Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany Geminate Recombination as a Photoprotection Mechanism for Fluorescent Dyes** Phil Holzmeister, Andreas Gietl, and Philip Tinnefeld* anie_ _sm_miscellaneous_information.pdf anie_ _sm_si_video_1.wmv

2 Table of contents: Materials and Methods o o o o o o Samples and surface immobilization Buffers Confocal single-molecule setup Widefield single-molecule setup Derivation of autocorrelation analysis Fitting model for AA dependence Photobleaching discussion Supporting Figures o Figure S1: Exemplary fluorescence transients and autocorrelations of Alexa568 molecules in presence of ROXS and for varying ME concentration o Figure S2: Exemplary analysis and determination of experimental error. o Figure S3: Triplet lifetime of Atto647N molecules for varying ME concentration compared to varying ph at constant concentration. o Figure S4: Confocal images of immobilized Alexa568 molecules in presence of ascorbic acid (AA) or ß-mercaptoethanol (ME) o Figure S5: Photon numbers from Atto647N before entering a triplet and radical state together with geminate recombination yield at varying AA concentration o Figure S6: Geminate recombination yield for Atto647N at varying glycerol concentration o Figure S7: ME dependent lifetime of the radical anion for Atto532, Alexa568 and Atto647N o Figure S8: Individual frames from Supporting Video 1. Alexa568 fluorescence for TX/TXQ, ME and TX/TXQ+ME buffers o Figure S9: UV activation of thiol induced long-lived dark state of Atto647N in TIRF and confocal measurements o Figure S10: Total photon count before apparent photobleaching from Alexa488, Alexa532 and Atto532 molecules for different buffer conditions o Figure S11: Photobleaching of Alexa568 under different buffer conditions. o Figure S12: Exemplary confocal images, fluorescence transients and autocorrelations of Atto647N molecules in presence of AA and varying DTT concentration References

3 Materials and Methods Samples and surface immobilization The dsdna samples consist of one strand labeled with biotin at the 3 end (5 -ATG CTA AGC TAA GGA ATG TGA ATA TAA TGT ATC GAT ATG C-3 ) and a complementary strand (5 -GCA TAT CGA TAC ATT ATA TTC ACA TTC CTT AGC TTA GCA T-3 ) with a fluorophore (Alexa488, Alexa532, Atto532, Alexa568 or Atto647N) attached to the 3 end. The dye modified strands were purchased from IBA, the biotin modified strand from Eurofins MWG. The samples are hybridized at 1 µm concentration in 1xTAE buffer with 12.5 mm MgCl 2 by heating to 90 C and slow cooling to 4 C over 1.5 h in an Eppendorf Thermocycler. For confocal measurements of individual molecules, Lab-Tek chambers (Thermo Scientific) are cleaned with 1 M KOH for 15 minutes. After each step of the surface preparation, the buffer is washed three times with 1x PBS. They are further incubated with a mixture of BSA and biotin labeled BSA for passivation (1 mg/ml and 0.1 mg/ml respectively). In the next step, neutravidin (0.1 mg/ml) is bound to the biotin on the surface for 5 min and washed away with 1x PBS. Finally, the chamber is incubated with dsdna until sufficient surface coverage is reached. Buffers 100 mm TRIS buffer (from Trizma base, chemicals from Sigma Aldrich), 50 mm NaCl with 1% (w/w) glucose is adjusted to the desired ph value with HCl (ph meter: Checker, Hanna Instruments). For concentration dependent ß-mercaptoethanol (ME) measurements, the same buffer is prepared with additional 1% (v/v) ME. The concentration is adjusted by mixing these buffers proportionally. For oxygen scavenging, 10% (v/v) of GOC (Glucose Oxidase and Catalase) buffer is added to the measurement buffer. GOC consists of 1 mg/ml glucose oxidase, 0.4% (v/v) catalase (50 µg/ml), 30% glycerol and 12.5 mm KCl in 50 mm TRIS ph 7.5. This buffer is used in all measurements unless states otherwise. The ROXS system Trolox/Trolox quinone (TX/TXQ) is prepared according to reference [1] and is used as current state of the art. Trolox is, however, not the optimal choice when a pure reductant is required since it quickly degrades to trolox quinone [1]. Because the reductants and oxidants can be easily exchanged regarding their photoprotecting properties [2], ascorbic acid was used when we intended to add a pure reductant. We frequently observe impurities when using methyl viologen with excitation below 600 nm and therefore employ trolox quinone as oxidant whenever possible. Confocal single-molecule setup The confocal single-molecule setup is equipped with an 80 MHz pulsed laser (640 nm, LDH-D- C-640, Picoquant) for excitation of Atto647N and a cw 532 nm laser (TECGL-30, World Star Tech) for excitation of Atto532 and Alexa568. The laser beam is coupled through the rear port of an inverted microscope (IX-71, Olympus) and focussed by the objective (UPLSAPO 100XO, NA 1.40 or UPLSAPO 60XO, NA 1.35, both Olympus) to a diffraction limited spot. The sample is scanned through this spot by a piezo stage (P-517.3CL, Physik Instrumente) to acquire an image of the molecules immobilized on the surface and the same stage is used to position molecules of interest in the laser focus for time-resolved measurements. Light emitted by the dye molecules is detected through the same objective and separated from the excitation light

