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Dimensionality Effects on Fluorescence Resonance Energy Transfer between Single Semiconductor Nanocrystals and Multiple Dye Acceptors Ido Hadar, Shira Halivni, Na’ama Even-Dar, Adam Faust, and Uri Banin* Institute of Chemistry and the Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: Colloidal semiconductor nanocrystals are outstanding donors in energy transfer processes due to their unique size and shape dependent optical properties, their exceptional photostability, and chemical processability. We examine the dimensionality effect in energy transfer between single heterostructure nanocrystals of spherical and rod shape, serving as donors, and multiple dye molecules attached to their surface acting as acceptors. Förster resonant energy transfer (FRET) to individual dyes attached to the surface of a single nanocrystal is identified via step-like changes in both acceptor and donor emission, enabling to calculate the efficiency of energy transfer and distance of each acceptor individually. This offers a unique tool to study the surface chemistry of various nanocrystals. The dimensionality of the nanocrystals is reflected by the acceptors distribution, which enables to study the inner geometry of these heterostructures, such as the location of the seed and shell thickness. Additionally, the nanocrystals serve as an optical antenna that enhances the excitation and emission of the dye molecules through the FRET interaction. These measurements enable to gain deeper understanding of the energy transfer process between semiconductor nanocrystals of various geometries and dye molecules and promote its utilization for extremely sensitive sensing applications at the single molecule level.

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INTRODUCTION The basic principles of Förster (or fluorescence) resonance energy transfer (FRET) were formulated in the pioneering work of Förster1 and have been extensively studied in molecular systems. In FRET, the energy is transferred by dipole−dipole interactions from an excited donor to a ground-state acceptor which consequently emits a photon. The rate and efficiency of the energy transfer are dependent on the spectral overlap between the donor emission and acceptor absorption, the relative orientation between their dipoles, and the donor− acceptor distance.2 The typical distances for FRET are on the nanometer scale, and thus it is widely used as a “nature-made” nanometric ruler in the study of molecular and biomolecular systems.3−5 In particular, single molecule FRET (smFRET) has emerged as a tool to investigate fundamental processes such as conformational changes in a variety of biological systems.6−10 smFRET is also being studied for advanced applications such as DNA sequencing.11−13 Colloidal semiconductor nanocrystals (NCs) can be used as excellent donors for FRET, with significant advantages over organic fluorescent dyes.14−22 As a result, there is a growing interest to utilize NCs as donors for various FRET-based applications such as sensing,23−25 biolabeling,26−28 and solar energy harvesting.29,30 This is due to their unique properties including their quasi-continuous above gap absorption spectra providing flexibility in the choice of the excitation wavelength, the ability to tune their emission wavelength by controlling © XXXX American Chemical Society

their size or shape, and thus tailor the spectral overlap with a specific acceptor, and their outstanding photostability. In smFRET, where single FRET couples are imaged for a relatively long time, at high excitation flux, the much higher photostability of the NCs in comparison to organic dye molecules provides a significant advantage as it enables much longer and stable measurements.31−35 Additionally, the broad absorption band of NCs and their high extinction coefficient enables to excite the sample at an energy and power that minimizes direct excitation of the acceptor thus reducing the non-FRET background emission signals in the measurements. Furthermore, the high number of binding sites on the surface of a NC allows conjugation of multiple dye molecules directly to the surface of the NC, enabling to study FRET between a single donor to multiple acceptors.36−40 In this scheme, the NC acts both as a donor and as the scaffold that determines the spatial distribution of the acceptors. The high sensitivity of FRET to the nanometric scale can be utilized to study the effects of the NC dimensions and dimensionalities on the FRET characteristics such as energy transfer rate and efficiency, as recently reported by us, in a study on the dimensionality effects in FRET in the ensemble level.36,37 Here we examine the energy transfer between single heterostructure NCs with various dimensions and dimensionReceived: December 19, 2014 Revised: January 29, 2015

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DOI: 10.1021/jp512678j J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

Ar flow was applied, and additionally the sample was excited only during the measurements.

