Real-Space Investigation of Energy Transfer through Electron

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Real-Space Investigation of Energy Transfer Through Electron Tomography Yohei Ishida, Ikumi Akita, Thomas Pons, Tetsu Yonezawa, and Niko Hildebrandt J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b10628 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Real-space Investigation of Energy Transfer through Electron Tomography Yohei Ishidaa,*, Ikumi Akitaa, Thomas Ponsb, Tetsu Yonezawaa,*, Niko Hildebrandtc

a

Division of Materials Science and Engineering, Faculty of Engineering, Hokkaido University, Kita

13 Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan b

Laboratoire de Physique et d’Etude des Materiaux; ESPCI Paris, PSL Research University; CNRS;

Sorbonne Universites, UPMC; 10 rue Vauquelin, F-75231 Paris Cedex 5, France c

NanoBioPhotonics, Institute for Integrative Biology of Cell (I2BC), Universite Paris-Saclay,

Universite Paris-Sud, CNRS, CEA, 91400 Orsay, France

ABSTRACT We herein report the first real-space investigation of Förster resonance energy transfer (FRET) in two different types of quantum dot (QD) supramolecular assemblies by observing their three-dimensional (3D) configurations through high-resolution electron tomography. Owing to its critical role in photosynthesis, artificial light-harvesting antennas, and investigation of protein-protein interactions, the mechanism of FRET has been intensively studied by monitoring its excited state dynamics via various spectroscopic techniques. The utilized electron tomography technique allowed the direct localization of 3D coordinates of individual QDs in self-assembled nanostructures and theoretical estimation of the FRET efficiency of a single fluorophore, domain, or supramolecular assembly. Moreover, the experimental value of the FRET efficiency determined by fluorescence spectroscopy was in good agreement with the magnitude obtained via electron tomography. We believe that the described strategy can be used in single-molecule FRET studies

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and will help to create a new bridge between material science and molecular/supramolecular photochemistry.

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INTRODUCTION Photochemical processes such as electron or energy transfer are distance-dependent phenomena involving donor and acceptor fluorophores.1 Förster (or fluorescence) resonance energy transfer (FRET) is a non-radiative energy transfer process from a fluorescent donor to a lower energy acceptor via long-range dipole-dipole interactions.2,3 Since the reaction rate constant of FRET is inversely proportional to the sixth power of the donor–acceptor distance, it generally occurs at distances ranging from 1 to 10 nm.4-7 Hence, FRET can be used as a highly sensitive spectroscopic measurement technique for processes occurring on the nanometer and sub-nanometer scales, which include various qualitative and quantitative biochemical applications such as DNA sequencing, intracellular protein-protein interactions, molecular binding studies, and clinical diagnostics.8 Moreover, the light-harvesting antennas in natural photosynthetic bacteria utilize highly efficient energy transfer processes during photosynthesis,9 which strongly motivated multiple research groups to investigate synthetic molecular or supramolecular FRET systems involving artificial photosynthesis and light-harvesting phenomena.10-12 As a result, the mechanism of FRET has been extensively studied using a large variety of donor–acceptor pairs including organic dyes, metal complexes, fluorescent proteins, and fluorescent quantum dots (QDs). The high brightness and photostability of QDs compared to organic dyes or fluorescent proteins as well as their large absorption cross sections and rigid structure, make them attractive materials for FRET for highly sensitive biosensing and long-term imaging.13 The purpose of this study was the first real-space investigation of FRET between two different types of QDs14,15 assembled via supramolecular interactions, using three-dimensional (3D) reconstruction images obtained via electron tomography. The scanning transmission electron microscopy (STEM) technique utilizing a high-angle annular dark field (HAADF) detector for collecting incoherently scattered electrons and aberration-corrected probe-forming optics is a powerful tool for analyzing nanostructured materials at the atomic scale.16 Electron tomography17-19 has become a standard 3D characterization technique for a broad variety of nanomaterials. Therefore, the 3D reconstruction of aberration-corrected HAADF!STEM images from the tilt series 3



