All-optical strategies to minimize photobleaching in reversibly switchable fluorescent proteins

Domain: physics
Published: 2025-12-01
Editorial outcome: major_revision

Abstract
Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Introduction
Reversibly switchable fluorescent proteins (RSFPs) have advanced the field of fluorescence microscopy by enabling new super-resolution imaging1–7 and spectroscopic approaches8,9, as well as allowing for kinetic-based imaging multiplexing10–13. The switching mechanism in RSFPs involves a light-induced transition between two distinct molecular species, an emissive (on-state) and a non-emissive (off-state). The nature of the photoswitching is generally associated with a cis/trans isomerization of the chromophore followed by a protonation/deprotonation14–18. Due to the stable off-state, it is possible to create enough sparsity to use RSFPs in single-molecule localization microscopy5,19 and super-resolution optical fluctuation imaging (SOFI)20,21, while the reversible nature of the switching mechanism makes them compatible with coordinate-targeted techniques, in particular, reversible saturable optical fluorescent transition 1 (RESOLFT) and non-linear structured illumination microscopy3 (NL-SIM). The diversity in rates of the off-switching kinetics associated with different variants has been used in imaging multiplexing at the cellular and subcellular levels11–13,22. In fluorescence-based spectroscopic techniques, such as fluorescence anisotropy, the long lifetime of the on-off states of RSFPs enables the extension of the mass limit inherent to conventional fluorescence anisotropy and allows for measuring the rotational diffusivity of molecules and complexes with larger masses and hydrodynamic radii8.

Regardless of the approach, the ability of RSFPs to withstand more than thousands of photoswitching cycles defines the resulting image quality. The progressive loss of fluorescence upon cycling is referred to as photoswitching fatigue and reflects the photobleaching profile of the fluorescent protein. In a broader sense, photobleaching results from the interplay between the local nano-environment of the probe and its complex photophysics upon excitation; thus, efforts to mitigate photobleaching in microscopy strived to optimize the conditions on those two fronts. Regarding the former, multiple studies have shown that refining the composition of the imaging solvent improved the photostability of the probe23–25. Fusing an enhancer nanobody to proteins of the rsGreen family increased their brightness and photostability26. At the same time, the development of RSFPs as fluorescent markers has been guided by recursively introducing mutations that favor faster and more robust photoswitchers27–29.

Studies in enhanced green fluorescent protein (EGFP) identify the triplet state as the gateway for photobleaching, being the leading pathway for oxidative chemistry in the chromophore30. Recent studies have characterized the triplet lifetime of rsEGFP2 as well as its absorption spectrum. The properties of the triplet state can be leveraged to reduce the photobleaching of fluorescent proteins31 (FPs). Modulation of the illumination light at a low repetition rate compared to the triplet lifetime efficiently extends imaging time32. Furthermore, given that the long-lived triplet state of FPs has a red-shifted absorption spectrum maximum compared to the fluorescent form, co-illuminating in this spectral window can promote the depopulation of the triplet state back to the ground singlet state. The major photobleaching pathway becomes less efficient, enabling longer time-lapse imaging31. Although optically-induced repopulation of the emissive state from the triplet has been known for years32,33, and even used in imaging with organic dyes34,35, this technique has not been applied to photobleaching reduction until recently31. NIR co-illumination has been demonstrated to reduce photobleaching up to 3–4 times for non-switchable FPs, such as EGFP, under widefield illumination (power densities between 1–100 W/cm2 and illumination time of 15–50 ms).

Here, we characterize the main pathways underlying photoswitching fatigue in RSFPs and identify strategies to minimize photobleaching. A mechanistic understanding of the photocycle enables us to maximize the fluorescence output of rsEGFP2 by modulating the on-switching dose. Furthermore, by co-illuminating the probes with orange/NIR light, we demonstrate a reduction in photoswitching fatigue across a library of green-emitting RSFPs. Based on this photophysical investigation, we propose a strategy to extend confocal and super-resolution time-lapse imaging, as well as multiplexing approaches. The optimized imaging scheme, which incorporates patterned light at different wavelengths, enables prolonged time-lapse imaging of light-sensitive organelles.

Results
Characterization of photoswitching fatigue in rsEGFP2
To study the fatigue in RSFPs, we focused on the negative photoswitcher rsEGFP236. This RSFP is a common marker for super-resolution imaging36–38, multiplexed bio-imaging11,12, as well as novel fluorescence anisotropy methods8. Illumination at 405 nm drives the off-to-on transition (on-switching), whereas 488 nm light excites rsEGFP2 and returns it to the off state (off-switching), inherently coupling illumination time and power density (Fig. 1a). As a first step, we quantified the loss of fluorescence as a function of illumination power density at these key wavelengths (405 and 488 nm; Fig. 1b, c, Supplementary Fig. 1).

