Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs

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Photoswitching of Green mEos2 by Intense 561- Nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy Daniel Thédié, Romain Berardozzi, Virgile Adam, and Dominique Bourgeois J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b01701 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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The Journal of Physical Chemistry Letters

Photoswitching of Green mEos2 by Intense 561-nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy Daniel Thédié, Romain Berardozzi, Virgile Adam, Dominique Bourgeois*. Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France

ABSTRACT:

Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561-nm light (~1 kW/cm2) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405-nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of

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anthozoan origin is substantially more complex than previously thought, and suggests that intense 561-nm laser light should be used with care, notably for quantitative or fast PALM approaches.


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PhotoActivated Localization Microscopy (PALM) has become a widely used super-resolution technique due to its high performance combined with a relatively simple implementation and because it offers perspectives for quantitative molecular counting.1,2 In PALM, the serial recording of the fluorescence signal from single emitters allows their localization to nanometer accuracy and the subsequent reconstruction of a high-resolution image. The most popular genetically encoded markers suitable for PALM are green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2, Dendra2, mKikGR, mClavGR2, mMaple and their various derivatives.3–5 These fluorescent proteins are all derived from anthozoan species such as reef corals or anemones and bear a histidine as the first amino acid of the chromophoric triad. They emit green light upon illumination with cyan (488-nm) light in their native state, which facilitates preliminary adjustments before PALM data acquisition such as cell selection and sample focusing. However, upon illumination with violet (405-nm) light, a β-elimination reaction occurs that results in a backbone chain break coupled to an elongation of the chromophore conjugated electron system, irreversibly shifting fluorescence emission to orange-red colors (for recent reviews, see ref 6,7). This red-shifted fluorescence may then be read out with yellow/green (561nm) light. Stochastic photoconversion of individual PCFPs and recording of their red fluorescence until photobleaching is thus a central concept in PALM microscopy. Many efforts have been dedicated recently to the engineering of bright and monomeric PCFPs.8–16 However, two major limitations have been found to always remain, seriously compromising the quality of PALM data, notably for quantitative studies aiming at counting molecules, e.g. to assess protein copy-numbers, oligomeric state or clustering tendency.17–20 The first obstacle is that, once photoconverted, PCFPs tend to blink, transiently entering into more or less long-lived dark states.16,21,22 Each PCFP may thus reappear several times along data


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acquisition, causing overcounting errors. Various blinking correction methods have been proposed to minimize such errors23–28 but none of them has been found fully reliable. The second obstacle to quantitative molecular counting is the limited signaling efficiency of PCFPs13. In an ensemble of PCFPs, not all of them fold nor maturate properly, and amongst those that do, only a fraction can be detected in the photoconverted state.13,27,29 Consequently, the number of detected molecules is lower than expected, limiting the effective labeling density and causing undercounting errors. Although reported numbers vary widely13,30, a careful study recently suggested a signaling efficiency of ~60% for mEos2, hinting at the possible role of thus far uncharacterized dark states29. Here, we investigate further the interplay of such dark states with the photoconversion mechanism of mEos2. Green-to-red photoconversion of PCFPs has traditionally been investigated by absorption spectroscopy using 405-nm light as the sole source of actinic light, despite the fact that intense 561-nm illumination is required to localize single molecules in PALM. Although absorption by green mEos2 at this wavelength is expected to be minimal due to a very small extinction coefficient, the common observation of “readout activation” in PALM experiments (i.e. residual photoconversion in the absence of 405-nm light) suggests that 561-nm light does interact to some extent with green mEos2. This prompted us to study the effect of strong 561-nm illumination of this PCFP in its green state at both the ensemble and singlemolecule levels. Thin layers of purified green mEos2 embedded in polyvinyl alcohol (PVA)31 were deposited onto a coverslip, placed on a PALM microscope, and submitted to illumination with either 488nm, 561-nm or 405-nm light (Supplementary Methods). Fluorescence readout was achieved with weak 488-nm excitation light (0.2 W/cm²), using an EMCCD camera (Figure S1). Moderate 488-