4 with a dichroic mirror (Dualband z532/633, AHF). After passing a 50 µm pinhole the emitted light ist split spectrally at 640 nm by a dichroic beam splitter (640DCXR, AHF) onto two APDs ( - SPAD 100, Picoquant) with appropriate filters (Bandpass ET 700/75m, AHF; RazorEdge LP 647, Semrock for Atto647N emission; BrightLine HC582/75, AHF; RazorEdge LP 532, Semrock for Atto532 and Alexa568 fluorescence). The signal from the APDs is registered with a PC-card (SPC-830, Becker&Hickl) for time-correlated single photon counting. Further analysis is carried out by home-made LabView software (LabView2009, National Instruments). Widefield single-molecule detection and analysis We use a homebuilt PRISM-TIRF setup based on an Olympus IX71 to perform widefield measurements. Fluorophores were excited according to Table 1. The fluorescence is collected by an 60x Olympus 1.20 N.A. water-immersion objective, further filtered with appropriate emission filter (Table S1) and recorded by an EMCCD camera (Andor IXon X3, pregain 5.1, gain 50, frame rate 10 5 Hz). Videos are analyzed by custom-made software based on LabVIEW bit (National Instruments). The fluorescent molecules are selected by an automated spotfinder from the first two frames of the video and the resulting transients are filtered with the built in cubic filter of LabVIEW The fluorescence intensities are background corrected by subtracting the surrounding pixels intensity. We calculate the total photon counts for each molecule by integrating the transient s intensity over time. Fluorophore Laser Excitation filter Emission filter Alexa ca. 0.5 kw/cm 2 Coherent Sapphire mW longpass RazorEdge, Semrock Alexa 532 ATTO ca. 1 kw/cm 2 Compact Laser Rapidus 10 W Clean-up filter 532/2 MaxLine, Semrock 582/75 BrightLine, Semrock Alexa ca. 1 kw/cm 2 Coherent Sapphire mW - 509/54 BrightLine, Semrock ATTO 647n 639 ca. 3 kw/cm 2 Toptica ibeam Smart 150mW Clean-up MaxDiode 640/8 Semrock 647 longpass RazorEdge, Semrock All filters were purchased from AHF Göttingen Table S1: Lasers and filters used for widefield TIRF measurements.

5 Derivation of autocorrelation analysis We consider a three level system with rate constants k mn for transition from state m to state n. State 1,2,3 are termed A,B and C for the general derivation. In our case, they will represent the singlet manifold S(t), triplet T(t) and radical R(t) states. The system is fully described by the following set of homogeneous first order differential equations: Assuming that the triplet dynamics occur on a significantly faster timescale than the radical dynamics (k12+k21)>>(k23+k31), the solution to this set of equations with the molecule initially in its ground state can be simplified to: This solution has been explicitly presented in ref. [3], only with different state assignment (ground state S 0 (t)=a(t), excited state S 1 (t)=b(t) and triplet state T 1 (t)=c(t)). The solution for our case describes the probability that an individual molecule resides in the singlet manifold, triplet state or in a radical state at a certain time t after it is reset to its ground state at t=0. The constant term is the steady state probability, while the terms with decay constants 2 = (k12+k21) -1 and 3 = (k12 k23/(k12+k21) +k31) -1 represent the triplet and radical state dynamics, respectively. Following ref. [4], the normalized autocorrelation function (AC) for three states can be defined as where P(m) is the probability of detecting a molecule in state m and P(m,t n,t+ ) the probability of subsequently detecting it in state m at time t and in state n at time t+. The formula greatly