We homogeneously illuminated a region (20×20 μm2) of a thin layer of rsEGFP2 embedded in polyacrylamide (PAA) gel with consecutive on- and off photoswitching cycles. The emitted fluorescence signal during each off-switching phase was recorded and the photoswitching fatigue curve was built by repeating the photocycle hundreds of times (Fig. 1a). The resulting fatigue curve for rsEGFP2 is characterized by two distinct components: (i) an initial drop, defined as a sharp loss of the fluorescence signal in the first few cycles, and (ii) a steady decrease over thousands of cycles, which we refer to as the fatigue fraction. An increase in the 405 nm light dose decreased the observed initial drop, corresponding to a higher fraction of on-switched fluorophores per cycle (Fig. 1a, b). Similarly, the fatigue fraction also increased at higher 405 nm, as well as 488 nm, illumination when monitoring two orders of magnitude in power density for both illuminations (~0.01 to ~1 W/cm2, Supplementary Fig. 1). We chose to study the bleaching as a result of a complete photoswitching cycle, thus we tuned the off-switching illumination time to match 80% decrease of the fluorescence signal (Fig. 1a). This choice is grounded in the general use of RSFPs, where the system is typically reset at the beginning of each cycle, allowing a comparison across power density values and among different RSFPs. The role of 405 and 488 nm illumination on bleaching is fundamentally entangled and depends on the balance between the pathways they predominantly drive. The 488 nm excitation light defines the access to the triplet state and, consequently, bleaching from it. The 405 nm light contributes to photoswitching fatigue through both an indirect effect, as it increases the on-switched population and thereby promotes triplet-mediated photobleaching, and a direct effect through its own absorption, which leads to photobleaching.

The photocycle of rsEGFP2 has been extensively studied by means of spectroscopy and crystallographic methods36,39, and the intermediate steps of the photocycle and their rates are well-reported in the literature8,14–16. Using this information, we built a minimal effective kinetic model for rsEGFP2 that could aid the mechanistic explanation of the observed photoswitching fatigue behavior (Supplementary Note 1). We considered the photocycle of rsEGFP2 as a 5-state kinetic model where the photoswitching reaction occurs from a light-induced isomerization followed by a protonation/de-protonation process. The thermodynamically stable cis anionic (C-) form is the emissive form of the chromophore. From there, the on-to-off photoswitching occurs via an excited state isomerization (cis-to-trans) reaction, followed by a ground state process to the trans neutral (TH) form40,41, with a characteristic time of ~50 μs at physiological pH as revealed by high-power time-resolved off-switching experiments8. To recover the C- form of the chromophore, the off-to-on transition is efficiently triggered by violet light, typically 405 nm, from TH. Similarly, the excited state isomerization (trans-to-cis, in this case), to the cis neutral (CH) form, is followed by the deprotonation of the chromophore. As previously reported14–16, the off-to-on photoswitching process can be described as a cascade of intermediates at different timescales, ranging from a few μs-to-ms. In the investigated experimental setting, we have identified an on-like intermediate, CInt, with a lifetime of ~1 ms and therefore compatible with the time resolution explored in this study and generally used for advanced imaging application. The detectable fluorescence suggests it relates to an anionic form of the chromophore41 and places it as the major gateway for bleaching pathways that stem from the on-switching process (Supplementary Notes 2, 3).

For rsEGFP2, and in general for all RSFPs, the off-switching provides a competitive channel to the triplet formation via intersystem crossing (ISC), which, combined with the spectral shift between on and off states, inherently shields the fluorophore from the adverse effects of prolonged excitation and decreases the probability of triplet-state accumulation. The triplet shielding introduces differences in the photobleaching profile of RSFPs and non-switchable fluorescent proteins. Given a constant 488 nm illumination, the former will reach a lower level of steady-state triplet population due to the competition with the off-switching process. RSFPs will converge back to the FPs photobleaching behavior in the case of constant and simultaneous 405 and 488 nm illumination, whereas a substantial steady-state on-population can be reached.