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nm illumination (10 W/cm²) was sufficient to rapidly switch off green fluorescence (Figure 1A), accounting for the rapid fading typically observed when cyan light is used to examine mEos2labeled cells prior to PALM experiments. Fluorescence swiftly recovered upon faint 405-nm illumination (0.03 W/cm²), and the process could be repeated several times with only moderate photofatigue (Figure S2), revealing that green mEos2 exhibits the typical behavior of reversibly switchable fluorescent proteins (RSFPs). Much more surprising was the observation that, when submitted to strong 561-nm light (~1 kW/cm²), the green fluorescence of mEos2 decreased in a similar manner (Figure 1B, D and Figure S3). Again, most of the fluorescence could be recovered upon illumination with weak violet light, demonstrating that despite a strongly redshifted wavelength relative to the absorption maximum of green mEos2 (Figure S4A), 561-nm light also induces reversible switching of this PCFP.


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Figure 1. Evidence for dark-state formation in green mEos2 at the ensemble level. mEos2 embedded in PVA (A, B)

or expressed in E. coli BL21 cells (C, D) exposed to 488-nm (A, C) or 561-nm (B, D) light (10 and 2400 W/cm2, respectively). Fluorescence at 525 nm is shown. After fluorescence on-off switching, the samples were illuminated with weak 405-nm light (0.03 W/cm2), causing a rapid recovery of fluorescence. Switching curves are well fitted with the model of Scheme 1 (red curve, on-off switching, and cyan curve, off-on recovery), suggesting that green mEos2 undergoes reversible transitions to two distinct dark-states. Colored bars above each panel are representative of wavelengths used for illumination.

Interestingly, upon switching by either 488-nm or 561-nm light, only little recovery can be observed in the absence of light over a period of hours (Figure S5), suggesting that mEos2 in its switched-off state is highly thermostable. Lowering the pH induces at both wavelengths a decrease of the switching rate, in agreement with the notion that off-switching involves the mEos2 anionic state (Figure S6). The strong sensitivity to violet light of switched-off mEos2, on the contrary, is consistent with a protonated chromophore, possibly twisted or isomerized in a trans configuration.16


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The fluorescence decays in Figure 1A or 1B display a biphasic-like behavior, suggesting a mixture of a reversible phase, due to switching, and of a nonreversible phase, due to either nonreversible bleaching or photoconversion. However, attempts to fit the decays with a corresponding model (Scheme S1A) result in large fractions of the fluorescent protein population ending up in a bleached or photoconverted state (> 50%, Figure S7A, D), a finding inconsistent with the high-level of recovery (> 80%, Figure 1) observed upon subsequent 405-nm illumination. Likewise, imposing a rate for bleaching and/or photoconversion matching this level of recovery did not allow to obtain a satisfactory fit of the experimental data (Figure S7B, E). We checked that the shape of the fluorescence decays was not significantly influenced by spurious effects such as residual diffusion or reduced tumbling of the mEos2 chromophores within the PVA matrix (see Supplementary discussion and Figures S8, S9). In contrast, the data support the hypothesis that, in addition to nonreversible photobleaching/photoconversion, not only one but (at least) two reversible dark states are formed. In fact, two dark states have been previously identified in red mEos2: a short-lived one attributed to chromophore distortion and a long-lived one attributed to twisting or isomerization.16 It is reasonable to assume that similar dark states can be visited by green mEos2. The corresponding kinetic model depicted in Scheme 1 (see also Scheme S1B) provide satisfactory fitting (Figure S7C, F), with rate constants summarized in Table 1.


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Scheme 1. Photophysical scheme representing the states visited by mEos2 during a PALM experiment. Rate constants used to fit the switching curves of Figure 1 are those driving the photophysics of green mEos2, within the green dashed box. The photophysics of red mEos2 have been described in ref 16. hν represents a lightinduced process, while kT stands for a thermal process independent from illumination conditions. Fitting of the switching curves in Figure 1 used a single constant for bleaching and photoconversion (k2 = kbleach, green + kphotoconversion) due to the impossibility to distinguish those two irreversible processes when monitoring only mEos2 green fluorescence.