of projection images acquired during sample rotation can potentially elucidate the atomic structure of nanomaterials in three dimensions. Recent pioneering works have reported STEM tomography studies of various inorganic nanomaterials at the atomic resolution.20-25 A significant advantage of the atomic-resolution electron tomography is the direct localization of individual atoms or nano-species in non-crystalline materials such as supramolecular assemblies, which cannot be analyzed by X-ray crystallography.17 Hence, this technique can be utilized to obtain the 3D real coordinates of supramolecular assemblies of QD nanoparticles with different sizes that exhibit FRET between each other (Figure 1a). During the last five decades, the investigation of FRET mechanisms has been primarily conducted using various spectroscopic techniques that allow deeper understanding of the bulk processes. Due to the recent advances in scientific instrumentation, single–molecule FRET can now be observed via high-resolution fluorescence microscopy26-29 or scanning tunneling luminescence microscopy30. However, high-resolution electron tomography is able to directly and precisely localize the 3D coordinates of individual nanoparticles in a self-assembled nanostructure and thus estimate the reaction efficiency of a single fluorophore, domain, or supramolecular assembly. Therefore, this technique can be used in single-molecule FRET studies as a new important complementary tool. In this work, spectroscopic measurements were combined with electron tomography observations for the first time to determine the real-space 3D configurations of supramolecular structures and fully characterize FRET in QD assemblies.

RESULTS and DISCUSSION Donor and acceptor CdSe/CdS/ZnS (core/shell/shell) QDs with different core diameters (denoted as QDs(D) for donor QDs and QDs(A) for acceptor QDs) were synthesized using the procedures outlined in the Experimental Section. Since the studied QD(D) and QD(A) exhibit different emission maxima at 560 and 640 nm, respectively (Figure 1b), FRET from the higher to the lower energy QDs can be anticipated. The diameters of the synthesized QDs were around 7.1 ± 0.9 and 8.5 ± 1.0 nm, respectively, as shown in Figure 1a. The obtained QDs were functionalized via a ligand exchange reaction, which converted initial neutral ligands to cationic thiol ligands 4


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((11-mercaptoundecyl)-N,N,N-trimethylammonium bromide). These cationic QDs were anchored on anionically charged nanosheets of montmorillonite-type clay minerals31,32 via Coulombic interactions, resulting in the self-assembly of densely populated QDs on the 2D clay surfaces (Figure 1a). The use of 2D clay nanosheets as supramolecular host materials has a significant advantage since the produced self-assembled QDs–clay structures retain the original anisotropy of the clay support (Figure 1a). During high-resolution STEM tomography observations, the clay nanosheet surface is irradiated by the electron beam, resulting in a limited thickness of the produced sample (which defined the maximum number of QD layers assembled along the electron beam direction). In contrast, large 3D assemblies of small inorganic species such as secondary aggregates of nanoparticles are very hard to observe by electron tomography due to the very strong scattering of electrons at different tilt angles over the entire structure. Hence, 2D QDs–clay supramolecular structures with limited thicknesses were successfully characterized by STEM tomography in this study. Moreover, multiple Coulombic interactions were able to strongly anchor the produced QDs on the clay surfaces, thus ensuring the stability and motionlessness of the supramolecular assemblies, which were required for electron tomography observations (in contrast, typical organic supramolecular assemblies are not stable enough and usually change their structure during STEM tomography measurements without cryo technology). First, the formation of supramolecular QDs–clay complexes was studied by recording their absorption spectra via the method outlined in the Experimental Section. Because the absorption spectra of QDs with and without clay were identical due to the lack of absorption or scattering of transparent clay nanosheets under the specified conditions, a special filtration technique utilizing polytetrafluoroethylene (PTFE) membranes with pore diameters of 0.1 !m was used to confirm the anchoring of QDs on the clay surfaces (free QDs could pass through the membrane filter, while the QDs adsorbed on the clay surfaces were retained by it). The absorption spectra recorded for the QDs with the concentrations [QD(D)] = [QD(A)] = 1.0 " 10–7 M mixed with a 60 mg L–1 clay solution before and after filtration are shown in Figure 1c. The absence of the absorption peaks originated from the presence of QDs in the filtered supernatant indicated that all QDs were 5