ality acting as donors to multiple dye acceptors chemically bound to their surface. In particular, we compare between spherical CdSe/CdS core/shell NCs41 (QDs) and elongated CdSe/CdS seeded rods (NRs),42,43 in which a spherical CdSe seed is embedded within CdS rod-shaped shells of various lengths. In both structures, upon excitation, the exciton is localized to the spherical seed,44 meaning that the relevant donor−acceptor distances are between the NC core and the dye molecules attached to the surface of the particle.45−47 Furthermore, as the emission wavelength is influenced mainly by the core characteristics, a careful synthesis can yield NCs with various dimensions but with similar emission wavelength and thus similar Förster distance, allowing to compare directly the effects of the geometrical differences on the FRET. By tracing the emission of single FRET couples (single donor and one or more acceptors), we were able to detect multiple acceptor photobleaching events on a single NC donor, indicating the energy transfer to multiple acceptors. These measurements enable the study of this complex scheme of energy transfer as well as the calculations of the FRET efficiency and distance to each acceptor individually, which provide a unique tool to study the inner structure of the heterostructure NC in addition to its dimensionality. Furthermore, these measurements also reveal that the NCs can serve as an optical antenna that enhances the excitation and emission of the dye molecules through the FRET interaction in comparison to direct excitation. Apart from reflecting the geometrical features of the NCs, similar measurements provide future means to gain deeper understanding of the surface chemistry of NCs and might help design complex heterostructures and self-assembled systems. Additionally, this study may open the path to utilize these systems for applications, in particular sensing based on smFRET.

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RESULTS AND DISCUSSION System Characterization. The NCs physical and spectroscopic properties are summarized in Table 1, Figure 1a,b, and Figure S1. Atto 590 dye with amine linker which enables direct binding to the surface of the NC was used as the acceptor. The NC samples synthesized for this study have comparable Förster distances of 5.4−5.7 nm, calculated from the overlap integral between the NC emission and Atto 590 absorption, which serve as the donor and acceptor, respectively (Figure 1c and Supporting Information). Effective conjugation between the NCs and the dye molecules was done in solution, verified by ensemble FRET photoluminescence measurement (Figure S2), and dispersed on a glass coverslip at low density, suitable for far-field smFRET study. Table 1. Summary of the Properties of the Different NCs sample

core diam (nm)

NC dimensions (nm)

ensemble QY (%)

calcd R0 (nm)

QDs short NRs longer NRs

2.1 2.2 2.2

4.1 4.2 × 9 4.2 × 18

50 43 60

5.4 5.7 5.6

A setup based on an inverted microscope, described in Figure 1d and the Experimental Methods section, was used for smFRET. This setup enables both imaging of single molecules at high rate in imaging mode or spectral measurement of a single particle through a grating in spectroscopy mode. To study the changes in the FRET efficiency, the camera was operated in continuous imaging mode at the highest rate that still enabled good signal-to-noise ratio. Single Particle FRET Study. Initially, measurements in spectroscopy mode were performed, in which the full emission spectrum is recorded from each single particle, allowing us to spectrally resolve the emission from the donor and from the acceptors simultaneously. The results of a typical spectrally resolved measurement are presented in Figure 1e, showing subsequent spectra recorded on a single FRET couple. The acceptor emission (above 590 nm) decreases, while the donor emission (below 590 nm) increases with time. The emission intensities of the donor and of the acceptor have been extracted by fitting a bi-Gaussian function, one for the donor (green) and one for the acceptor (red), allowing to generate a time trajectory of the emission for each independently. These trajectories are fitted to a rising step and to a falling step function for the donor and acceptor channel, respectively. This analysis is presented in the upper panel of Figure 1f. At early times there is a moderate FRET indicated by the stronger emission from the donor channel than in the acceptor channel. After 35 s there is a sharp, antiparallel step in both channels, indicating a photobleaching event of a single dye acceptor accompanied by increase in the donor NC emission. Additionally, the emission in the acceptor channel is dropped to zero, meaning that this structure was composed from a single NR conjugated to a single dye acceptor.31 The increase in the donor emission intensity is a clear indication that upon photobleaching the donor recovers to its unperturbed emission intensity. In the lower panel of Figure 1f we present the results of such analysis performed on the particle shown in Figure 1e displaying in this measurement two sharp, anticorrelate steps.

