Article pubs.acs.org/JPCA
Probing the Absorption and Emission Transition Dipole Moment of DNA Stabilized Silver Nanoclusters Emma N. Hooley, Miguel R. Carro-Temboury, and Tom Vosch* Nanoscience Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark S Supporting Information *
ABSTRACT: Using single molecule polarization measurements, we investigate the excitation and emission polarization characteristics of DNA stabilized silver nanoclusters (C24-AgNCs). Although small changes in the polarization generally accompany changes to the emission spectrum, the emission and excitation transition dipoles tend to be steady over time and aligned in a similar direction, when immobilized in PVA. The emission transition dipole patterns, observed for C24AgNCs in defocused wide field imaging, match that of a single emitter. The small changes to the polarization and spectral shifting that were observed could be due to changes to the conformation of the AgNC or the DNA scaffold. Although less likely, an alternative explanation could be that several well aligned spectrally similar emitters are present within the DNA scaffold which, due to Förster resonance energy transfer (FRET) processes such as energy hopping, energy transfer, and singlet−singlet annihilation, behave as a single emitter. The reported results can provide more insight in the structural and photophysical properties of DNA-stabilized AgNCs.
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INTRODUCTION In recent years, the spotlight has been cast on DNA-scaffolded silver nanoclusters (DNA-AgNCs) as new and promising fluorophores due to their unique properties, including their small size, biocompatibility, tunability, and high fluorescence output.1−5 As a result of this, there has been increasing interest in adapting these fluorophores for further practical application.6−11 However, the structural and electronic properties of DNA-AgNCs have proven to be difficult to pin down, as they can adopt a wide variety of shapes, sizes, charges, and conformations.12−16 Synthesis and purification methods have significantly improved15−21 the characterization and composition determination. The question of the shape of these clusters does not yet have a definitive answer, although recent literature22−24 suggests that the clusters could adopt an elongated, thread-like shape that clings closely to the DNA scaffold. The symmetry of a chromophore can be probed, to an extent, using polarization methods.24 Linear chromophores show a strong polarization dependence as the transition dipole tends to lie along a specific axis of the emitter, commonly the long axis. Polarization anisotropy measurements have been used to determine the linear but bent nature of chromophores in conjugated polymers.25 One method for investigating the emission dipoles of chromophores is defocused widefield microscopy.26−30 Using this technique, the emission transition dipole of an individual chromophore is imaged, allowing the orientation of the emission transition dipole to be determined and dynamics to be studied. Defocused widefield has been successfully used to study fluorescent dyes,26,28,31−36 conjugated polymer emission dynamics,37 and the emission polarization dependence of metal nanorods.38,39 In this paper, we employ defocused widefield microscopy to investigate the effect of changing polarization on the absorption © XXXX American Chemical Society
and emission of a subset of NIR emitting silver nanoclusters in an all cytosine single stranded DNA scaffold. By rotating the polarization of the excitation light, we can investigate the effect on both the absorption efficiency and the emission transition dipole. Additionally, using confocal microscopy techniques, we investigate whether spectral shifting in these spectrally selected C24-AgNCs can be linked to small changes to the emission polarization.
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MATERIALS AND METHODS Sample Preparation. The C24-AgNCs were prepared following the method described elsewhere,40 using single stranded DNA dC24 (RP-Cartridge-Gold purification, Eurogentec/Standard desalting, IDT technologies), sodium borohydride (99.99%, Sigma-Aldrich), and silver nitrate (99.9999%, Sigma-Aldrich). The DNA was diluted in citrate buffer (pH 6.2), mixed with silver nitrate (Milli-Q water), and reduced with sodium borohydride (Milli-Q), in a molar ratio of 1:12:12. The sample was further diluted in Milli-Q to achieve an absorbance below 0.1 along the excitation range above 500 nm for bulk spectroscopic measurements. Steady state analysis of the absorption and emission of the C24-AgNCs can be found in Figure SI1. Samples were prepared for confocal and widefield measurements by dilution in a saturated poly(vinyl alcohol) (PVA, Sigma-Aldrich) solution and spin-cast onto an oven cleaned glass slide (Menzel, 22 mm × 22 mm, no. 1.5). The emitter of interest was spectrally selected by the excitation sources and has been characterized in a previous study.41 Briefly recapping our previous findings, we showed that with excitation at 633 nm, two emissive species were observed at the single Received: November 18, 2016 Revised: January 9, 2017
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Figure 1. Defocused widefield patterns of a two single C24-AgNCs (a, c) as the excitation polarization is rotated. The top row of each indicates the collected data, while the fitted theoretical patterns are below. The summed patterns over the entire measurements are shown alongside (b, d). The static, bilobed pattern characteristic of single emitter is clear.
