Dynamics of Single Molecule Stokes Shifts: Inﬂuence of Conformation and Environment

Martin Streiter,†,‡ Stefan Krause,∗,†,‡ Christian von Borczyskowski,† and Carsten

Deibel†

†Institut für Physik, Technische Universität Chemnitz, 09126 Chemnitz, Germany ‡These authors contributed equally to this work

E-mail: [email protected] arXiv:1806.08951v1 [physics.chem-ph] 23 Jun 2018

1 This document is the Accepted Manuscript version of a Published Work that appeared in ﬁnal form in The Journal of Physical Chemistry Letters, copyright c American Chemical Society after peer review and technical editing by the publisher. To access the ﬁnal edited and published work see DOI: 10.1021/acs.jpclett.6b02102 https://pubs.acs.org/articlesonrequest/AOR-JggM5eevqNDv9ddqMPhM

Abstract

We report on time dependent Stokes shift measurements of single molecules. Broadband

excitation and emission spectroscopy were applied to study the temporal Stokes shift

evolution of single perylene diimide molecules (PDI) embedded in a polymer matrix

on the time scale of seconds. The Stokes shift varied between individual molecules as

well as for single molecules undergoing diﬀerent conformations and geometries. From

the distribution and temporal evolution of Stokes shifts, we unravel the interplay of

nano-environment and molecular conformation. We found that Stokes shift ﬂuctuations

are related to simultaneous and unidirectional shifts of both emission and excitation

spectra.

Graphical TOC Entry

Keywords

Single Molecule Spectroscopy, Molecular Dynamics, Excitation Spectroscopy, Stokes Shift

2 The first experiments on optical detection of a single molecule were based on resonant excitation at low temperatures.1,2 The excitation spectra measured by the sweep of a narrow- band dye laser provided an unexpected wealth of information such as fluorescence lifetimes,3 quantum jumps,2 spectral diffusion processes,4 photon antibunching,5 blinking dynamics6 and magnetic resonance effects.7 Single molecule spectroscopy has developed into a stan- dard technique in many aspects of science.8–11 Extending the applicability of single molecule spectroscopy to room temperatures and living cells supported this progress.12–14 Since these first experiments, the obtainable information such as emission spectra, photon distributions, positions and orientations of individual emitters have become accessible in the lab as well as by commercial setups.15–17 However, measuring excitation spectra of a single molecule at room temperature remains challenging. Especially for strong phonon broadening, the red shifted fluorescence signal is weak, increasing the acquisition time for an excitation spectrum. Furthermore, at room temperature, the excited molecule is vulnerable to photo-oxidation. Nevertheless, the Stokes shift of a single molecule can be determined by measuring emission and excitation spectrum simultaneously and has been reported re- cently.18–20 Stopel et al. applied a tuneable laser source to excite single molecules at discrete steps of different wavelengths to reconstruct the excitation spectrum. They found that single molecule Stokes shifts are not inherently constant for chemically equivalent dye molecules. The experimentally determined Stokes shifts formed a broad distribution.19 In the most re- cent study by Piatkowski et al., excitation spectra were acquired for about two minutes by a broadband Fourier approach.20 The method’s advantage is the robustness against blinking and bleaching of the observed molecule. While these studies already revealed unexpected information on the photo-physics of single molecules, the temporal evolution of the Stokes shift during spectral diffusion processes remained unknown. It is worthwhile to push the temporal resolution of excitation and emission acquisition to the time scale of seconds and below where spectral diffusion can be observed. This information is not only desirable from a photo-physical point but is of great importance for single molecule Förster resonance energy