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EXPERIMENTAL METHODS Sample Preparation. The QDs and NRs samples used as donors have been synthesized following known methods, described in the Supporting Information. Atto 590 dye with amine linker which serves as the acceptor has been bought from ATTO-TEC GmbH. In order to achieve effective conjugation between the donor and acceptor, excess ligands were removed from the NCs samples by successive precipitation and dissolution. The clean NCs were dissolved in chloroform and mixed with the dye solution (in chloroform) at molar ratio of approximately 6 dye molecules per NC. Upon conjugation, for smFRET measurements, the samples were diluted and immediately spin-coated on a clean glass coverslip, resulting in homogeneous distribution of single NCs on the surface with density well below 1 particle per μm2. smFRET Experimental Setup. For the single particle optical measurements, a setup based on an inverted optical microscope (Nikon Eclipse-Ti) was used. The sample was excited with a white LED (Prizmatix) spectrally filtered at a wavelength of 470 ± 20 nm in order to minimize direct excitation of the dye. Emission was collected through a spectrograph (Acton SP2150) enabling to switch between grating (for spectroscopy) or mirror (for imaging) configuration and imaged by an EM-CCD (Andor iXon3) enabling both high sensitivity and fast imaging. In order to separate between the donor and acceptor channels in imaging mode, we added suitable filters before the spectrograph (550 ± 20 nm band-pass for donor channel, 590 nm long pass for acceptor channel). To minimize photobleaching of the dye acceptor, an B


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Figure 1. Transmission electron microscope (TEM) images of CdSe/CdS seeded NRs with average size of 4.2 × 9 nm (a) and of CdSe/CdS seeded nanorods with average size of 4.2 × 18 nm (b). (c) Absorption and emission spectra of typical NC donor (blue curves, NRs shown in (b)) and Atto 590 dye (black curves); the arrow indicates the excitation wavelength in which the dye absorption is minimal. (d) Schematic illustration of the experimental setup. (e) Consecutive spectra of a single FRET couple between 10 s (bottom) to 60 s (upper), indicating the stepwise decrease in the FRET efficiency upon photobleaching of acceptors. Black dots are experimental; green and red lines are fits for the donor and acceptor emission, respectively. (f) Time trajectory for single FRET couples extracted from spectral time traces. Upper panel show FRET between single NR and single acceptor. Lower panel (corresponding to the same particle of (e)) showing FRET between single NR and two acceptors.

Initially a strong FRET can be seen; after the first step there is still signal from the acceptor channel, indicating moderate FRET efficiency; after the second step the emission in the acceptor channel decreases to the background level. The presence of the sharp photobleaching events clearly indicates that this structure was composed of two dye acceptors conjugated to a single NR. The spectral measurements of both donor and acceptor emissions indicate the FRET process and unequivocally present the ability to study energy transfer to few single acceptors attached to the surface of the NC simultaneously. In order to quantify the data obtained from such measurements, the FRET efficiency (E) is calculated at each step by the ratio between the emission intensity of the donor with (ID−A) and without (ID), the acceptor: E=1−

ID − A ID

emission, which simplifies the measurement and enables much faster acquisition rates, suitable for tracing acceptor kinetics. FRET Study by Imaging the Donor Channel. Measurements of the donor channel in imaging mode were carried out, with a band-pass filter (550 ± 20 nm), to select only the donor emission. A typical measurement can be seen in Figure 2a. At the beginning of the irradiation (left panel), only five particles are evident in the donor channel due to the strong energy transfer. 150 s later, approximately 20 particles are observed in the same region due to photobleaching of the acceptors. Each particle is now imaged over only few pixels in comparison to few hundreds in the spectral mode, and since the undispersed signal is much stronger, the acquisition time for each frame was reduced down to 20 ms, a suitable time scale to observe dynamic features in the emission and to distinguish between close-by occurring photobleaching events. Home-written code was used to automatically detect single particles and to extract the intensity of each particle at each frame, resulting in a detailed time trajectory of the donor intensity. These were fitted to a multistep function according to the number of steps observed. Figure 2b presents such analysis for a NR with a single acceptor. The fit was used to determine the emission intensity of the donor during FRET (ID−A), and upon photobleach of the acceptor (ID), before and after the step, respectively. From these values the rate of energy transfer to the acceptor (kT) can be calculated according to

(1)

ID is taken as the emission intensity upon photobleaching of all the acceptors, and ID−A is the intensity at each step. From these, the donor−acceptor distance (r) is calculated to each acceptor separately using Förster distance (R0) and E: ⎛1 ⎞1/6 r = R 0⎜ − 1⎟ ⎝E ⎠

(2)

