Ionization and electron excitation of C60 in a carbon nanotube: A variable temperature/voltage transmission electron microscopic study

Significance The destruction of specimen molecules by an electron beam (e-beam) is either beneficial, as in mass spectrometry capitalizing on ion formation, or deleterious, as in electron microscopy. In the latter application, the e-beam not only produces the specimen image, but also causes information loss upon prolonged irradiation. However, the atomistic mechanism of such loss has been unclear. Performing single-molecule kinetic analysis of C60 dimerization in a carbon nanotube (CNT) under variable-temperature/voltage conditions, we identified three reactive species—that is, radical cation, singlet, and triplet excited states—reacting competitively as the voltage and the properties of the CNT were changed. The key enabler was in situ continuous recording of the whole reaction process, suggesting an upcoming new era of “cinematic chemistry.”

Since the time of the Knoll/Ruska invention of transmission electron microscopy (TEM) (1), electron microscopy has suffered from information loss during observation often ascribed to the structural changes of a specimen into a different substance, known as radiation damage (2). As summarized recently by Egerton (3), the electron-beam (e-beam) damage in organic matter predominantly occurs via processes triggered by ionization (radiolysis). Although radiolysis is highly important, studies on radiolysis have largely been descriptive and qualitative because of the complexity of the process and the difficulty in quantifying the changes under variable-temperature and variablevoltage (VT/VV) conditions. The first step of the process involves electron-impact ionization (EII), which removes an electron to form a radical cation (RC) of the specimen, and also electron-impact excitation (EIE), where the electron does not fly away to the vacuum, but stays in a higher antibonding state in the system. The processes were recently studied in depth for the first time by a thorough quantum chemical study (4). There has been, however, a paucity of experimental mechanistic information-that is, how a specimen is transformed to what product with what level of activation energy and frequency at what acceleration voltage. Single molecules encapsulated in a carbon nanotube (CNT) have often been observed either stably or undergoing well-defined chemical transformations (5)(6)(7)(8)(9), and it is primarily because the damage due to secondary electrons is minimal (10). Drawing an analogous specimen stabilization by a thin metallic coating or deposition on a conductive indium tin oxide substrate in scanning electron microscopy (SEM) (11), we can consider that the electron-rich CNT interacts with the specimen molecules and protects them from ionization by filling in the electron vacancy in the RC. In light of the recent characterization of singlet and triplet excitons of CNTs (Fig. 1A) (12), we can also consider that the CNT exciton would excite a molecule in the CNT via energy transfer (ENT) ( Fig. 2A) (13,14). Electron excitation of graphene under TEM conditions has also been suggested recently (15,16).
We report here the VT/VV kinetic study of the dimerization of a van der Waals (vdW) dimer [60]fullerene (C 60 ) to C 120 in a CNT (17)(18)(19), in which we found five

Significance
The destruction of specimen molecules by an electron beam (e-beam) is either beneficial, as in mass spectrometry capitalizing on ion formation, or deleterious, as in electron microscopy. In the latter application, the e-beam not only produces the specimen image, but also causes information loss upon prolonged irradiation. However, the atomistic mechanism of such loss has been unclear. Performing single-molecule kinetic analysis of C 60 dimerization in a carbon nanotube (CNT) under variabletemperature/voltage conditions, we identified three reactive species-that is, radical cation, singlet, and triplet excited states-reacting competitively as the voltage and the properties of the CNT were changed. The key enabler was in situ continuous recording of the whole reaction process, suggesting an upcoming new era of "cinematic chemistry." competing reaction pathways that serve as model pathways of radiolysis damage ( Fig. 1 B-F). We found a marked influence of the temperature, the acceleration voltage, and the properties of the CNT-pristine CNT (prCNT), oxidized CNT (oxCNT), or damaged CNT (dmCNT) (Fig. 2B). They have been distinguished by the pre-exponential factor (PEF) and the Arrhenius activation energy (E a ). Singlet (S 1 ) and triplet (T 1 ) species generated by ENT from a CNT exciton account for frequently occurring reactions ( Fig. 1 B and C) (3). The triplet reaction also occurs when we use an oxCNT (Fig. 1D), which is known to form a triplet exciton. Electrons of 60 keV in energy cannot energize prCNT to either the singlet or the triplet state, but instead directly ionize C 60 into the RC. The RC is then reduced by the CNT to a mixture of S 1 and T 1 states (Fig. 1E). This process illustrates how prCNT protects the specimen from radiolysis by "grounding" (Fig. 2C) and accounts for the stability of a variety of molecules having lowlying highest-occupied molecular orbitals, such as saturated hydrocarbons (20), amides (21), alcohols (22), and inorganic salts encapsulated in a CNT (23,24). We observed the RC when the reaction was performed in dmCNT (Fig. 1F). The VT/VV behavior of the kinetics agrees with the competitive occurrence of electron excitation and ionization, but not with the atomic displacement damage mechanism, which is the major cause of radiation damage in conductive inorganic materials (25).