3 transfer (FRET) based experiments where the spectral overlap between emission of donor and absorption of acceptor strongly impact the transfer efficiency.21–24 Here we present an experiment that allows us to study the temporal evolution of emission, excitation and Stokes shift of photo-stable single PDI molecules in a polymer matrix. PDI molecules of this type have been shown to exhibit strong and temperature-dependent fluctuations between different conformations.15,25–29 As a consequence, these molecules show strong shifts in emission energies while the impact on the absorption remains unknown. We explored differences between individual molecules and their time-dependent behavior, revealing the influence of nano-environment and molecular conformation. Single PDI molecules were studied at room temperature under ambient conditions and at 77 K in vacuum by confocal microscopy as explained in the experimental section. At room temperature, 38 single molecule time-traces with a length of t = 10−160 s were recorded. At 77 K, 32 time-traces were measured. The accessible length of time-traces varied depending on the photo-stability between t = 20 − 2000 s. Figure 1 shows a representative example of 400 consecutive emission and excitation spectra of a single PDI molecule at 77 K recorded for 37 minutes. We determined the Stokes shift from the difference of the main emission and excitation peaks (marked as red dots in the Figure). Both emission and excitation spectra show simultaneous and discrete energetically different transitions. The investigated PDI molecules are known to form different conformations depending on the orientation of the four substituents in bay positions.15,25,27,29 The chemical structure of PDI is shown in the experimental section. Transitions between different molecular conformations have been extensively studied and are observable even for molecules embedded in polymer matrices beyond the glass transition temperature.15,25,28,29 In the present example, several distinct spectral jumps were observed. The emission spectrum shifts over an energy range of about 50 meV which is comparable to the overall difference in transition energies for all possible conformations of 88 meV calculated by Fron et al.27 Additionally, we would like to mention that the energy range of the emission and excitation peak positions covered by all measured

4 molecules in this study is about 100 meV. For t = 630 − 1050 s (state A) and t = 1050 − 1520 s (state B) in Figure 1 the averaged emission and excitation spectra are shown above the time-traces. The main emission peak appears at 2.003 eV for state A and at 1.977 eV for state B. The corresponding excitation spectrum shows a comparable main peak at 2.036 eV for state A and 2.000 eV for state B, which is assigned to transitions from the S0 ground state to S1 states. Additionally, emission and excitation spectra feature at least one distinct vibronic sideband. The energy diﬀerences for two consecutive states A and B, either in emission or excitation, were calculated from

the peak energy diﬀerences ∆E = EB − EA. From emission and excitation maxima follows a Stokes shift of 33 meV in state A, which decreases to 23 meV in state B. This change in Stokes shift of 10 meV is small in comparison to the change in the peak position of emission

∆Eem = 26 meV and excitation ∆Eex = 36 meV. These comparably small changes of the Stokes shift apply for all spectral fluctuations in Figure 1 and are typical for every measured single molecule. For a detailed discussion of how conformational changes influence emission and excitation properties, we evaluated for all molecules every spectral jump above a threshold of 5 meV. Smaller spectral fluctuations were observed but neglected due to their high fluctuation fre- quency and therefore insufficient evaluation accuracy. We observed only minor changes of spectral shape and intensity during these jumps. Spectral jumps were found to occur simultaneously within our experimental time resolution (3 s at 300 K and 5.5 s at 77 K for one emission and excitation spectrum). Figure 2 (top) shows a nearly linear and temperature- independent correlation between ∆Eem and ∆Eex. It can be seen that emission and excitation always shift synchronously to higher or lower energies, respectively (quadrants i and iii). No anti-correlation or opposite dynamics could be observed (quadrants ii and iv).

We further examined the variations of the Stokes shift ∆EStokes as a function of emission

energy fluctuations ∆Eem in Figure 2 (bottom). At 300 K, increasing and decreasing Stokes shifts were measured during spectral fluctuations. They were caused by different jump widths

5 emission excitation 1 B A B A 0.5 intensity 0 2000

1500

B / s t 1000 A

500 B B A A

1.8 1.9 2.0 2.1 2.0 2.1 2.2

Eem / eV Eex / eV

Figure 1: Time dependent emission and excitation spectra of a single PDI molecule at 77 K. Red dots indicate the peak positions gained from ﬁtting the spectra. Changes between discrete states of emission and excitation are simultaneously occurring. The averaged and normalized spectra of conformation A and B (marked by dashed lines) are presented above the time-trace.

of emission or excitation, respectively. These processes are reversible. Consequently, spectral jumps appear in quadrants I to IV. At 77 K, spectral jumps appear only in quadrant II and IV. This means that shifts of emission energy are larger than shifts of excitation energy at low temperatures. We assume a temperature dependent correlation between emission and

excitation shifts. For both temperatures, the largest Stokes shift changes were ∆EStokes = ±15 meV.