pulsed light selected from the continuum laser (SuperK EXTREME EXB-6 with a SuperK SELECT wavelength selector from NKT Photonics, 10 MHz and 45 nW on the sample for all experiments). The laser beam is passed through an excitation filter (Semrock LL01-633-25) and a Berek Compensator in λ/4 retardation mode (Newport, 5540M) in order to achieve circular polarization and focused through an Olympus IX71 microscope by the oil immersion objective (Olympus UPLFLN 100×, 1.3 NA). Scatter from the excitation source was removed using a dichroic mirror (Semrock LPD01-633RS) and two long-pass filters (Semrock LP02-633RU-25). The fluorescence signal was split using a 50−50 beamsplitter (Thorlabs), with half collected by two avalanche photodiodes (APDs, PerkinElmer CD3226) arranged perpendicularly to each other and separated by a polarizing beam splitter (Thorlabs) and the other half by a spectrograph and cooled CCD camera (Princeton Instruments SPEC-10:100B/LN_eXcelon CCD camera, SP 2356 spectrometer). The signals from the APDs were processed by a SPC-830 card (Becker & Hickl) with one APD delayed by a DG535 delay generator (Stanford Research Systems).
molecule level, one dim emissive species with an emission maximum centered around 660 nm and a bright emitter with an emission maximum centered around 690 nm. In the defocused wide field measurements reported here, we most likely only are able to detect the bright emitter. So despite the large number of emissive species present in the sample (see Figure SI1), we are mainly monitoring one emissive species in the defocused widefield experiments, when exciting at 638 nm. The previous results also showed that all clusters behaved as single photon emitters at the excitation conditions used and that there was no correlation between emission maximum and fluorescence decay time. Of the measured molecules, 27% showed significant spectral changes over time. Steady State Absorption and Emission Spectroscopy. Steady state fluorescence measurements were made on a QuantaMasterTM 400 (PTI) using a slit width of 5 nm, and absorbance spectra were recorded on a Lambda 1050 UV/vis/ NIR spectrometer (PerkinElmer) using a slit width of 1 nm. Defocused Widefield Microscopy. The widefield microscope is built around an inverted microscope frame (IX83, Olympus) with a CMOS camera (Orca Flash 4.0 V2, Hamamatsu) at the detection port. Excitation is provided by a 638 nm continuous wave diode laser (LaserBoxx, Oxxius, linearly polarized 1000:1), run at 6.1 mW for all experiments. The laser beam is passed through a beam expander to achieve a beam diameter of approximately 1 cm and is then passed through a λ/2 plate (zero order, 633 nm, Thorlabs WPH05M633) in a motorized holder (Thorlabs). No additional cleaning up after the λ/2 plate with a polarizer was performed. The beam is focused on the back of the objective lens (UPlanSApo 100x/1.4 NA oil, Olympus) using a focusing lens (f = 50 mm, Thorlabs) to achieve Koehler illumination. This results in an excitation area of approximately 0.1 mm2, with an approximate energy density of between 0.5 and 1 kW cm−2.37 The emitted light is collected by the objective; scattered excitation light is eliminated by a dichroic mirror (Semrock, Di02-R635) and two long pass filters (Semrock, BLP01-635R). Images were captured using a 2 s exposure time. Defocused patterns are created using the methods described elsewhere.33 The objective lens is moved approximately 1 μm from the focal plane, resulting in the observed patterns.26,32 The patterns were matched to theoretical patterns32,42 using a least-squares algorithm. Patterns were selected for analysis using the following criteria: the clusters were isolated from the other clusters such that the defocused patterns do not overlap, and there was a suitable signal-to-noise ratio to the background. All fitting was performed using a routine written in MATLAB. Confocal Microscopy. Confocal microscopy measurements were made on a home-built confocal microscope described previously.43 The excitation source was 633 nm
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RESULTS AND DISCUSSION Defocused Widefield Imaging. By defocusing a typical widefield image, the emission direction can be spatially resolved.32 Emission from a single emitting chromophore shows a characteristic bilobed shape that is dependent on the 3D orientation of the emission transition dipole. By comparing these patterns to those derived theoretically,32,42 any significant change to the emission transition dipole, due to, for example, a change in emitting chromophore, energy transfer (homoFRET), or rotation within the host matrix, can be viewed as a change to the defocused pattern. The bilobed pattern described above is only clear and can be fitted to the theoretically derived pattern if the emitter in question has a single transition dipole moment. Figure 1 shows two examples of the C24-AgNC defocused patterns, with the fitted theoretical patterns below. Both examples clearly show the characteristic bilobed pattern of single emitting species. All the clusters we measured showed the same clear patterns, suggesting that C24-AgNCs emit from a single chromophore with a well-defined emission transition dipole. Most measured molecules showed an emission transition dipole moment aligned in-plane, indicating that the out of plane ones are not efficiently excited under our experimental conditions. Moreover, of the 63 clusters that exhibited sufficient signal-to-noise and longevity, none showed any significant change to the fitted angle of the emission transition dipole over the experiment time. B