successfully anchored on the clay surfaces at the specified ratios between the concentrations of QDs and clay nanosheets. To determine the maximum population density of QDs on clay nanosheets, absorption spectra of the aqueous solutions with various clay concentrations (ranging between 0 and 30 mg L-1) were recorded at constant QD concentrations before and after filtration. In Figure 1d, the absorbance of the supernatant is plotted against the concentration of clay nanosheets. It clearly shows that the absorption after filtration was almost negligible at a clay concentration of above 16 mg L–1, indicating that QDs were fully anchored on the clay surfaces in this concentration range (at lower concentrations, the supernatant solution contained some amounts of free QDs). Taking into account that a surface area of 16 mg L–1 clay nanosheets (with 760 m2 g–1) is occupied by 2 " 10–7 M of QDs (1 " 10-7 M for each type), the maximum estimated adsorption density of QDs on the clay surface was around 1 QD per 100 nm2. Assuming the hexagonal distribution of QDs, the calculated average center-to-center distance between adjacent particles was around 10.8 nm, which might be short enough to demonstrate FRET between different QD pairs. Hence, the HAADF!STEM and 3D tomography studies were performed for the QDs-clay complexes characterized by the maximum adsorption densities.

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The 3D real-space configurations of the produced QDs-clay supramolecular assemblies were investigated using the aberration-corrected HAADF!STEM technique. Figures 2a and b show the representative HAADF!STEM images of QDs-clay complexes obtained at different magnifications, which contain spherical QDs densely assembled on the clay nanosheets. The high-magnification image depicted in Figure 2b clearly reveals the overlaps of different QDs along the electron beam direction, suggesting the existence of QDs on both sides of the nanosheet surface. To confirm the real-space 3D configuration of QDs across the nanosheet surface, a 3D tomogram was reconstructed from the HAADF!STEM observations obtained during sample rotation from – 64º to +56º at a tilt increment of 2º (the detailed procedure is described in the Experimental Section). Figure 2c contains the volume-rendering images projected onto the yz plane. It shows the existence of two different layers of QD assemblies with a very small spatial gap, suggesting the presence of a clay nanosheet between them. It is important to note that this nanosheet is tilted at an angle of approximately 8° with respect to the electron beam direction (for a better demonstration of the observed effect, the alternative x’, y’ and z’ axes corresponding to the clay nanosheet orientation are displayed in Figure 2c as well). It should be also noted that the geometrical resolution of the reconstructed images is generally not isotropic. Because reconstruction is typically performed slice-by-slice along the tilt axis (x), the resolution along the x direction may be as good as the Nyquist frequency of the pixel in the input image, while the resolution along the z (z’) axis is degraded by an elongation factor due to the “missing wedge” in the tilt data.33,34 These inevitable phenomena typically cause the appearance of a spherical phantom along the z axis. Judging from Figure 2c the particle shapes observed are nearly spherical. Figures 2d!f show the digital slices of the reconstructed images obtained along the z’ axis at different heights (varied at increments of 5.5 nm). Both Figures 2d and f contain the 2D assemblies of QDs; however, their distributions were different. Moreover, no apparent contrast between the two QD layers was observed in Figure 2e, strongly suggesting the existence of a clay nanosheet at this particular height. Thus, it can be concluded that the obtained HAADF!STEM 8


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tomography data contain the successfully reconstructed 3D real configuration of the self-assembled QDs on the clay nanosheet (in the further discussion, the two layers displayed in Figures 2d and f are denoted as Surface I and Surface II, respectively). Owing to the high resolution of the utilized STEM tomography technique, the QD(D) and QD(A) can be distinguished using the difference in their particle diameters. The particle diameter (determined by the reconstruction images at different heights) histogram plotted for all QDs in Figure S1 indicates the existence of two different distributions corresponding to the smaller (donor) and larger (acceptor) QDs. The surface-rendering image of the QDs–clay complexes shown in Figure 2g contains four different colors, which are used to distinguish the QD(D) and QD(A) assemblies on Surfaces I or II (see the corresponding legends). The total number of the displayed QDs was 73. The obtained images also indicate that only two QD layers were assembled on Surfaces I and II, while the multi-layer deposition of QDs was inhibited. To the best of our knowledge, this is the first successful real-space investigation of 2D supramolecular assemblies of nanoparticles performed using the electron tomography technique. Since the clay nanosheet could not be observed due to its relatively low contrast in the resulting HAADF-STEM images, it was denoted by the light-blue square in Figure 2g. As indicated by the surface-rendering image, the QD(D) and QD(A) assemblies with different sizes were anchored on the clay surface without segregation, which represented a well-known phenomenon observed during domain formation on solid surfaces. Segregation structure is typically preferred by the strong guest-guest interaction such as hydrophobic or van der Waals interactions.35 Because segregation causes a decrease in the adjacent probabilities between two different species on 2D surfaces, the related rate constant or efficiency of inter-molecular processes (such as FRET) tends to be very low. Therefore, a uniform (random) assembly without segregation observed for the studied QDs-clay complexes is preferable for FRET demonstration. The center positions of each QD plotted along the plane perpendicular to the z’ axis are shown in Figure 2h. The center-to-center distances for all QD pairs were determined from the 3D tomography images (see Table S1 in the Supporting Information section), while the numbering of 9