Now we discuss the Stokes shift variations ∆EStokes of individual single molecule time- traces with respect to the overall Stokes shift distribution of all molecules measured. Figure 3 shows the distribution of average Stokes shifts. Each value results from calculating the mean

6 40 300 K 77 K 20 ii) i)

/ meV 0 ex

E -20 ∆

-40 iii) iv) 20 10

/ meV II) I) 0 Stokes

E -10 ∆ -20 III) IV) -40 -20 0 20 40

∆Eem / meV

Figure 2: Quadrants i) to iv): spectral jumps ∆Eex vs. ∆Eem above a threshold of 5 meV at 300 K (red) and 77 K (black). Quadrants I) to IV): Stokes shift variations ∆EStokes for the spectral jumps above.

Stokes shift of a complete time-trace. At 300 K we found a broad distribution of Stokes shifts centered around 65 meV. The width of the distribution shows that Stokes shift variations of

∆EStokes ≈ 70 meV are possible despite chemically identical molecules. This value far exceeds the maximal Stokes shift variations of 15 meV which have been found within individual spectral time-traces. From the broad and temperature-dependent Stokes shift distribution, we conclude that the nanoscopic environment of the molecules is responsible for large differences in the Stokes shift. This can be explained by a different strength in interaction of individual molecules either to their own phenoxy side groups27 or the surrounding polystyrene matrix. Large spectral jumps in emission and excitation up to 80 meV as observed for individual molecules within a spectral time-trace do not influence the Stokes shift to the same extent. Vallée et al. have shown that geometrical fluctuations can occur within the volume available for geometrical rearrangements of the molecule in a polymer below the glass transition temperature.28 These changes do not require a large rearrangement of the chromophoric PDI backbone with respect to the polymer environment. We suggest that the resulting impact on

7 10 300 K EStokes 8 T = 300 K 77 K 1.0 6 emission excitation 4 2 0.8 0 10 0.6

occurrence T = 77 K 8

6 normalized intensity 0.4 4 2 0 0.2 20 40 60 80 100 1.8 1.9 2.0 2.1 2.2 EStokes / meV E / eV

¯ Figure 3: Occurrence of average Stokes shift values EStokes of single molecule spectral time- traces at 300 K and 77 K. With higher temperature, the distribution of Stokes shifts is broader and located at higher values. The right graph shows the average of all spectra measured. The average Stokes shift increases due to increased line-broadening (indicated by arrows) at 300 K.

coupling is comparably small, leading to minor Stokes shift variations during measurement of an individual molecule. This is reasonable since the phenoxy side groups are immobilized by the rigid polymer below the glass transition temperature. Temperature lowering to 77 K results in a narrowed distribution of Stokes shifts centered at 50 meV. The smaller Stokes shifts at 77 K can be assigned to a reduction of the phonon wing by annihilation of phonons at low temperatures.30 The phonon wing is responsible for the line-broadening of the main emission and excitation peak at elevated temperatures resulting in a dislocation of peak centers and therefore larger Stokes shifts as shown in Figure 3. In conclusion, we performed time dependent emission and broadband excitation spectroscopy of single perylene diimide molecules on the time scale of seconds. Varying states of emission and excitation energies were observed and attributed to different fluctuating molecular geometries caused by interactions with the heterogeneous environment. We found that emission and excitation spectra change at the same time. Emission and excitation spectra showed simultaneous red or blue shifts, respectively. Spectral jump widths of emission and excitation were found to be different, leading to Stokes shift variations with a maxi-

8 mum difference of 15 meV within single molecule spectral time-traces. Since the molecules are immobilized within the polymer matrix below glass transition temperature, we assume that these minor variations are caused by twisting of the chromophoric PDI backbone.28 In contrast, stronger variations of the average Stokes shifts between different molecules were observed, covering an energy range of 70 meV. This can be explained by different coupling of the static nanoscopic environment and the phenoxy side groups to the PDI chromophoric backbone.