As can be seen from the analysis presented above, the meaningful data regarding the FRET efficiency, and thus the acceptor distribution, lies chiefly at the donor channel. Moreover, the much higher photostability of the NCs in comparison to the dye molecules ensures that the acceptors will bleach well before the donor, meaning that the intensity at the donor channel at the end of the measurement is indeed ID (the unperturbed donor intensity). This unique property of NCs allows studying this process solely by following the donor

⎛ I ⎞ k T = kD⎜ D − 1⎟ ⎝ ID − A ⎠

(3)

where kD is the natural decay rate of the donor (upon photobleach of the acceptor). The calculation of the rate will enable to determine the energy transfer to each acceptor C


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the distance and the number of photons emitted for each acceptor dye separately (a full derivation of such an analysis is presented in the Supporting Information). Dimensionality Effect on FRET. A few hundred particles from each sample of NCs were measured and analyzed as described above. In Figure 3 we present three typical time trajectories obtained for donors with various dimensions. At the acceptor dye concentrations used in this study, for most of the NCs we detected one or two acceptors, but for some we could detect up to four single acceptors. The time trajectory presented in Figure 3a is a typical example for dye acceptors attached to spherical QD when three individual acceptors have been detected. The analysis shows that all have fairly similar distances from the seed (3.3, 4.2, and 4.5 nm), as expected for a donor with close to spherical symmetry. Note that although the energy transfer to each acceptor is almost identical, the height

Figure 2. Measurement and analysis for imaging of the donor channel. (a) Optical image at the beginning of the irradiation (left) and at the end of the measurement (right). Initially only few particles can be seen due to the strong FRET. 150 s later, upon photobleaching of the acceptor dyes, much more NRs can be seen in the same region. (b, c) Time trajectory for a single FRET couple with fit to step functions. (b) FRET between single NR and single acceptor at a distance of 3.9 nm. (c) FRET between single NR and two acceptors at distances of 3.8 and 7.8 nm. The color shaded boxes in (b, c) represent the photons emitted from the donor (green) and from the acceptors (red, pink).

individually in the cases where multiple acceptors are attached to the surface of the nanocrystal. The FRET efficiency is calculated as well using the relation E=

kT k T + kD

(4)

Now, knowing the FRET efficiency and R0, the donor− acceptor distance (r) is calculated using eq 2. The number of photons emitted by the acceptor until photobleach is calculated as well from the fit by multiplying the number of “missing” photons from the donor emission and the time of the step (red square in Figure 2b). An example for a time trajectory with two steps, corresponding to a single NR with two acceptors, is presented in Figure 2c. The analysis is similar, but now the intensity of the first step reflects energy transfer to two acceptors, while the second step represents energy transfer to one acceptor and the last step is the donor emission alone. The energy transfer rate to each acceptor is calculated individually, enabling to calculate

Figure 3. Typical time trajectories of NC donor emission manifesting FRET to multiple acceptors. (a) FRET between a single QD to three acceptors located at similar distances of 3.3, 4.2, and 4.5 nm. (b) Time trajectory for a short NR and two remote acceptors, located at distances of 6.9 and 7.7 nm. (c) FRET between a longer NR and three acceptors located at distances of 4.8, 5.7, and 6.9 nm. D


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Figure 4. Donor−acceptor distance distributions for the various NCs studied (left panels) and the corresponding simulation for the expected distributions, considering only the geometry of the donor (right panels). (a, d) Spherical QDs, (b, e) short NRs, and (c, f) longer NRs. The insets in (d−f) portray the different facets of the NCs: body (blue), near edge (green), and far edge (red).

these histograms also reflect the inner structure of the NCs, enabling to study the concealed geometry of the heterostructure. For the QDs, the distribution peaks around the short distances, reflecting the average shell thickness. The tail is correlated with a fairly broad shell thickness distribution in this sample and also might indicate a distribution in the seed location within the shell. The extended acceptors distribution for the short NRs also displays high probability at fairly short distances, but the tail to larger acceptor distances is clearly extended. For the longer NRs, the distribution is even wider, extending and shifting to longer distances. Considering the utility of this approach to learn about the inner structure of the NCs, it is important to evaluate the limitation in the distance measurements for both short and long distances. For acceptor at distance of 0.5R0, the FRET efficiency is exceeding 0.98, meaning that ID−A is in the level of the noise. This sets the lower donor−acceptor detectable distance limit to ∼3 nm. For acceptors located at long distances, steps with intensity differences of approximately 10% can be detected, correlated to distance of approximately 1.4R0, thus setting the upper limit for donor−acceptor distances to approximately 8 nm. In order to correlate between the acceptor distance distribution and the physical dimensions of the NCs, we