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all QDs is shown in Figure S2, Supporting Information. Since FRET is a distance-dependent phenomenon, and its reaction rate constant (kFRET) is inversely proportional to the sixth power of the donor-acceptor distance, the efficiency of FRET in the studied assembly can be determined using Förster’s equation36: k FRET

9 ln10$ 2# D = J, 128" 5 n 4 N! D r 6

(Eq. 1)

where ! is the orientation factor, "D is the fluorescence quantum yield of QDs(D) determined via an absolute method using an integrating sphere photometer (0.22), n is the refractive index of the bulk medium (1.33 for water), N is Avogadro’s number, #D is the lifetime of the excited QDs(D) determined via time-resolved fluorescence spectroscopy (23 ns), and r is the center-to-center distance between the QD(D) and QD(A). In the current study QDs randomly anchored on clay nanosheets and there should be no preferential orientations during FRET. For a static 3D system containing donors and acceptors with isotropically oriented dipoles, the calculated magnitude of !2 was equal to 0.667.37,38 According to the analysis of the overlap between the QD(D) fluorescence and QD(A) absorption, the magnitude of the spectral overlap integral (J)39 (3.4 " 1016 M-1 cm-1"nm4 or 3.4 " 1033 nm6 mol–1) was calculated using the following formula: J = " FD (! )" A (! )! 4 !! ,

(Eq. 2)

where # is the wavelength in nm, $A (#) is the extinction coefficient of QDs(A) at wavelength # in M–1"cm–1, and FD(#) is the normalized fluorescence intensity of QDs(D) (its integral is equal to unity). The computed J value was very large as compared to typical magnitudes for molecular FRET systems, owing to the high extinction coefficient of QDs(A). The Förster distance (R0) of 7.2 nm, corresponding to the distance between fluorophores, at which the energy transfer rate was equal to the radiative decay rate, was calculated using the following equation: R0 = 0.0211(n !4! D" 2 J )1/6 nm.

(Eq. 3)

Using the obtained parameters, the FRET efficiency (%) of each donor can be determined as $=

" k FRET ( D ! Ai )

#D

!1

,

(Eq. 4)

+ " k FRET ( D ! Ai )

where #kFRET(D–Ai) denotes the sum of kFRET obtained from donor to neighboring acceptors. For 10