Experimental

The perylene diimide derivate N,N’-Dibutyl-1,6,7,12 - tetra (4- tert-butylphenoxy) perylene- 3,4:9,10-tetracarboxylic acid bisimide (PDI) was synthesized by Würthner et al.31 and embedded in a poly(styrene) (PS) matrix with a molecular weight of 20 kg/mol. A PS toluene solution was doped with 10−10 mol/l PDI and spincoated onto silicon dioxide substrates. Single molecule confocal microscopy and emission spectroscopy as a function of obesrvation time were performed as previously described.32 For excitation spectra acquisition, a tuneable supercontinuum white light laser source (SuperK EXR-15 with SELECTplus filter, NKT Photonics) was used. Excitation spectra were measured by detecting the photoluminescence above 640 nm with a longpass filter. Emission spectra were measured with an excitation wavelength of λex = 560 nm. A non-polarizing 50:50 beam splitter separated the fluorescence signal into two equal fractions for subsequent excitation and emission measurements. A scheme of the experimental setup is given in Figure 4. At 300 K, emission spectra were acquired for 0.5 s (1 s at 77 K) and excitation spectra for 2.5 s (4.5 s at 77 K, longer acquisition times due to cryostate and objective with lower numerical aperture). Fast switching (10 ms) between acquisition of emission and excitation was realized by utilizing an automated bandpass filter and a spectrograph shutter.

9 Figure 4: Scheme of the experimental setup. A supercontinuum laser provides a tuneable excitation wavelength λex. In combination with achromatic beam splitters, fast switching between emission (spectrograph) and excitation acquisition (ADP) was realized.

Author Information

Corresponding Author

Email: [email protected]

Notes

The authors declare no competing ﬁnancial interest.

Acknowledgement

This work was partly funded by the DFG (project DFG DE830/13-1). We thank Frank Würthner (Julius-Maximilian-University Würzburg) for a kind donation of PDI.

10 References

(1) Moerner, W. E.; Kador, L. Optical Detection and Spectroscopy of Single Molecules in a Solid. Phys. Rev. Lett. 1989, 62, 2535–2538.

(2) Orrit, M.; Bernard, J. Single Pentacene Molecules Detected by Fluorescence Excitation in a p-Terphenyl Crystal. Phys. Rev. Lett. 1990, 65, 2716–2719.

(3) Pirotta, M.; Gütter, F.; Gygax, H.; Renn, A.; Sepiol, J.; Wild, U. P. Single Molecule Spectroscopy. Fluorescence-Lifetime Measurements of Pentacene in p-Terphenyl. Chem. Phys. Lett. 1993, 208, 379–384.

(4) Ambrose, W.; Moerner, W. E. Fluorescence Spectroscopy and Spectral Diﬀusion of Single Impurity Molecules in a Crystal. Nature 1991, 349, 225–227.

(5) Basché, T.; Moerner, W. E.; Orrit, M.; Talon, H. Photon Antibunching in the Flu- orescence of a Single Dye Molecule Trapped in a Solid. Phys. Rev. Lett. 1992, 69, 1516–1519.

(6) Moerner, W. E. Those Blinking Single Molecules. Science 1997, 277, 1059–1060.

(7) Wrachtrup, J.; Von Borczyskowski, C.; Bernard, J.; Orritt, M.; Brown, R. Optical Detection of Magnetic Resonance in a Single Molecule. 1993, 363, 244–245.

(8) Moerner, W. E. A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics. J. Phys. Chem. B 2002, 106, 910–927.

(9) Kulzer, F.; Orrit, M. Single-Molecule Optics. Annu. Rev. Phys. Chem. 2004, 55, 585– 611.

(10) Lu, H. P.; Xun, L.; Xie, X. S. Single-Molecule Enzymatic Dynamics. Science 1998, 282, 1877–1882.

11 (11) Wöll, D.; Braeken, E.; Deres, A.; De Schryver, F. C.; Uji-i, H.; Hofkens, J. Polymers and Single Molecule Fluorescence Spectroscopy, What Can We Learn? Chem. Soc. Rev. 2009, 38, 313–328.

(12) Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Near-Field Spectroscopy of Single Molecules at Room-Temperature. Nature 1994, 369, 40–42.

(13) Xie, X. S. Single-Molecule Spectroscopy and Dynamics at Room Temperature. Acc. Chem. Res. 1996, 29, 598–606.

(14) Xie, X. S.; Trautman, J. K. Optical Studies of Single Molecules at Room Temperature. Annu. Rev. Phys. Chem. 1998, 49, 441–480.

(15) Krause, S.; Aramendia, P. F.; Täuber, D.; von Borczyskowski, C. Freezing Single Molecule Dynamics on Interfaces and in Polymers. Phys. Chem. Chem. Phys. 2011, 13, 1754–1761.

(16) Schmidt, R.; Krasselt, C.; Göhler, C.; von Borczyskowski, C. The Fluorescence Inter- mittency for Quantum Dots Is Not Power-Law Distributed: A Luminescence Intensity Resolved Approach. ACS nano 2014, 8, 3506–3521.