Interestingly, while the recovery rate of the short-lived dark state Dshort appears essentially light independent, that of the long-lived dark state Dlong increases linearly with the 561-nm illumination power (Figure 2 and Figure S3). This finding is consistent with the notion that the distorted chromophore in Dshort could be sp3-hybridized and not absorb visible light, whereas the protonated chromophore in Dlong still absorbs violet light.32,33 Dshort could correspond to the redox-induced blinking mechanism characterized in green IrisFP, a single mutant of EosFP engineered to display efficient switching in both its green and red states.32,33


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Figure 2. Switching rates of green mEos2 as a function of 561-nm illumination intensity. mEos2 embedded in PVA was exposed to different intensities of 561-nm light. The resulting on-off switching curves were fitted with the model of Scheme 1, and the rates for reversible dark-state formation (k1,long and k1,short), green state recovery (k-1,long and k-1,short) and photoconversion+bleaching (k2) were extracted. These rates all evolve linearly with the intensity of the 561-nm light used during the experiment, except k-1,short, which is thus expected to result from a thermal process. Error bars are standard deviations over triplicate experiments.

Table 1. Summary of the rates fitted with the photophysical model of Scheme 1 for green mEos2 switching in PVA under 488-nm (10 W/cm2) or 561-nm illumination (2400 W/cm2). Rates from ensemble data are fitted from the switching curves of Figure 1A and 1B, while rates from single-molecule data are fitted from the cumulative curve of Figure 3a. Quantum yields for on-off switching at 488-nm excitation could be derived based on the knowledge of the green mEos2 extinction coefficient at this wavelength. Switching “brightness” refers to the product (switching quantum yield × extinction coefficient). Different back switching rates k-1,short were fitted in ensemble experiments at 488 nm and 561 nm, which could be attributed to residual photosensitivity of Dshort or to different temperatures of the samples, possibly due to a heating effect of the 488-nm laser. ND: Not Determined

488 nm Ensemble

Long-lived darkstate

Switching quantum yield φ [-] -1

Switching brightness [M .cm ] -1

-1

1.2x10

-1

1.6x10 -2 (± 3.6x10 )

Switching rate k1, short [s ] -1

Back switching rate k-1, short [s ]

-3

1.2x10 -4 (± 3x10 )

-3

3.0x10 -4 (± 1.3x10 )

-5

-1

-1

Short-lived darkstate

2.6x10 -5 (± 6.0x10 )

1.58

Back-switching brightness [M .cm ]

Singlemolecule

-2

2.6x10

-1

Ensemble 8.7x10 -3 (± 2.1x10 )

1.1x10 -3 (± 1x10 )

-1

Back switching rate k-1, long [s ]

561 nm

-1

1.4x10 -2 (± 3.2x10 )

-1

Switching rate k1, long [s ]

561 nm

-1

1.8x10 -2 (± 2.1x10 )

-2

-3

ND 6.1x10

-4

8.4x10

-4

1.9x10

-4

2.6x10

-4

-2

1.1x10 -3 (± 2.9x10 )

ND

-2

3.8x10 -3 (± 5.1x10 )

ND


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Switching quantum yield φ [-] -1

-1

Switching brightness [M .cm ] Bleaching and photoconversion

k2 [s ]

Photoconversion

kphotoconversion [s ]

3.0x10

-1

-5

1.78 -2

-1

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ND 7.6x10

-4

1.0x10

-3

-3

1x10 -3 (± 5x10 )

1.9x10 -4 (± 1.2x10 )

ND

ND

ND

4.9x10 -5 (± 2.5x10 )