In 2011, C 60 dimerization in CNTs was reported to take place even with 20-keV irradiation, although it then requires a 100-times-larger electron dose that at 80 kV (36). The 20-keV energy is far lower than the threshold voltage of carbon atom displacement (CAD; knock-on displacement), and hence CAD is an unlikely mechanism of the C 60 dimerization. A 60-keV e-beam was recently reported to cause reactions of C 60 sandwiched between two graphene sheets-loss of one carbon atom to form a C 118 (quasi) dimer via C 59 (37). This may suggest a difference between a one-dimensional (1D) CNT and gapless two-dimensional (2D) graphene that lacks van Hove singularities (Fig. 1A). This is briefly examined in the present study.

Results
Variable-temperature single-molecule atomic-resolution timeresolved electron microscopic (VT-SMART-EM) imaging is an emerging experimental tool for the study of kinetics and thermodynamics of individual chemical events (19,22,38), and it has provided direct experimental proof of the Rice-Ramsperger-Kassel-Marcus theory (39). In this study, we monitored several hundred individual events of the [2 + 2] reaction under VT/VV conditions at 103 to 493 K and 60 to 120 keV (10,19). Following the reaction conditions developed previously (19), we encapsulated the fullerene molecule in a bundle of prCNTs, oxCNTs, or dmCNTs, 1.3 to 1.4 nm in diameter (40), by heating them together at 823 K for 15 h in vacuo. A specimen was deposited on a TEM grid, and the time evolution of the [2 + 2] cycloaddition of a (C 60 ) 2 @CNT was visually monitored under e-beam irradiation at 120, 100, 80, and 60 kV, with a constant electron dose rate (EDR) of 3.1 × 10 5 e -Ánm À2 Ás À1 for 120 kV and 5.0 × 10 6 e -Ánm À2 Ás À1 for 60 to 100 kV throughout this study. We examined frame rate ranging from 533 frames per second (fps) to 2 fps (40) and employed the 2-fps rate because the higher frame rates do not reduce the magnitude of error that originates largely from the inhomogeneity of the specimens. Note that the reaction rate per electron measured in the present work is not affected by the variation of EDR as previously reported, indicating that the observed reaction events occur independently (19).
The cycloaddition event was characterized by the change in intermolecular distance from 1.00 nm for the vdW dimer to 0.90 nm for the cycloadduct (Fig. 3B), and the E a and PEF were determined (19). The [2 + 2] cycloadduct features a strained cyclobutane ring in the middle and reverts to two molecules of C 60 upon heating at >500 K, thus providing compelling chemical evidence that the adduct is the C 120 cycloadduct (Fig. 3A).
Voltage-Dependent Singlet and Triplet Dimerization of a (C 60 ) 2 @prCNT. Unlike photo-excitation of π-conjugated molecules, in which the photo energy is efficiently transferred to the molecule via an EDT mechanism, electron excitation of a molecule by momentum transfer from an e-beam is very inefficient. To assess how inefficient it would be, and to know the consequence of poor efficiency, we performed the kinetic analysis at an acceleration voltage decreasing stepwise from 120 keV to 60 keV. In Fig. 4, we show the kinetics data of the dimerization in prCNT at 60 to 120 keV at 443 to 493 K. The raw data at 443 K are shown in Fig. 4A, where we plot the number of dimerization events observed at intervals of 8.0 × 10 6 e À Ánm À2 irradiation against the total electron dose (TED) up to 3.0 × 10 8 e -Ánm À2 (for 60 s). After in situ monitoring of the reactions of 39 to 55 C 60 dimerization events at acceleration voltages of 120 (black), 100 (red), 80 (blue), and 60 kV (green; Fig. 4A), we observed three features. First, each reaction event takes place stochastically (41). Second, the occurrence of the events follows the first-order kinetics shown in Fig. 4 B and C, where the 1 -P and ln(1 -P) values are plotted against TED (P = normalized conversion of C 60 ). Third, we find three different kinetic profiles (Fig. 4E).