6 simplifies, since in our case only state 1, the singlet state S, is fluorescent (I m I n =I 1 2 m1 n1, with Kronecker delta ). P(1) is the steady state probability of the singlet population and because after each detected photon the system is reset to its groundstate, P(m,t n,t+ ) can simply be expressed as presented above. This can be summarized to The parameters of the autocorrelation function relate to the macroscopic on- and off-times as follows: The experimental observables relate to the rate constants from Figure 1 of the main text as The indices represent the triplet (T) or radical (R) dynamics. Here fl is the fluorescence lifetime of the excited state and the expression for k12 is only valid in the low excitation regime we work -1 in (k ex << fl ). Experimentally, the correlations G ( ) are calculated from the fluorescence transients I(t) as where <> denotes temporal averaging and I(t)=I(t)-<I(t)>. Compared to the conventional AC definition, we subtract the constant offset of value 1. The AC is fitted with the biexponential decay model presented above. Before further analysis, we multiply the background correction factor C to the amplitudes of the decays to account for the influence of uncorrelated background signal. [5] The time-averaged background signal B is determined for each molecule after the fluorophore bleached.

7 Together with the mean intensity I m = <I(t)> we extract the number of detected photons before radical blinking occurs N on as well as the number of photons before a transition to the triplet state N T. The macroscopically observed intensity I on during t on,r represents the intensity in the fluorescence transient, but is averaged over multiple singlet-triplet cycles with I T during t on,t For convenience, the triplet lifetime t is termed t off,t in the main text. T

8 Fitting model for AA dependence In absence of ascorbic acid, the geminate recombination yield can be described as =1- gr N T /N on, where N T and N on are the number of detected photons before the dye enters the triplet and radical state respectively. Hence, the on-counts are Here k gr and k t are the geminate recombination and intrinsic triplet rates, k 0 is the reduction red rate in absence of AA. Since already 1% ME is present (Figure 3c, main text), k t is negligible. We now introduce an additional reduction rate proportional to the AA concentration The proportionality factor describes the interaction strength of AA with the dye. With this, we can describe N on dependent on the concentration of AA: where t 0 T is the triplet lifetime in absence of AA. This function is used to obtain and gr by fitting the data in Figure 3c (main text). These values are subsequently used to calculate the expected reduction of the on-counts, when AA is added to a buffer with 0.1% ME in Figure S11.

9 Photobleaching discussion The off-switching with thiols to long-lived dark states (Figure S9) explains why tc-roxs can be more efficient for photoprotection than GR-ROXS with thiols. But why is GR-ROXS more efficient for some dyes and likely for all dyes if there was not the thiol-induced off-switching? For the improved photostabilization with GR-ROXS we envision two possibilities. If photo-oxidized states are dominant intermediates on the photobleaching pathway, the advantage of GR-ROXS is obvious because photo-oxidized intermediates are avoided. Alternatively, it is possible that ROXS components themselves induce photobleaching, e.g. if the formed radical ions of the dye and the ROXS component have a probability to chemically react with each other. To test whether e.g. AA enhances photobleaching we take into account two scenarios. In the first scenario we assume that photobleaching proceeds through reactive intermediate states such as triplet and radical anionic states. In this case, the photobleaching tendency should scale with the overall time spent in any of these intermediate states. Direct reaction with AA intermediates would then be indicated if the photostability in the presence of AA is lower than expected from this correlation. We calculate the expected reduction of N on in presence of 0.1% ME, when 50 µm AA (0.20 N on ) and 1 mm AA (0.02 N on ) is added using the results from the fits in Figure 3c (main text) together with t T and N on for the 0.1% ME measurement (Figure 3a-c). If bleaching occurred from the radical anionic state, the total photon numbers should be reduced by the same amount. In fact, the photostability is reduced less significantly (Figure S11), which suggests that the addition of AA has a positive effect on photostability predominantly by further quenching the triplet state, i.e. we find no evidence that AA enhances photobleaching pathways directly. MV, on the other hand, dramatically reduces the total photon number already at 50 µm but we cannot differentiate whether this is due to the tendency of photo-oxidized states for photobleaching or selectively induced photobleaching by MV intermediates (Figure S11).

10 Supporting Figures Figure S1. Fluorescence transients of Alexa568 (left) and corresponding autocorrelation functions (right, offset subtracted G ( )=G( )-1) with biexponential fits (blue, red). a) Fluorescence from an Alexa568 molecule (all measurements with enzymatic oxygen scavenging). The intensity is constant (no significant AC amplitude) but the dye quickly bleaches. b) The fluorophore bleaches fast and does not emit at a constant intensity level (inset) for 0.01% ME. The AC reveals that this is caused by pronounced triplet blinking which is faster than the binning (5 ms). c) Higher ME concentration of 0.1% results in longer fluorescent transients, a reduced triplet component in the AC and clearly separated on and off states of the intensity (inset). d) These effects are even more pronounced for 1% ME. e) A combination of 2 mm TX/TXQ and 1% ME offers a trade-off between blinking and bleaching of the Alexa568 molecules. The total number of detected photons before bleaching of these exemplary molecules are (a), (b), (c), > (d) and (e).