(17) Krause, S.; Neumann, M.; Fröbe, M.; Magerle, R.; von Borczyskowski, C. Monitoring Nanoscale Deformations in a Drawn Polymer Melt with Single-Molecule Fluorescence Polarization Microscopy. ACS nano 2016, 10, 1908–1917.

(18) Blum, C.; Schleifenbaum, F.; Stopel, M.; Peter, S.; Sackrow, M.; Subramaniam, V.; Meixner, A. J. Room Temperature Excitation Spectroscopy of Single Quantum Dots. Beilstein J. Nanotechnol. 2011, 2, 516–524.

(19) Stopel, M. H.; Blum, C.; Subramaniam, V. Excitation Spectra and Stokes Shift Mea- surements of Single Organic Dyes at Room Temperature. J. Phys. Chem. Lett. 2014, 5, 3259–3264.

12 (20) Piatkowski, L.; Gellings, E.; van Hulst, N. F. Broadband Single-Molecule Excitation Spectroscopy. Nat. Commun. 2016, 7, 10411.

(21) Roy, R.; Hohng, S.; Ha, T. A Practical Guide to Single-Molecule FRET. Nat. Methods 2008, 5, 507–516.

(22) Schuler, B.; Eaton, W. A. Protein Folding Studied by Single-Molecule FRET. Curr. Opin. Struct. Biol. 2008, 18, 16–26.

(23) Ha, T.; Enderle, T.; Ogletree, D.; Chemla, D.; Selvin, P.; Weiss, S. Probing the Interac- tion Between two Single Molecules: Fluorescence Resonance Energy Transfer Between a Single Donor and a Single Acceptor. Proc. Natl. Acad. Sci. 1996, 93, 6264–6268.

(24) Beljonne, D.; Curutchet, C.; Scholes, G. D.; Silbey, R. J. Beyond Förster Resonance Energy Transfer in Biological and Nanoscale Systems. J. Phys. Chem. B 2009, 113, 6583–6599.

(25) Kowerko, D.; Schuster, J.; Von Borczyskowski, C. Restricted Conformation Dynamics

of Single Functionalized Perylene Bisimide Molecules on SiO2 Surfaces and in Thin Polymer Films. Mol. Phys. 2009, 107, 1911–1921.

(26) Osswald, P.; Leusser, D.; Stalke, D.; Würthner, F. Perylene Bisimide Based Macrocy- cles: Eﬀective Probes for the Assessment of Conformational Eﬀects on Optical Prop- erties. Angew. Chem. 2005, 44, 250–253.

(27) Fron, E.; Schweitzer, G.; Osswald, P.; Würthner, F.; Marsal, P.; Beljonne, D.; Müllen, K.; De Schryver, F. C.; Van der Auweraer, M. Photophysical Study of Bay Substituted Perylenediimides. Photochem. Photobiol. Sci. 2008, 7, 1509–1521.

(28) Vallée, R. A.; Cotlet, M.; Van der Auweraer, M.; Hofkens, J.; Müllen, K.; De Schryver, F. C. Single-Molecule Conformations Probe Free Volume in Polymers. J. Am. Chem. Soc. 2004, 126, 2296–2297.

13 (29) Hofkens, J.; Vosch, T.; Maus, M.; Köhn, F.; Cotlet, M.; Weil, T.; Herrmann, A.; Müllen, K.; De Schryver, F. Conformational Rearrangements in and Twisting of a Single Molecule. Chem. Phys. Lett. 2001, 333, 255–263.

(30) Friedrich, J.; Haarer, D. Photochemical Hole Burning: a Spectroscopic Study of Relax- ation Processes in Polymers and Glasses. Angew. Chem. 1984, 23, 113–140.

(31) Würthner, F.; Sautter, A.; Schmid, D.; Weber, P. J. Fluorescent and Electroactive Cyclic Assemblies from Perylene Tetracarboxylic Acid Bisimide Ligands and Metal Phosphane Triﬂates. Chem. Eur. J. 2001, 7, 894–902.

(32) Krause, S.; Kowerko, D.; Börner, R.; Hübner, C. G.; von Borczyskowski, C. Spec- tral Diﬀusion of Single Molecules in a Hierarchical Energy Landscape. ChemPhysChem 2011, 12, 303–12.