The rate constants (k) at 100 kV are summarized in Fig. 4D. The error is arguably large for several reasons. The CNT is a mixture of entities having different chirality indexes (i.e., the diameters) (42) and different physicochemical environments. In addition, molecular packing in CNTs changes as the dimerization reaction progresses.
The slope of the Arrhenius plot of the 80-kV reaction (blue) is half as steep as those of the 120-and 100-kV reactions and the E a values of 13.4 ± 3.6 kJÁmol À1 , indicating that the reactive species is more reactive than the one at 120-and 100-kV. The PEF value of 1.2 × 10 À7 (e -) À1 Ánm 2 is far smaller than those at 120 and 100 kV, indicating that the reaction occurs 1,000 times less frequently. Overall, the reaction at 80 kV is slower than at 120 and 100 kV (Fig. 4B).
The reaction at 60 kV was markedly slower at ∼400 K than the reaction at 100 to 120 kV ( Fig. 4B and SI Appendix, Fig.  S3). The Arrhenius plot (green, Fig. 4E) deviates from linearity, indicating that the mechanism changes as the temperature changes. We averaged the data over 443 K and 493 K and obtained an E a value of 20.8 ± 6.1 kJÁmol À1 and PEF = 3.7 × 10 À7 (e -) À1 Ánm 2 . These values fall between those at 100 to 120 kV and at 80 kV.
The Arrhenius plot indicated the formation of one type of reactive species at 100 to 120 kV, another at 80 kV, and probably a mixture of the two at 60 kV. We assign the species to the S 1 state, the T 1 state, and a mixture of the two, respectively. While the details will be discussed below, we point out here that EIE with high-energy e-beam (e.g., 120 keV) occurs with conservation of spin-angular momentum (30) and hence generates the singlet exciton of CNT and singlet excited state of C 60 . Second, we also note that the E a and PEF values at 80 kV are essentially the same as those in the reaction in oxCNT, which can only generate the T 1 species by acting as a triplet sensitizer (see below). In Fig. 4F, we show a schematic orbital diagram of the concerted singlet cycloaddition, which is a reference standard of the [2 + 2] cycloaddition governed by the Woodward-Hoffmann orbital symmetry rule.
Triplet Dimerization of a (C 60 ) 2 @oxCNT. The oxCNT (Fig. 2B), prepared by using KMnO 4 oxidation of a CNT (43), has both the πand σ-carbon skeletons destroyed by chemical oxidation, as demonstrated by infrared (IR) absorption (due to benzophenonelike groups) (44). It is reported to be a triplet sensitizer in solution, as efficient as benzophenone, and has a triplet energy lower than ∼2.5 eV (45), and hence we expect that oxCNT will generate the T 1 C 60 species. We encapsulated C 60 in oxCNT and studied 30 to 52 vdW C 60 dimers [(C 60 ) 2 @oxCNT].
The time course of the dimerization events at 120 kV, with a constant EDR of 3.1 × 10 5 e -Ánm À2 Ás À1 , is shown in Fig. 5A and the frequency integrated over time in Fig. 5B. The semilogarithmic plot in Fig. 5C gives the reaction rates at temperatures between 378 and 453 K, and the Arrhenius plot gives E a and PEF values, both of which are small in magnitude (Fig. 5D). These values ascribable to T 1 are similar to the data found in a prCNT at 80 kV (blue line, Fig. 4E). In Fig. 5E, we show at a schematic orbital interaction in the triplet cycloaddition reaction.