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formation was recently suggested in double stranded DNA.47 A recent study also hinted that a supramolecular complex of several smaller AgNCs embedded in the DNA could be possible.48 Despite the fact that we experimentally cannot exclude the second model, the first model seems to be the most plausible one, also since the multichromophoric model would potentially support the presence of stepwise photobleaching, depending on the number of emitters.46 Previously we also showed that spectral shifting was random, while one would expect a dominating blue shift as a function of time for the second model. In Figure 2, the cluster’s emission transition dipole was fitted to a φ (in plane) angle of 102° and a θ (out of plane) angle of 71°. The maximum intensity of the cluster corresponds to an in plane polarization angle of approximately 100°, suggesting that the excitation and emission transition dipole lie along a similar direction. Indeed, when the angle of the excitation polarization that gives the maximum intensity for each silver cluster is plotted against the in-plane emission angle of the fitted defocused image, we can see a clear correlation between the two, as shown in Figure 3. The lack of a clear offset between the
Combining defocused widefield microscopy techniques with rotating polarized excitation light allows us to examine both the excitation and emission transition dipoles simultaneously. During the fitting of the defocused pattern, the intensity of the image is also calculated and can be used to determine at which angle the C24-AgNC is most efficiently excited. A λ/2 plate was mounted in a motorized and programmable holder, and the polarization was rotated 20° between each recorded frame. The intensity trace in Figure 2 shows the
Figure 2. Normalized intensity of a single C24-AgNC with a changing excitation polarization angle. This image shows the same AgNC through two full rotations of the polarization. Intensity was calculated after background subtraction (see Figure SI2 and SI3). Two additional representative examples can be found in Figures SI4 and SI5.
characteristic waxing and waning of the intensity that one expects with a single excitation transition dipole as the λ/2 plate rotates. Deviations in intensity from a clear periodic function are most likely due to experimental noise on the intensity determination and fluorescence blinking.41 Of the clusters we measured that were photostable enough to be recorded for >180°, all showed similar behavior. The defocused patterns clearly show a single emission transition dipole. The above fitted defocused patterns and previous confocal work41 have shown that a single emitting chromophore is most likely for this spectrally selected C24-AgNC species at 638 nm. If multiple similar chromophores are present within one DNA strand, very efficient homo-FRET must be present. Energy transfer to the lowest energy chromophore for emission would still give a strong emission modulation. 44 This was demonstrated previously for identical organic chromophores that by homoFRET could transfer energy to a single emissive site that was slightly lower in energy due to specific interaction with the surrounding.45 As the emitting chromophore photobleaches, the next lowest energy chromophore takes over the emission.37 For chromophores that have different alignments to one another this would results in a very abrupt change in the emission transition dipole, as the emission changes from one chromophore to the other.45 In the case of very closely aligned chromophores, this would not be the case.46 Based on our findings two models are theoretically possible, (1) each DNA strand most likely only contains one AgNC emitter with a well-defined excitation and emission transition dipole moment or (2) there are a number of well aligned emitters that by FRET communicate with each other and emission occurs from an emissive trap. Multiple emitter
Figure 3. Excitation polarization angle that gives the maximum emission intensity for each C24-AgNC emitter is plotted against the fitted in plane emission angle. If more than 1 full cycle was recorded, the average of all maxima was used.
