially lower because the cross-sectional area electroformation technique (28). Diblock copolymer poly(buta- of the chamber is much larger than the cross-sectional area in the diene-b-ethylene oxide) PBd(1800)-b-PEO(900) was purchased channel region. The low flow velocity in the chamber region from Polymer Source (Dorval, PQ, Canada). A mixture of the allows the appropriate chemicals to reach the nanotube without diblock copolymer at 20 mg͞ml solution in chloroform, 8 wt % destroying it. (The process of adding chemicals directly to the pluronic F-127 (Sigma), and 10 mM 3,3Ј-dihexadecyloxacarbo- chamber by pipette turned out to be too violent and destroyed cyanine perchlorate (DiO-C16) membrane dye (Molecular any nanotubes before stabilization by cross-linking could take Probes) was prepared. The detergent-like triblock copolymer place.) PEO-poly(propylene)-PEO, Pluronic F-127, which incorporates into the membranes, was added to increase membrane fluidity. Optical Tweezers and Scalpel. Optical manipulation was performed F-127 was chosen because very low concentrations (0.06 wt %) on a Zeiss Axiovert 100 inverted microscope, which was modi- can permanently disrupt liposome membranes at room temper- fied to include optical tweezers that can be moved across the field ature (29). Because of the structural similarity between diblock of view, and an optical scalpel (30). The trapping light is from a PBd-PEO and triblock F-127, polymersome formulations incor- continuous-wave, ytterbium fiber laser (IPG Photonics, Oxford,

1176 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510803103 Reiner et al. Downloaded by guest on September 28, 2021 MA) emitting at a wavelength of 1.07 ␮m. The trap power can vesicles and nanotubes. Fluorescence video microscopy and laser be varied up to Ϸ2 W and is focused by a 100ϫ, 1.3 NA, scanning confocal microscopy was performed by using laser Plan-Neofluar objective lens (Zeiss). The optical scalpel, which excitation of either the membrane dye or the encapsulated dye. is at 355 nm, comes from the 3rd harmonic of a pulsed Nd:YAG In addition, some samples of cross-linked nanotubes were im- laser (Continuum, Santa Clara, CA). The focus of this beam is aged by using TEM. To prepare for TEM imaging, the nanotubes kept at a fixed position with respect to the objective lens. The were washed several times with milli-Q water to remove the pulse duration is Ϸ5 ns, and the energy per pulse can be varied buffer, which contains salts, placed on a TEM grid, and then left up to 5 mJ; however, we typically used only 10 ␮J per pulse. The to dry in air at room temperature. Before placement in the microscope also was modified to include fluorescence micros- transmission electron microscope, the sample was stained with copy excited by an air-cooled, tunable argon ion laser (Melles uranyl acetate to enhance contrast. Griot, Carlsbad, CA) and imaged by a Sunstar 300, low-light- level charge-coupled device video camera (Electrophysics, Fair- We thank Richard Leapman at the National Institutes of Health for field, NJ). assistance in obtaining the TEM images. We also thank Laurie Locascio, Ksenia Brazhnik, and Jack Douglas for useful discussions. This work was Microscopy. Polymer vesicles and nanotube structures were ob- supported in part by the Office of Naval Research. Certain commercial equipment, instruments, or materials are identified in this work to specify served by using either brightfield or fluorescence video micros- the experimental procedure adequately. Such identification is not in- copy on the same optical microscope apparatus that contained tended to imply recommendation or endorsement by the National the optical tweezers and scalpel. In some cases, laser scanning Institute of Standards and Technology, nor is it intended to imply that confocal microscopy (Pascal LSM, Zeiss) with a 63ϫ, 1.4 NA, the materials or equipment identified are necessarily the best available Plan-Apochromat objective lens (Zeiss) was used to image the for this purpose.

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