The E a value close to zero suggests an RC, which is formed by ionization and is expected to be extremely reactive (Fig. 6E). Ionization is the standard damage mechanism of radiolysis (3), and our observation shows that ionization of C 60 does occur, when the π-conjugation in the surrounding CNT is destroyed and has lost its grounding function (Fig. 2C ). Table 1 summarizes the E a and PEF data in prCNTs, oxCNTs, and dmCNTs of the five reaction types (Fig. 1 B-F ). The kinetic profiles are color-coded in black, blue, green, and purple. We consider that the path with E a values of 32.9 to 33.5 ± ∼6 kJÁmol À1 in Table 1 (black) took place via S 1 , first because a high-energy e-beam excites CNT with conservation of spinangular momentum (30) and second because the values compare favorably (within experimental error) with an E a value of 28 kJÁmol À1 (a value calculated from figure 5 of ref. 47) reported theoretically for S 1 [2 + 2] cycloaddition in gas phase (47). We assign the E a values of 11 to 15 kJÁmol À1 in Table 1 (blue), as obtained for the oxCNT to the T 1 pathway (48) because an oxCNT is an effective triplet sensitizer due to aromatic ketone residues that accelerate relaxation of singlet to triplet (49). The low values of E a agree with the biradical character of the T 1 excited state of C 60 . Similarly, we assign T 1 to the 80-keV experiment in a prCNT (Table 1) because the kinetic data agree with those for an oxCNT in Table 1 (blue). The value of 20.8 kJÁmol À1 at 60 keV in Table 1 (green) coincides with a value of 23 kJÁmol À1 determined for photodimerization, which likely reflects the consequence of ISC from singlet to triplet in a 1:3 ratio (50).

Discussion
The SMART-EM study on the electron-impact-promoted [2 + 2] cycloaddition mediated by CNTs (Fig. 7) is unique in that we can study in situ the individual reaction events one by one as they take place. The first stage is a fast EIE reaction, characterized by the PEF data. The second stage is a slow, thermally driven reaction of excited C 60 going across an energy barrier with a frequency of exp(-E a /RT ). We determined the kinetic parameters separately for the two steps by visually monitoring the individual events of the forward cycloaddition of vdW complexes, which excludes the contribution of cycloreversion and reversible collisions from the kinetic data analysis.
The ln(PEF) values represent ln(k) at T = infinite, and they vary widely between -7.9 and -16.4 [PEF = 3.9 × 10 À4 to 1.2 × 10 À7 (e -) À1 Ánm 2 ]. They are also extremely low in absolute magnitude, indicating that a large number of electrons (1.0 × 10 3 to 3.0 × 10 6 electrons) are required to form one excited or ionized C 60 molecule (area of 0.396 nm 2 ) that produces the dimer. The E a values (slope) reflect the reactivity of these species in the thermal dimerization reaction (Fig. 7,  second step).
To describe the efficiency of the reaction, we borrow the concept of external quantum efficiency (EQE) used to evaluate the efficiency of photovoltaic devices-the ratio of the number of electrons and holes generated by a device to the number of incident photons shining on the device from outside. Similarly, we can define the EQE based on the number of dimers formed relative to the TED shining on the CNT. The EQE values of the S 1 reaction in a prCNT and the T 1 reaction in an oxCNT at 120 kV are 9.8 × 10 À4 and 2.1 × 10 À6 , respectively, indicating that the latter is nearly 1,000 times less efficient because of the infrequent formation of the triplet exciton of the CNT. The very low value of the energy-attenuation factor (∼10 À5 ; from ∼100 keV to ∼2 eV) reflects the lack of a mechanism for efficient ENT from the e-beam to the CNT and the loss of energy to phonon vibration of the CNT and physicochemical processes.
The Arrhenius plots for the four representative reactions in Fig. 8 summarize the present finding. In accordance with the accepted mechanism of radiation damage, the ionization pathway operates in dmCNT (purple). In prCNT encapsulating C 60 (at >300 K), the ionization is suppressed, and much faster excited-state pathways dominate when the energy of the e-beam is >80 keV (black and blue). When the e-beam energy is 60 keV, it does not excite the CNT and hence C 60 .