of the steps is not equal. This is due to the competition between the acceptors, resulting in overall lower efficiency than simple addition of the energy transfer for each acceptor individually. Figure 3b presents an example for a short NR with two small steps (or low FRET efficiency), indicating that both acceptors are attached at relatively long distances (6.9 and 7.7 nm). In Figure 3c we present a trajectory of a longer rod, with three steps, reflecting acceptors at various distances from the seed of approximately 4.8 nm for the first, 5.7 nm for the second, and nearly 7 nm for the third acceptor. We note that the order of photobleaching of the acceptor dyes followed the distance and the rate of energy transfer to each. This is typical in many particles, although due to the random nature of photobleaching, sometimes the order is flipped. In all of these trajectories, a temporarily switching to OFF state of the donor NC (blinking) can be seen, clearly indicating that we are indeed measuring single particles. Statistical Analysis of FRET. The analysis of acceptor distances allows us to uniquely compare the acceptors distribution on the surface of the different NC donors. Figure 4 shows histograms of the donor−acceptor distances for each of the samples with clear differences between them, reflecting the dimensionality of each sample. Moreover, as the relevant distances are between the seed and the acceptor on the surface, E


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The Journal of Physical Chemistry C apply a simplified model that assumes homogeneous shell of dye molecules that cover the NC surface with certain thickness (see ref 37) and then calculate the distribution of distances between the seed and the possible sites for the dye in this layer (Figure 4d−f and Supporting Information). For all the structures there is a peak in the distribution which is correlated to the smallest distance between the seed and the middle of the dye layer. For the QD, this model predicts a symmetric distribution with width due to the thickness of the dye layer. For the NRs, due to the asymmetry in the seed location,42−44,48 we should consider three regions: the body, the near edge, and the far edge. Still, a peak representing the smallest distance to the body is expected. Additionally, at the edges, the distance from the seed is similar at all sites, and hence it should also induce peaks at the typical distances of the edges. For the short NRs, the near edge is partially overlapping the peak related to the distance of the seed to the side wall, and the far edge is expected to appear at approximately 7 nm. In comparison to the experimental histogram, indeed it appears that the peak centered at 4 nm is wider than expected from the side wall sites alone, yet there is no clear evidence for an additional peak around 7 nm. For the longer NRs, the near edge is expected to appear at 7 nm and the far edge is at distances that exceed the experimental detection limit. The distribution is shifted to longer distances, and a peak appears at 5.5 nm, which is in between the peaks predicted by our simulation and in addition wider than expected. The origin for this discrepancy might be related to distribution in the distances of the near edge due to distribution in the rods length and in the position of the seed. Alternatively, it might be related to errors in the calculated distances. In order to convert the measured FRET efficiency to distance, we assumed certain constant value for R0. However, R0 for a specific FRET couple is dependent also on few factors that might differ from one couple to the other. One major factor is the relative orientation between the donor and acceptor (κ2), which is usually taken as an average over all possible orientations. It is reasonable to assume that for a molecule conjugated to the surface, the mobility is restricted and thus it is expected to show values that differ from the average angular value. This might lead to larger or smaller values of R0 for acceptors at specific orientations and result in larger or smaller distances than those calculated accordingly (such a model is presented in the Supporting Information). Nonetheless, our results clearly indicate a utility for smFRET as a mean to investigate the inner structure of core/shell NCs in a unique manner. Comparing FRET and Direct Excitation of the Dye. In addition to the measurements of the donor channel, we also conducted measurements of the same systems in the acceptor channel by replacing the band-pass filter with a long pass filter. A typical time trajectory of single NR with three acceptors can be seen in Figure 5a. In this trajectory, the initial intensity is high, and upon each photobleach event the intensity is abruptly decreased. From these measurements the number of photons that were emitted until photobleach is directly extracted, which yield similar results to those obtained from the donor channel, further indicating that the measurements in the donor channel can indeed be used as a measure for the number of photons emitted by the acceptor dye. Comparing the measurements of acceptor dye excited by FRET with measurements of single dye molecules at direct excitation (as presented in Figure 5b) reveals an additional