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example, QD(DI–4) in Figure 2h is surrounded by several QDs(A) located either on the same (Surface I) or on the opposite surface (Surface II). The center-to-center distances between QD(DI–4) and the neighboring QDs(A) determined by STEM tomography are summarized in Table 1. The magnitude of kFRET calculated for DI–4 and its nearest acceptor QD(AI–13) by using Eq. 1 was 24.7 " 106 s–1, while for other acceptors the value of kFRET decreases as the distance increases. Thus, the FRET efficiency of QD(DI–4) determined via Eq. 4 was around 49%. It should be noted that only the kFRET values obtained for the interactions between QD(DI–4) and the QD(AI–13), QD(AI–5), QD(AI–14), and AII–17 were significant since all other acceptors were positioned at distances exceeding 2 " R0 (14 nm). The relatively longer distances to the QDs(A) located on the opposite surface (Surface II) can be explained by the following two reasons: (i) the presence of the clay nanosheet with a thickness of 1 nm between Surfaces I and II, and (ii) possible entanglement of the cationic thiolate ligands on the same surface due to the existence of long hydrophobic carbon chains (about 2 nm), which is prohibited for the QDs located on the opposite surfaces because of the spatial separation. In a similar manner, all possible combinations between QDs(D) and the neighboring QDs(A) were examined, and the obtained results are summarized in Table S1, Supporting Information. The total FRET efficiency (%total) of the studied assembly (equal to around 27±2%; see the determination of error range described in the Supporting Information) was calculated by averaging the values of % obtained for all 36 donors. Note that the QDs(D) located on the outer side of the assembly exhibit smaller % values (3!22%) due to the lower number of neighboring QDs(A) as compared to the structures present on the inner side of the nanosheet surrounded by other QDs. To the best of our knowledge, this work demonstrates for the first time the efficiency of FRET via electron tomography. It should be noted that the contribution of the energy migration process (homo-FRET between donors) is neglected in the utilized assumption. The R0 for energy migration calculated via Eq. 3 after taking into account the spectral overlap J between donor’s absorption and donor’s fluorescence (2.6 " 1015 M-1 cm-1"nm4) was 4.5 nm. This value was much smaller than the diameter of the observed QDs, indicating that the contribution of homo-FRET was negligible. The same reasoning can also be applied to the backtransfer from QD(A) to QD(D) with an R0 of 2.3 nm 11




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and z’ axes are alternatively inserted along the nanosheet orientation. d!f, Digital slices obtained along the z’ axis at height increments of 5.5 nm. g, A surface-rendering image. The clay nanosheet (represented by the light-blue square) was sketched for guidance. The four colors represent the QD(D) and QD(A) assemblies on Surfaces I and II. h, Coordinates of the center positions plotted along the z’ axis. The highlighted QD(DI–4) with adjacent QDs(A) is discussed as a typical example for calculating FRET efficiency in the main text and Table 1.

Table 1. Distances from QD(DI–4) to the neighboring QDs(A) estimated using the STEM tomography images depicted in Figure 2 and the related energy transfer rate constants (kFRET) calculated using Eq. 1. Distance from DI–4 Acceptor

kFRET 6

–1

/ nm

/ 10 s

AI–13

7.9

24.7

AI– 5

9.4

8.8

AI–14

10.8

3.9

Surface

AI–15*

16.5

0.3

I

AI–18*

17.2

0.2

AI– 3 *

17.6

0.2

AI– 8 *

17.8

0.2

AI– 4 *

19.8

0.1

AII–17

12.7

1.5

Surface

AII–13*

14.0

0.8

II

AII–16*

15.0

0.5

AII–10*

19.7

0.1

*The distances to these QDs(A) exceed 2 ! R0 (14 nm).

13



In order to verify the obtained electron tomography results, the FRET process was also investigated by fluorescence spectroscopy. The control experiments confirmed that the homogeneous solutions of free QDs(D) and QDs(A) without clay nanosheets did not exhibit FRET due to the very long distances between individual QDs. Figure 3a shows the steady-state fluorescence spectra recorded for QDs(D), QDs(A), and their mixtures on the nanosheet surfaces at different clay concentrations, which corresponded to average QD-per-nanosheet densities of 1 QD per 100 nm2 (1.6 mg L–1 of nanosheets per 2 " 10-8 M of QDs), 1 QD per 200 nm2 (3.2 mg L–1 of nanosheets per 2 " 10-8 M of QDs), and 1 QD per 500 nm2 (6.4 mg L–1 of nanosheets per 2 " 10-8 M of QDs). The excitation wavelength was set to 405 nm, which excites both QDs. When the density of QDs was increased, the fluorescence intensity of the donor QDs decreased, while that of the acceptor QDs increased, suggesting the possible occurrence of FRET from the excited QDs(D) to the ground-state QDs(A). The fluorescence spectra obtained for the mixed samples can be expressed by the sum of individual QD(D) and QD(A) spectra. The isoemissive point was observed at a wavelength of 609 nm (denoted by the arrow in Figure 3a), indicating different efficiencies of FRET. The decrease in the donor fluorescence intensity was enhanced at higher adsorption densities and was equal to around 28% under the highest adsorption conditions (see the green line in Figure 3a), when the ratio between the QD and clay concentrations was identical to that utilized during electron tomography measurements (1 QD per 100 nm2). The relative increase in the acceptor fluorescence intensity was around 9%, which represented a good evidence for FRET sensitization, but could not be used for calculating the FRET efficiency. To verify the steady-state FRET investigation (which may be difficult to quantify due to the direct excitation of both the donor and acceptor QDs), time-resolved fluorescence measurements were conducted using the samples with a density of 1 QD per 100 nm2 nanosheet. Fluorescence of QD(D) and QD(A) was detected at 560 and 640 nm, respectively. The decay curves of individual QD(D) and QD(A) were recorded, and their average lifetimes were equal to 23.0 and 20.1 ns, respectively (see Figures 3b and S3 in the Supplementary Information section). The average lifetime of QD(D) in the QDs-clay mixture was 17.3 ns, which corresponded to a decrease by 14