Putting together the above experimental data and the literature information, we suggest, in Fig. 9, the two mechanistic possibilities of radiolysis/ionization of the specimen (Fig. 9A) and excitation ( Fig. 9 B and C), where the schematic energy levels are based on the reported S 0 /S 1 energy difference of ∼2.5 eV for C 60 and the S 0 /T 1 energy difference of ∼1.5 eV for C 60 . These schemes are admittedly incomplete, but may serve as a cornerstone for future quantitative studies of radiation chemistry under TEM observation conditions using a high-energy e-beam. Fig. 9 A, 1 shows ionization of the π-rich C 60 , the standard mechanism of radiation damage (Fig. 1F) (52). We found this path at 103 to 203 K in a dmCNT and consider that it also accounts for the CNT damage at low temperatures (compare Fig. 6 A and  B). Fig. 9 A, 2 illustrates C 60 ionization followed by charge neutralization by prCNT and generation of S 0 , S 1 , or T 1 C 60 (Fig. 1E). We observed this path at 60 kV (36). In prCNT and with 100-to 120-keV electrons (Fig. 9B), the e-beam generates singlet exciton of CNT, and ENT forms S 1 C 60 (Fig. 1B). However, the 80-keV electron can only form a less-energetic triplet exciton to generate T 1 C 60 (Fig. 1C). In Fig. 9C, oxCNT generates a triplet exciton and then forms T 1 C 60 (Fig. 1D). Note that oxCNT has been known to form triplet excitons, probably because of rapid singlet-to-triplet relaxation.
In summary, the kinetics data summarized in Fig. 8 have shown the importance of VT/VV kinetic analysis in the studies of radiation damage. The data also showed that chemical ionization and electron excitation are inseparable, but different, mechanisms of the radiation damage, which have often been classified loosely under the single term "ionization." The conducting prCNT, with its high-lying filled orbital, not only protects the molecule from radiolysis (8) (Fig. 2C), but can cause selective chemical reactions if suitable orbital interactions between the molecule and the CNT are available. We can account for the mechanism of the five observed reaction types with the standard chemical reaction mechanisms of ionization and excitation without invoking bond scission due to atom displacement, which is known to occur so infrequently that it contributes negligibly to the observed reactions (3) (Fig. 8). The complexity of the kinetics of EII and EIE suggests a risk in making any mechanistic interpretations of chemical events seen using TEM without performing VT/VV kinetic analysis. Enabled by continuous monitoring of chemical reaction events at atomic resolution with variable frame rate (e.g., between millisecond and second), the present research illustrates the potential of "cinematic chemistry," microscopic imaging of dynamic chemical events, for elucidation of the mechanisms of chemical reactions (24,40,53).

Materials and Methods
Materials. Single-walled CNTs (Meijo Arc SO, produced by arc-discharge using Ni and Y catalysts, >99% purity, average diameter 1.4 nm, lot no. 6601316) were purchased from Meijo Nano Carbon Co. Ltd. C 60 powder (nanom purple ST, >98% purity) was purchased from Frontier Carbon Corporation. TEM grids precoated with a lacey microgrid (RO-C15, for VT experiments; pore size 3 to 8 μm and carbon thickness 70 nm) were purchased from Okenshoji Co., Ltd. Toluene was purchased from Wako Pure Chemical Industries and purified by using a solvent-purification system (GlassContour) (54) equipped with columns of activated alumina and supported copper catalyst (Q-5) prior to use. Potassium permanganate was purchased from Tokyo Chemical Industry Co., Ltd., and sulfuric acid was purchased from Wako Pure Chemical Industries.
General. The water content of the solvent was determined by using a Karl Fischer moisture titrator (CA-21, Mitsubishi) to be <10 ppm. Bath sonication for the dispersion of CNTs in toluene was carried out with a Honda Electronics WT-200-M instrument. Oxidative removal of the terminal caps of CNTs was carried out in an electric furnace, ASH ARF-30KC. Encapsulation of C 60 into CNTs was carried out in an electric furnace, ASH AMF-20, equipped with a temperature-controller AMF-9P. IR spectra were recorded on a JASCO FT/IR-6100 instrument with attenuated total reflection. X-ray photoelectron spectroscopy analysis was carried out on a JPS-9010MC instrument by using Mg Kα X-rays (1,253.6 eV).