11 Figure S2. Exemplary data analysis for Atto647N in 1% (v/v) ME after oxygen depletion. a) Detected photons before entering a radical state N on =(7.1±2.3) b) Lifetime of radical state t off,r =(22.8±8.1) ms. c) Detected photons before entering a triplet state N T =60±16. d) Lifetime of triplet state t off,t =(141±36) µs. The histograms of values obtained from 49 molecules are fitted with a Gaussian to reduce the influence of outliers. The mean value and standard deviation of the fitted Gaussian are used for data points and error bars.

12 Figure S3. Triplet lifetime of Atto647N depending on the concentration of dissociated thiolate RS - when [ME] is changed at ph 8.6 (red triangles), and for constant [ME], but the ph is varied (ph 7.6, 8.1, 8.6, 9.1, black triangles). For higher ph values, we interestingly observe a similar quenching of the triplet state even though the absolute concentration of ME remains constant. We conclude that the active triplet quenching species is the dissociated thiolate ion RS - (ME RS - + H + ) in accordance with ref. [6], since at higher ph values more ME is deprotonated. The thiolate concentration is calculated with the pk a value of ME (pk a = 9.7 [6b,7] ) and the rearranged Henderson-Hasselbalch equation [RS - ]=[ME] 0 /(1+10^(pK a -ph)).

13 Figure S4. 10 µm x 10 µm confocal scans of immobilized Alexa568 molecules acquired with 2 ms/px, 1 px=50 nm, scale bar=2 µm, colour scale counts/px. Excitation at 532 nm with 10 µw. a) In presence of oxygen, molecules are clearly identified even in presence of high AA concentrations (10 µm shown). b) Enzymatic removal of oxygen leads to longer off-times (triplet and radical anion), which reduces the number of photons detected from an individual molecule. c) The effect is enhanced with 1 mm AA, since the longer-lived radical off-states are built up faster. d-f) The presence of increasing ME concentration exhibits the opposite effect: while at 0.01% ME the triplet off-states still lead to a rather blurry image (d), these triplet states are efficiently quenched with 0.1% (e) or 1% ME (f). Due to geminate recombination, the number of emitted photons before entering a radical off-state is increased, which facilitates the identification of individual molecules in absence of oxygen.

14 Figure S5. a) Number of photons collected from Atto647N before entering a radical state (N on, black squares) and before entering the triplet state (N T, red circles). Data from measurement of Figure 3c in the main text, measurement in 100 mm Tris ph 8.6, 1% (v/v) ME and enzymatic oxygen scavenging. The N on are significantly reduced with increasing AA concentration starting at approximately 10 µm, while the N T are only slightly affected at high concentrations, which means that the reduced N on at moderate concentrations are not caused by reaction of AA with the S 1 state. b) The yield of geminate recombination calculated from the photon counts in a) ( gr =1-N T /N on ). The yield is significantly reduced with increasing AA concentration.

15 Figure S6. Geminate recombination yields for 1% ME and Atto647N for different viscosities. The buffer consists of 100 mm Tris ph8.6, 1% (v/v) ME, 0.5% (w/v) glucose, 10% (v/v) GOC and up to 38% (v/v) glycerol. The viscosity of the buffer increases with glycerol concentration, which goes along with an increase of the geminate recombination yield. Figure S7. Lifetime of the radical dark states for the dyes Alexa568 (black squares), Atto647N (red triangles) and Atto532 (blue circles) at different ME concentrations from the datasets of Figure 3 in the main text. The data clearly show that the enhanced photostability is not caused by an oxidizing species that is supplied with ME as would be required for two component tc-roxs like TX/TXQ [1]. An oxidant would clearly depopulate the radical anion state and therefore reduce t off,r. [8] The slight increase of the off-time for higher concentrations can be caused by oxygen scavenging effects reported for thiols. [6a,9]

16 Figure S8. Individual frames from the Supporting Video 1. The figure shows the first frame and the frames after 5, 15 and 30 seconds, respectively, for measurements in tc-roxs buffer (2 mm TX/TXQ, left column), with 1% (v/v) ME (center), and a combination of both (right column). The molecules show homogeneous brightness in the tc-roxs buffer, but bleach very fast. With ME, they survive longer, but the intensity is generally lower and inhomogeneous due to blinking (low ms range), which is faster than the integration time of the camera (200 ms). For the combination of both, the dyes are bright and bleach significantly slower compared to measurement without ME. Excitation intensity is 1 kw/cm² (568 nm). All measurements were carried out in 100 mm TRIS ph 8.6 and with enzymatic oxygen scavenging. Scale bar: 10 µm, color scale: Hz. The last row shows transients from a confocal microscope with better temporal resolution (data from Figure S1) that illustrate how the photophysics on shorter timescale affects the homogeneity of imaging with longer integration time.