-4

Using the extinction coefficient (ε = 79000 M-1cm-1) at 488 nm reported for mEos2 at pH 7.4,16 quantum yields of Φlong ≃ 2.6x10-5 and Φshort ≃ 3.0x10-5 for off-switching to the long-lived and the short-lived dark-states at this wavelength can be deduced, respectively. This results in an apparent overall switching-off quantum yield for mEos2 at 488 nm of Φ = 5.6x10-5. This value is much smaller than the switching-off yield found for IrisFP (Φ = 0.014), but only five times smaller than the value found for Dronpa (Φ = 3.2x10-4)34, highlighting that EosFP derivatives intrinsically bear substantial switching capacity. Interestingly, the model predicts that, upon prolonged illumination (e.g. 250 s, as in Figure 1A and Figure 1B) the Dshort dark state rapidly depopulates (see Figure S7C, F), resulting in a limited number of molecules in this state (< 10%) at the end of such long exposure. Therefore, in Figure 1, recovery upon subsequent 405-nm illumination essentially proceeds from Dlong and is close to monophasic. Likewise, thermal recovery after prolonged illumination at 488 or 561 nm (Figure S5) is very limited. Comparable recovery rates k405 ≃ 0.25 s-1 after off switching by either 488-nm or 561-nm light were found, consistent with the notion that the same long-lived off state is reached upon illumination at either wavelengths. Following off-switching by 561-nm light at 2400 W/cm2 for 250 s, ~10% of the green fluorescence signal gets irreversibly lost, due to both photoconversion to the red state and photobleaching in the green state. The rate of appearance of the red-state fraction remains however out of reach at the ensemble level, as red molecules get quickly bleached by the strong


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561-nm light and therefore cannot be reliably detected. Thus, single-molecule experiments were required to evaluate the relative contributions of green mEos2 photobleaching and photoconversion by 561-nm light (see below). Overall, it is striking to see that, under illumination by 561-nm light with intensities typical of PALM microscopy, in the absence of 405-nm light, green mEos2 gets largely switched-off in ~250 sec, a time of the order of typical PALM data collection times. Similar photoswitching by 561-nm light was observed in PVA with mEos3.2 and mEos4b (Figure S10A, B), suggesting that the effect is not a peculiarity of mEos2, but rather a common property to all EosFP derivatives. In contrast, the switching propensity of Dendra2 was much less pronounced (Figure S10C), in accordance with recent findings under 488-nm illumination35 or in the red state of this PCFP.16 When expressed in bacterial cells, mEos2 exhibited a photoswitching behavior similar to that observed in vitro (Figure 1C and 1D). However the presence of Dshort was not required to fit the data in a satisfactory manner, suggesting that mEos2 green-state photophysics is dependent on the local environment. This is consistent with the notion that PVA restricts oxygen accessibility and exerts redox effects on organic dyes,36 which in mEos2 may affect the buildup of Dshort. Intrigued by the 561-nm light-induced switching of green mEos2, we turned our attention to single-molecule experiments so as to investigate how switching may affect photoconversion kinetics during PALM data collection. To this aim, we conducted PALM experiments on mEos2 sparsely embedded in PVA so as to obtain precise cumulative curves of photoconverted molecules along time. Such curves can be obtained after reconstruction of the fluorescence trace from each single photoconverted molecule, and correcting for blinking, so as to prevent repeated counting of the same molecule.23,26 In the absence of 405-nm light, “readout photoconversion”


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occurs upon illumination of the sample by 561-nm light, leading to a clear biphasic cumulative photoconversion curve (Figure 3A). Qualitatively, this biphasic curve can be explained by the rapid decay of the green mEos2 reservoir due to off-switching, as shown above, decreasing the photoconversion rate by a shelving effect. On the contrary, in presence of moderate 405-nm light (0.8 W/cm2), the photoconversion rate increases, and the curve is more monophasic (Figure 3B). A similar behavior could be observed for mEos2 expressed in E.Coli (Figure 3C, D). Like for the ensemble data, before attempting a quantitative analysis based on mEos2 photophysics, we eliminated the possible involvement of spurious effects that could cause a deviation from a monophasic growth of the red molecules (Supplementary discussion and Figure S11, S12, S13, S14). Fitting the cumulative curve of Figure 3A with the model of Scheme 1 gives rates that are highly consistent with those measured at the ensemble level (Table 1). Importantly, the difference between the photoconversion rate extracted at the single-molecule level and the overall nonreversible fading of green mEos2 measured at the ensemble level allows to estimate that, under conditions of readout photoconversion, only ~25% of the molecules may eventually reach the red state. Thus, 561-nm readout light contributes significantly to the limited signaling efficiency of mEos2.