Preparation of Samples for SMART-EM. The C 60 @CNTs prepared above are in solid form and thus difficult to deposit directly on a TEM microgrid. We therefore first dispersed samples in toluene (0.01 mgÁmL -1 ) in vials, which were then placed in a bath sonicator for 1 h. The aim was to soften the samples so that we could secure intimate contact between the CNTs and the carbon surface of the grid. A 10-μL solution of the dispersion was then deposited on a TEM grid placed Fig. 7. PEF and E a , representing EIE/ENT and cycloaddition, respectively. Of two possible mechanisms of ENT, the F€ orster mechanism of ENT is shown. The E 33 transition is shown as an example of transitions responsible for C 60 excitation. The E 33 transition followed by thermal relaxation is shown as an example of the processes involved in the C 60 excitation.  Table 1 via S 1 . Blue: the 120-kV data in Table 1 via T 1 . Green: the 60-kV data in Table 1 via direct EIE. Purple: the 120-kV data in Table 1 via RC. Gray band: a range of ln(k) values for temperature-independent CAD estimated from ln(PEF) of radiolysis. The x indicates the ln(k) value for dimerization of C 60 sandwiched between graphene sheets estimated from figure 4 in ref. 37. on a paper that absorbs excess toluene. The resulting TEM grid was dried in vacuo (60 Pa) for 2 h.
SMART-EM Observation. Atomic-resolution TEM observations were carried out on a JEOL JEM-ARM200F instrument equipped with an aberration corrector and cold-field emission gun (point resolution 0.10 nm) at acceleration voltages of E = 60, 80, 100, and 120 kV, under 1 × 10 À5 Pa in the specimen column, and with typical spherical aberration values of 1 to 3 μm. Calibration of the EDR was conducted following a method described in a previous report (19). C 60 dimerization at 60 to 120 kV and C 60 dimerization in oxCNT were monitored at the temperatures mentioned in the Results section of the main text and an EDR (the number of electrons per second per nm 2 ) of circa 3.1 × 10 5 e -Ánm À2 Ás À1 for 120 kV and 5.0 × 10 6 e -Ánm À2 Ás À1 for 60 to 100 kV at 800,000× magnification. The imaging instrument was a complementary metal oxide semiconductor camera (Gatan OneView, 4,096 × 4,096 pixels), operated in binning-2 mode (output image size 2,048 × 2,048 pixels, pixel resolution 0.20 nm at 1,000,000×). A series of TEM images was recorded every 0.5 s as a superposition of 25 consecutive images of 0.04-s frames (automatically processed on Gatan DigitalMicrograph software) over 5 to 15 min.
We first surveyed C 60 encapsulated in CNTs on the screen at 200,000× magnification to identify CNTs for reaction monitoring. Having found bundles of CNTs suitable for kinetic studies, we stopped the beam irradiation and changed the magnification to 800,000×. After waiting for 1 min, until thermal drift of the grid ceased or it was at least relatively relaxed, we commenced observation and movie recording. Focusing was carried out during the collection of images, which was recorded at slightly underfocus conditions (defocus value 10 to 20 nm). At 80, 100, and 120 kV, we continuously focused on 25 to 70 molecules in total, with a frame rate of 1.0 s for 5 to 15 min, until most of the C 60 molecules oligomerized to form an inner nanotube. At 60 kV, the recording time was set to be 15 to 20 min, following the results of kinetic studies at 80 kV.
Temperature Control. The temperatures were controlled by using a heating holder (JEOL EM-21130). The accuracy of the grid temperature was two to three kelvins (according to the instrument specifications). After the stage temperature was raised to the setting value, we waited at least 30 min before commencing observations, in order to stabilize the stage for minimization of thermal drift.
Image Processing. The images were collected as a .dm3 or .dm4 format file on Gatan DigitalMicrograph software and processed by using ImageJ 1.47t software for .dm3 files (55).
Visual Data Analysis for Counting Reaction Events of C 60 dimerization.
The products of C 60 dimerization were visually identified following a protocol described in a previous report (19), where molecular structures of [2 + 2] cycloadducts were studied thoroughly by using atomic-resolution TEM imaging combined with TEM simulations. The progress of the reactions was studied by analyzing the movies backward, from the end of the reaction, to identify C 60 dimerization. This procedure eliminates complications due to the intervention of equilibrium caused by thermal cycloreversion. The kinetics of cycloaddition between the fused dimer of C 60 molecules and C 60 was excluded from the analysis because the resultant product could possess very different properties.