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CONCLUSION In conclusion, this work represents the first real-space investigation of the excited energy transfer in QD supramolecular assemblies conducted through electron tomography. The utilized HAADF!STEM technique was able to successfully localize the 3D coordinates of individual QD nanoparticles of the self-assembled nanostructures on clay nanosheet surfaces. These coordinates were used to determine the energy transfer efficiency using the Förster equation; as a result, the reaction efficiency of one fluorophore, one domain, or one supramolecular assembly could be estimated. Moreover, the experimental energy transfer efficiency values determined via steady-state and time-resolved fluorescence spectroscopies (28% or 25%) were in good agreement with the results of the electron tomography (27±2%). Although only QD nanoparticles were used in this study, the described strategy can potentially be applied to 3D imaging of molecular systems to analyze the related energy transfer processes via cryo–electron tomography. We believe that the proposed method can be used as an important complementary tool in single-molecule FRET studies and helps to create a new bridge between material science and molecular/supramolecular photochemistry.

EXPERIMENTAL SECTION Materials and methods (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (Aldrich) and dichloromethane (TCI, HPLC grade) were used as received without purification. Deionized water (> 18.2 M$) was prepared using an Organo/ELGA Purelabo system. Montmorillonite (a typical anionic clay mineral) was acquired from Kunimine Industries (its chemical structure corresponded to the formula Na0.66(Al3.34Mg0.66)Si8O20(OH)4 with the cation exchange capacity equal to 0.90 meq. g–1) and was purified using the following procedure. First, raw clay dispersion (300 mL, 10 g L–1) was placed in centrifuge bottles, which were then centrifuged at a rotation speed of 7000 rpm for 15 min. The obtained supernatant was collected and poured into a container with 1500 mL of ethanol. After 1 h of mixing at a temperature of 70 °C, the resulting colloidal solution was filtered using a PTFE 16


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membrane with a pore size of 0.1 !m (Millipore). The produced cake was collected and dried under vacuum by rotary pump overnight. The dried cake was dispersed in water, and the suspension was stored overnight to achieve complete exfoliation. Absorption spectra were obtained using a Lambda 35 UV/Vis spectrometer (PerkinElmer). Photoluminescence spectra and decay curves were recorded using a FluoTime 300 fluorescence lifetime spectrometer (PicoQuant) equipped with a continuous-wave Xe lamp for spectra acquisition and a 405-nm diode laser (Edinburgh Instruments). Fluorescence quantum yield was measured by an absolute method using a JASCO FP!8600 spectrometer containing a JASCO ILF!533 integrating sphere unit.

Synthesis of donor and acceptor QDs CdSe/CdS/ZnS core/shell/shell QDs were synthesized using previously established protocols. Briefly, CdSe cores were prepared from cadmium myristate and selenium powder via the injection-free synthesis route described elsewhere.40 To obtain cores with relatively small sizes, their growth process was terminated at a temperature of 200 °C (alternatively, reinjection was performed for larger cores until the emission wavelength reached the magnitude of 610 nm). The prepared CdSe cores were purified by centrifugation in ethanol and resuspended in hexane (their concentration was estimated from the absorbance measured at a wavelength of 350 nm).41 Shell growth was performed following the SILAR protocol.42 To synthesize QD donors emitting at 580 nm, smaller CdSe cores were coated with 2 monolayers of CdS, 3 monolayers of Cd0.5Zn0.5S, and 2 monolayers of ZnS. To obtain QD acceptors emitting at 640 nm, larger CdSe cores were coated with 3 monolayers of CdS, 2 monolayers of Cd0.5Zn0.5S, and 2 monolayers of ZnS. The resulting core/shell QDs were finally centrifuged in ethanol, redispersed in hexane, and stored at around 20ºC in the dark.