polarization angle and the in plane angle of the emission shows that the excitation and emission transition dipoles are similarly aligned. The spread on the difference between in plane emission angle versus excitation polarization angle is most likely due to experimental conditions and fitting accuracy since for an organic fluorophore (terrylene diimide in PS) we observe a similar spread (data not shown). Confocal Microscopy. A drawback of defocused widefield microscopy is that the time resolution is relatively poor (generally seconds per frame). As a result, changes to the emission transition dipole on a faster time scale may be concealed or averaged out, especially if these changes are small, dim, or short-lived. To overcome this, we conducted polarized confocal microscopy on the C24-AgNC sample, in order to obtain spectral information along with polarization data at higher time resolution in order to investigate a potential correlation between the spectral fluctuations observed in a previous paper and orientation changes.41 Using circularly polarized excitation light, we split the fluorescence from the sample between a spectrometer and two avalanche photodiodes (APDs). A polarizing beam splitter C
DOI: 10.1021/acs.jpca.6b11639 J. Phys. Chem. A XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry A was used to split the fluorescence between the two APDs into perpendicular and parallel polarization components. Although two APDs are not sufficient to determine the exact 3D orientation and provide exact changes in angle,49 they can provide qualitative info if reorientations take place. The latter is here more important since we want to investigate if there is a link between reorientation and the spectral changes. C24-AgNCs show some spectral shifting, both red and blue, over their emissive lifetimes as we demonstrated previously.41 We have previously suggested that this shifting is due to small changes in the DNA scaffold (in line with the first model). The defocused widefield studies have shown that large rotations or translations of the emitters do not occur under these conditions, which does not exclude small changes in the scaffold conformation, having a large impact on the photophysics of the emitter. Here we have defined the polarization (P) of the collected light according to eq 1. P = (Ipar − Iper)/(Ipar + Iper)
the excitation light was aligned with the emission transition dipole, suggesting that for the set of C24-AgNC emitters that were spectrally selected at 638 nm, under these experimental conditions, the excitation and emission transition dipole moment are aligned in a similar direction. Using confocal microscopy to probe the clusters at higher temporal resolution, we determined that although large movements of the emission transition dipole do not occur, some small changes to the polarization occur concurrently with changes in the emission spectra. Based on the presented data, the single emissive cluster model seems the most plausible. Although the second model cannot be experimentally excluded, it would require a number of specific conditions in order to match the observed results. The multiple spectrally similar chromophores should be aligned with respect to each other and should be within homo-FRET and singlet−singlet annihilation range, and both long off states and photobleached emitters should act as a nonemissive trap for the other spectrally similar emitters in the strand.
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(1)
An example of the data from a single spectrally selected C24AgNC at 633 nm is shown in Figure 4. The C24-AgNC shown
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.6b11639. Bulk spectral data, polarization dependent background and laser power data, and additional defocused widefield and confocal single molecule data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tom Vosch. E-mail:
[email protected]. ORCID
Miguel R. Carro-Temboury: 0000-0003-2817-9172 Tom Vosch: 0000-0001-5435-2181
Figure 4. A single molecule trace of an isolated C24-AgNC showing (a) the counts over time in the two polarization channels, parallel (black) and perpendicular (red), (b) polarization of the emission, and (c) the fitted emission maxima (nm). An additional representative example can be found in Figure SI6.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS T.V. gratefully acknowledges financial support from the “Center for Synthetic Biology” at Copenhagen University funded by the UNIK research initiative of the Danish Ministry of Science, Technology and Innovation (Grant 09-065274), bioSYNergy, University of Copenhagen′s Excellence Programme for Interdisciplinary Research, the Villum Foundation (Project number VKR023115), and the Danish Council of Independent Research (Project number DFF-1323-00352). The authors also thank Dr. Hiroshi Uji-I for providing analysis tools.
in Figure 4 exhibits both a blue and red shift, occurring at 170 and 220 s, respectively. This corresponds with a change in the emission intensity, as well as a slight change in the polarization of the emitted light. Of the 150 molecules measured, some had enough signal to detect a degree of spectral shifting and showed concurrent changes to the polarization. There were also instances where either the spectra shifted without change to the polarization or the polarization changed without affecting the spectra. Overall, the changes in the polarization are relatively small. This result fits with the findings of the defocused widefield imaging that no large changes are present.
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CONCLUSION Two methods of examining the polarization behavior of silver nanoclusters, defocused widefield and polarized confocal microscopy, were used to probe the excitation and emission polarization of AgNCs. With defocused widefield, C24-AgNCs show clearly defined emission transition dipole moments, consistent with the behavior of a single emitter. Rotating the excitation light during the measurement led to characteristic changes in the emission intensity for each C24-AgNC but had no clear effect on the angle of the emission transition dipole. The maximum emission intensity of each trace occurred when D
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DOI: 10.1021/acs.jpca.6b11639 J. Phys. Chem. A XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jpca.6b11639 J. Phys. Chem. A XXXX, XXX, XXX−XXX