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Figure 3: Dark-state formation at the single-molecule level induces a shelving effect during PALM experiments. Cumulative photoconversion curves obtained in PVA (A,B) or E. coli BL21 cells (C,D) under 561-nm readout light only (2400 W/cm2) (A,C) or with additional moderate 405-nm light illumination (~0.8 W/cm2) (B,D). Data are well fitted with the kinetic model presented in Scheme 1 that includes the formation of and recovery from both dark states in the mEos2 green form (red curves). Nevertheless, to prevent overfitting, rates for formation and recovery of Dshort (which has the least impact on the shape of the cumulative curves) were fixed to their values derived from ensemble experiments. Light-grey areas show standard deviations over triplicate experiments. Curves in (C) and (D) are normalized to their highest point, as the numbers of mEos2 molecules in different bacteria significantly differ, preventing a strict comparison.

In the presence of increasing amounts of 405-nm light to prompt photoconversion, as typically performed during PALM data collection, the biphasic character of the cumulative curves progressively gets less pronounced (Figure S15). This observation is fully consistent with rapid back switching of green mEos2 molecules by 405-nm light, strongly limiting shelving, followed by green-to-red photoconversion. However, at constant 405-nm illumination, increasing the readout laser power density increases again the biphasic character of the cumulative curve, in


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line with increased shelving (Figure 4). It is striking to note the overall slow-down of the appearance of red molecules as the power of the 561-nm laser is increased. These data clearly indicate that the advantage of using a highly intense readout laser to speed up PALM data collection is strongly offset by very substantial shelving due to both Dlong and Dshort.

Figure 4. Influence of the 561-nm illumination intensity on cumulative photoconversion curves. Cumulative photoconversion curves of mEos2 embedded in PVA under weak 405-nm illumination (0.03 W/cm2) and varying 561-nm illumination (darker shades correspond to higher power densities: 1200, 2400, 3600 and 4800 W/cm2). Stronger intensities of 561-nm light slow down photoconversion while increasing the biphasic behavior of the cumulative curves, which is particularly visible at short times at high 561-nm illumination intensity (inset).

Violet-light-induced green-to-red photoconversion in PCFPs has been shown to proceed from the protonated cis state of the chromophore.37 As the Dlong dark state visited by green mEos2 also most likely corresponds to a protonated state, one may wonder whether photoconversion may as well proceed from this state. To investigate this hypothesis, we performed 2-color experiments at the single-molecule level on mEos2 embedded in PVA, so as to monitor green “ancestors” of newly appearing red molecules. Similar experiments have been conducted in the case of Kaede38, concluding that a surprisingly low fraction of red molecules that had immediate green ancestors could be detected. In the case of mEos2, we also found that only a very small fraction of


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appearing red molecules (~8%) could be matched with a disappearing green molecule in the same or the preceding frame (Figure S16). This observation could point towards a direct photoconversion path from Dlong. To better understand these experimental data, we simulated 2color experiments in-silico thanks to an in-house made Matlab-based software (Supplementary methods) faithfully reproducing both the experimental parameters and the complete photophysical model of mEos2 depicted in Scheme 1 (in which photoconversion can only proceed from the green state). Analysis of these data resulted in a similarly low detection of immediate green ancestors (8%, Figure S17). Based on the knowledge of the ground-truth behavior of the single molecules, this could be mainly attributed to the rather poor switching contrast of green mEos2 (Supplementary discussion). This analysis, together with the satisfactory fit of our cumulative curves assuming only direct green-to-red photoconversion (Scheme 1), lead us to propose that mEos2 cannot photoconvert from Dlong. Interestingly, during two-color experiments using alternate 488-nm and 561-nm illumination in the absence of 405-nm light, we observed that substantially more red molecules appeared than during one-color experiments where only 561-nm illumination is employed (Figure S18). This provides evidence that 488-nm light, in addition to switching green mEos2 molecules, is able to photoconvert them at a rate that largely exceeds the 561-nm induced readout photoconversion rate. It has been reported in the literature that Dendra2 exhibits 488-nm induced photoconversion, but not mEos2. Our data allows refining this view. We propose that 488-nm induced photoconversion is mechanistically possible in both proteins, but is more pronounced in Dendra2 due to substantially less photoswitching in this protein. This is in accordance with the mechanism of primed photoconversion, observed in Dendra2 to a larger extent than in mEos239,