Cationization of donor and acceptor QDs via ligand exchange reactions Cationization of donor and acceptor QDs was conducted through the ligand exchange reaction of 17



trioctylphosphine

oxide

(TOPO)

ligands

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with

cationic

(11-mercaptoundecyl)-N,N,N-trimethylammonium bromide. Briefly, the initial TOPO-stabilized QDs (4 nmol) were dissolved in 1 mL of dichloromethane followed by the addition of an aqueous solution (1 mL) containing 10 mg of cationic thiol ligand (the resulting mixture was stirred overnight). Subsequently, the prepared solution was centrifuged at a rotation speed of 5000 rpm for 5 min, and the resulting colored aqueous phase was collected, washed with pure dichloromethane several times, and used in further experiments (the QD concentration remained unchanged during the entire process, as indicated by the constant absorbance value measured before and after the ligand exchange reaction). The described procedure was used for the preparation of both donor and acceptor QDs.

Preparation of QDs–clay complexes Supramolecular complexes composed of anionic clay nanosheets and cationic QDs were prepared by mixing their corresponding aqueous solutions under stirring (see Figure 1a). The adsorption of QDs on the clay nanosheets was investigated via absorption spectroscopy and electron microscopy as described in the main text.

STEM and 3D tomography studies HAADF!STEM and 3D tomography studies were conducted at an acceleration voltage of 300 kV using a FEI TITAN III G2 Cubed instrument. STEM samples were prepared by dropping a solution containing well-dispersed QDs-clay complexes onto a carbon-coated Cu grid. The obtained tilt series consisted of 61 HAADF images acquired at tilt angles ranging from –64° to +56°, tilt increment of 2°, and magnification of 160 k. The image size was 2048 " 2048 pixels; thus, one pixel corresponded to 0.27 nm. Spatial alignment of the obtained HAADF images was performed using a cross-correlation method of the FEI Inspect3D software package. Tilt axis alignment was also undertaken by Inspect3D followed by using a simultaneous iterative reconstruction technique (SIRT) to perform the reconstruction with 25 iterations. Amira software (FEI) was utilized for 3D 18


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volume rendering and the analysis of the QD(D) and QD(A) assemblies.

DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon request.

SUPPORTING INFORMATION Supplementary electron microscopy images, summary of inter-particle distances, and time-resolved fluorescence decays.

CORRESPONDING AUTHORS E-mail addresses: [email protected], [email protected]

AUTHOR CONTRIBUTIONS YI conceived and designed the project. YI and IA performed all experiments and analysis. TP prepared the QD samples. TY and NH participated in the initiation and supervision of the project. YI wrote the manuscript using the input provided by the other authors.

NOTES The authors declare no competing financial interest.

ACKNOWLEDGMENT YI acknowledges the financial support from Building of Consortia for the Development of Human Resources in Science and Technology, Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant-in-Aid for Young Scientists from JSPS (14448322), The Foundation for The Promotion of Ion Engineering, Foundation Advanced Technology Institute, 19



Inamori Foundation, TonenGeneral Sekiyu K.K. Foundation, Izumi Science and Technology Foundation, Yashima Environment Technology Foundation, Hokkaido Gas Co., Ltd., and JGC-S Scholarship Foundation. This work also partially supported by Hokkaido University, microstructural characterization platform as a program of Nanotechnology Platform of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Authors thank to Mr. Shashi Bhuckory (Universite Paris-Saclay, Universite Paris-Sud) for his experimental support for time-resolved fluorescence measurements, Mr. X. Xu (PSL Research University; CNRS; Sorbonne Universites, UPMC) for TEM experiments, and Mr. R. Oota (Hokkaido University) for STEM tomography experiments.

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