17 Figure S9. UV-activation of Atto647N. a) Number of detected fluorescent molecules on a surface in 100 mm Tris ph 8.5, enzymatic oxygen scavenging and 1% (v/v) ME. The surface with initially 130 molecules in the field of view was illuminated with 639 nm until the majority of molecules switched to a dark state, but they can be efficiently recovered by brief UV flashes (405 nm, illumination wavelength is indicated on top). b) With a confocal microscope, this can be used to repeatedly switch the same Atto647N molecule. c) Camera frames immediately before (i) and after (ii) a 405 nm activation flash (positions indicated in panel a). d) If ME is replaced by 1 mm AA, no reactivation with UV light can be observed under otherwise identical conditions to a).

18 Figure S10. Photon counts before apparent bleaching for the dyes Atto532 (a), Alexa532 (b) and Alexa488 (c). In case of Atto532, the addition of ME increases the total number of detected photons, while for Alexa532 and Alexa488 no significant difference between tc-roxs (TX/TXQ) and a combination of TX/TXQ with ME is observed.

19 Figure S11. Photon counts before apparent bleaching for Alexa568 under various buffer conditions. All measurements were carried out under oxygen depletion and in presence of 0.1% ME. The addition of 50 µm AA or 1 mm AA reduces the number of detected photons, but less than expected from the reduction of the on-counts, which indicates that the triplet state is more involved in photobleaching pathways than the radical anion state (discussion above in section Photobleaching discussion ). The stronger effect of 50 µm MV compared to the AA measurements and compared to the measurement in presence of 1 mm MV supports that the two main contributors to photobleaching are the triplet and the radical cation state. The data also provide no clear evidence of active bleaching through MV or AA. The improvement from 50 µm MV/AA to 1 mm MV/AA is in accordance with efficient depopulation of intermediate states, while completely avoiding radical cationic states (0.1% ME + GOC without MV/AA) still yields higher photon counts. The discrepancy to Figure 4a in the main text, where a combination of TX/TXQ with 1% ME performs better than 1% ME alone results from the 10fold higher ME concentration used for the measurements shown in Figure 4.

20 Figure S12. Confocal images (left, 2.9 x 1.8 µm², 2 ms/px, 50 nm/px, color scale counts/px, 2 µw excitation 640 nm), fluorescence transients (center) and corresponding autocorrelation functions (right) with biexponential fits (red, blue) for Atto647N molecules in 100 mm TRIS ph 8.6 with enzymatically removed oxygen. a) At 10 mm DTT concentration, triplet blinking dominates the fluorescence, which leads to patchy images and fast blinking in the transient. b,c) When the DTT concentration is increased to 50 mm and 200 mm, the triplet component of the AC (red) is reduced, while the radical component remains similar (blue). This is reflected in the scans and transients, where the noise caused by fast dynamics is clearly reduced. d) The reductant AA completely depletes the triplet state already at 1 mm concentration, but the dye frequently enters dark radical states.

21 References [1] T. Cordes, J. Vogelsang, P. Tinnefeld, J. Am. Chem. Soc. 2009, 131, [2] T. Ha, P. Tinnefeld, Annu. Rev. Phys. Chem. 2012, 63, [3] J. Widengren, U. Mets, R. Rigler, J. Phys. Chem. 1995, 99, [4] H. D. Kim, G. U. Nienhaus, T. Ha, J. W. Orr, J. R. Williamson, S. Chu, Proc. Natl. Acad. Sci. USA 2002, 99, [5] J. Bernard, L. Fleury, H. Talon, M. Orrit, J. Chem. Phys. 1993, 98, [6] a) S. van de Linde, I. Krstic, T. Prisner, S. Doose, M. Heilemann, M. Sauer, Photochem. Photobiol. Sci. 2011, 10, ; b) G. T. Dempsey, M. Bates, W. E. Kowtoniuk, D. R. Liu, R. Y. Tsien, X. Zhuang, J. Am. Chem. Soc. 2009, 131, [7] W. M. Haynes, Handbook of Chemistry and Physics, 93rd ed., CRC Press, Boca Raton, [8] J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, P. Tinnefeld, Proc. Natl. Acad. Sci. USA 2009, 106, [9] P. Schafer, S. van de Linde, J. Lehmann, M. Sauer, S. Doose, Anal. Chem. 2013, 85,