Figure 5. Analysis of the emission from the dye acceptors. (a) FRET between short NR and three dye acceptors, recorded in the acceptor channel. Each photobleaching event is indicated as a sharp decrease in the acceptors emission. (b) Emission from a single dye molecule attached to the glass surface and excited at the wavelength of its maximum absorption (594 nm). After the photobleach, the emission drops to the background noise level. Notice that due to the significantly lower direct excitation efficiency of the dye, the time scale and emission intensity are an order of magnitude longer and lower, respectively, in comparison to panel a (excitation through FRET). (c) Distribution of the number of photons emitted until photobleach for a dye molecule excited directly (black) and for a dye molecule excited by FRET from NRs (blue) or QDs (red).

unique advantage of the FRET process. Because of the much higher extinction coefficient of NCs in comparison to dye molecules, the excitation of the dye molecules by FRET is much higher, which is reflected in much higher emission intensity. This is clearly seen when comparing the trajectories in Figure 5a (excitation by FRET) and Figure 5b (excitation at the absorption peak of the dye). The excitation flux is the same in both measurements, yet the emission intensity is an order of magnitude higher and the time to photobleach is an order of magnitude shorter for excitation by FRET in comparison to direct excitation (see Supporting Information for additional details). This behavior suggest that the NCs can be used as F



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antennas for effective excitation of single dye molecules which can be beneficial for applications based on single molecule fluorescence such as super-resolution microscopy and single molecule sensing. We also compare in Figure 5c the statistics for photons emitted until photobleaching for dye molecules excited directly, with dye molecules excited by FRET on NRs and on QDs. It can be seen that the dyes excited through FRET emitted less photons prior to photobleaching, in comparison to dye molecules that have been excited directly, in particular for QDs. This may indicate that the dye molecules attached to the NC are less stable due to the environment of the organic ligands that surrounds them or due to possible charge transfer from the NC to the dye molecule which result in decomposition of the dye molecule, as reported previously.35 Additionally, this phenomenon might be related to the fact that for these dye molecules the excitation was much stronger. It has been shown previously that for Atto590 dye the photobleaching process is sequential and involving additional intermediate state prior to the permanent bleach of the dye; this is in contrast to most dyes, in which the photobleaching is initiated directly from the singlet excited state.49 In such case the rate of photobleach is dependent on the excitation flux, and thus as the excitation became stronger, the dye molecules tend to photobleach faster.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis protocol, absorption and emission spectra, and TEM images for all the samples studied; ensemble FRET measurement; measurement of the QY for the NCs; calculation of R0 for all systems studied; full derivation of the analysis for multiple acceptors and detailed explanation for the simulations done to model the donor−acceptor distance distribution; calculation of the excitation intensity of the dye through FRET and by direct excitation. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (U.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007−2013)/ERC Grant Agreement No. 246841 and the Israel Science Foundation (ISF) Grant No. 811/13. U.B. thanks the Alfred & Erica Larisch memorial chair.

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CONCLUSIONS We have performed a study of energy transfer between single NC and multiple acceptors, distinguishing between the individual dye molecules attached to the surface of a NC. The dimensionality of the donor NC was well reflected by the extended donor−acceptor distance distribution, meaning that the FRET efficiency distribution is strongly influenced by the dimensions and dimensionality of the donor NC. These results open the path to design FRET systems with specific, desired FRET properties by controlling the geometry of the NC. For example, in order to develop specific smFRET based sensing application for very low analyte concentrations, one can use small NCs, showing significant change in the emission upon conjugation of single acceptor molecule. On the other hand, larger NRs, enabling conjugation and detection of multiple acceptors may be used for quantitative sensing of relatively high concentrations and detecting changes in concentrations. The observation that NCs as donors can be used as antennas for much more efficient excitation of the dye molecules through FRET compared with direct excitation, which also causes faster photobleaching, suggests that this scheme might be used for applications requiring short and strong bursts of emission, such as fast sensing of multiple signals and super-resolution microscopy. Additionally, the ability monitor the attached dye molecule for long time (through direct excitation) or to burn it quickly (by excitation of the NC) may be beneficial for applications that requires reusing of the same probe. Furthermore, the distribution of FRET efficiencies can be used to study the concealed inner geometry of heterostructure NCs, which cannot be easily resolved by direct electron microscopy such as the distribution of seed location within the NC or the shell thickness. Moreover, this type of measurement will enable to measure even more complex FRET systems in which the competition between the acceptors is not determined solely by the distance but also by other factors such as the surface of the NC, the binding chemistry of the dye molecules, and the relative orientation.

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