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which has recently been proposed to compete with reversible photoswitching in the green state of these proteins35. In conclusion, we have shown that green mEos2 is highly sensitive to the excitation light used in localization microscopy. Not only 488-nm, but also strong 561-nm light populates at least two reversible dark states and generate residual green-to-red photoconversion, greatly complicating the kinetics of red molecule appearance in one- or two-color PALM experiments. The sensitivity of green mEos2 to 561-nm light, a wavelength longer than the 0-0 electronic transition of the anionic chromophore, can be assigned to the “hot band” or “Urbach tail” effect40, by which a molecule in a vibrationally excited state can be pumped into an electronically excited state. This effect has recently been put forward as a mean to perform single-wavelength localization microscopy.41,42 Four main consequences follow from our findings: (i) photoconversion kinetics of PCFPs measured at the ensemble level by monitoring absorbance spectra (Figure S4B) are generally not representative of actual kinetics in the conditions of single-molecule localization microscopy ; (ii) absorption of 561-nm (or 488-) and 405-nm light in the green state of mEos2 prior to photoconversion results in substantial photobleaching, contributing to the reduced signaling efficiency noticed for practically all PCFPs.27,29 (iii) dark-state shelving delays photoconversion of mEos2 in proportion to the intensity of the used 561-nm light. This constitutes a strong impediment to video-rate PALM data collection in which high excitation power densities are needed to rapidly collect photons.43 In such experiments, delayed photoconversion combined with increased green-state photobleaching is expected to strongly reduce the effective localized emitter density needed to provide high resolution images. (iv) typical “pre-bleaching” by 561-nm light, often applied to eliminate the fraction of red molecules already present at the start of experiments, results in substantial shelving and photobleaching of


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green molecules, thus altering the kinetics of photoconversion during subsequent PALM data acquisition. The influence of 561-nm light on both the green- and red-state photophysics of PCFPs should be carefully considered when designing specific PALM data collection schemes. Green-state shelving is typically advantageous when addressing high fluorophore densities, so as to stretch in time the available pool of green molecules. An intense 561-nm laser, without 405-nm light, can be preferred in such case, considering that this will minimize the lifetime of photoconverted red molecules. However, a high 561-nm laser intensity will also increase readout photoconversion, in addition to causing photobleaching and phototoxicity in living cells. In contrast, to perform quantitative molecular counting, a low 561-nm intensity should be preferred to minimize the bleaching of green and red fluorophores and thus maximize the signaling efficiency. Due to their reduced switching propensity in both the green and red states, we suggest that PCFPs of the Dendra2 sub-family16 are more suitable for quantitative investigations. Future studies will reveal the exact nature of the dark states visited by green mEos2 and other PCFP variants currently employed in super-resolution microscopy. One may foresee that PCFPs in general intrinsically bear the combined switching and photoconversion properties initially discovered in IrisFP.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Supplementary methods, Supplementary discussion, Supplementary figures S1 to S17 (PDF) AUTHOR INFORMATION


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Corresponding Author *Dominique Bourgeois ; Tel : +33 (0)4 57 42 86 44 Email: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS We thank Joël Beaudouin for insightful discussions and Michel Sliwa for pointing to us publications describing the Urbach tail effect. This work used the platforms of the the Grenoble Instruct-ERIC Center (ISBG : UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).

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Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs

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