We incorporated the triplet-mediated photobleaching pathway in our kinetic modeling of rsEGFP2 (ii in the simplified scheme of Fig. 1e) and simulated the fatigue fraction response over the explored experimental conditions. As shown in Fig. 1f, the experimental data are well supported by the simulation for 405 nm power densities below 300 W/cm2 and diverge hereafter. The experimental behavior suggests an additional photobleaching pathway stemming from the off-to-on transition (ii in Fig. 1f, e, Supplementary Note 4). The need for an additional photobleaching channel is further supported by the dependence of the fatigue on the waiting time between sequential cycles (Fig. 1g). Despite leaving time for the triplet population to relax, the fatigue can never be recovered fully, indicating that not all the irreversible fluorescence loss stems from a single mechanism (i.e., the triplet-based photobleaching). Increasing the dwell time from 10 to 100 ms resulted in ~20% photoswitching fatigue recovery after 1600 cycles (Fig. 1g). The time dependence of such relaxation exceeds the expected triplet state lifetime, both reported42 and measured in our data, suggesting that the triplet state acts as a precursor to longer-lived photoproducts, which lead to reversible and irreversible photobleaching (Supplementary Note 5). Increasing the interval between successive photocycles prevents the accumulation of these intermediate long-lived dark states.

Maximizing on-off cycles in rsEGFP2
The global photon budget of each RSFP is set by the combination of the initial drop of the fluorescence and the number of cycles the probe can undergo. Supported by the outlined kinetic model, we investigated strategies to maximize the detected fluorescence photons by modulating the illumination.

The initial drop in the fluorescence signal arises from the kinetics of the on-switching mechanism in rsEGFP243. At low power, this drop is a consequence of the 405 nm absorption of C-43. However, under the investigated power densities, both the kinetics and absorption properties of the intermediate state CInt account for an additional loss (Supplementary Note 3). At pH 7.5, rsEGFP2 is expected to have ~20% of the concentration of the fluorophore trapped in TH (Supplementary Notes 2, 3). The formation of the TH state can be mitigated by extending the on-switching times at a fixed total energy, which reduces CInt accumulation and lowers the probability of CInt-to-TH conversion, a transition promoted by 405 nm absorption. Experimentally, a constant dose of 64 mJ/cm2 was delivered by progressively increasing the illumination time (0.3–20 ms) while proportionally decreasing the 405 nm power density (200–3.3 W/cm2) as shown in Fig. 2a. Under these conditions, the fluorescence loss was reduced to ~7% when the activation dose was stretched to 20 ms. Figure 2a, b shows the enhancement for both rsEGFP2 embedded in the PAA layer and linked to actin in eukaryotic cells. Our simulations confirm that this is compatible with preventing the quasi-equilibrium among the on-switching intermediates (Supplementary Note 6) and, therefore, a more efficient turnover between the on and off-states upon 405 nm irradiation. Altogether, we found that the initial drop observed in the photoswitching fatigue curves is not irreversible and can be efficiently recovered by modulating the way the on-switching light dose is delivered.

Reverse inter-system crossing (RISC), i.e., the backward process from the triplet state to the singlet manifold, was shown to be optically modulated and thereby effectively increasing the brightness of organic dyes32 and preventing photobleaching in EGFP31. Since the protein scaffold shields the chromophore from the immediate local environment, the triplet lifetime in FPs is relatively long (~5 ms for EGFP30) and RISC transition can be triggered with lower light doses compared to dyes (0.1–40 MW/cm2 for Eosin Y32, compared to ~kW/cm2 for GFP-like proteins31,44). We measured the time evolution of a dark state relaxing in the μs-ms window by monitoring the increase in the integrated fluorescence signal between two 488 nm pulses separated by a variable delay. We observed a build-up in the signal of the second pulse with a raising time of ~2.5 ms (Fig. 2c, Supplementary Note 7). Since the thermal recovery for rsEGFP2 (thermal relaxation from TH to C-) has a timescale of several hours39, the increase in the concentration of the fluorescence emissive state is likely to originate from the relaxation of a dark state created upon excitation (Fig. 2d). The timescale of such relaxation is comparable to the reported triplet state lifetime in rsEGFP2 at 260 K42.

To see if and to which extent the optically-induced RISC could help mitigate the photoswitching fatigue of RSFPs, we used the same experimental framework of the previous section with a modified pulse scheme which included 592 nm co-illumination (Fig. 2e). At 592 nm the absorption cross-section for the triplet state of rsEGFP2 was reported to be ~20% of the peak at 900 nm at 100 K42. We examined the effect of 592 nm co-illumination as a function of power density (~0.5–8 kW/cm²; Fig. 2f) and of the delay time relative to the 488 nm excitation pulse (Fig. 2g). Introducing 592 nm co-illumination into the pulse scheme led to a reduction in photoswitching fatigue (Fig. 2e), with recovery increasing by up to ~15% at higher power densities. When 592 nm co-illumination was applied, fluorescence signals were restored to similar levels regardless of the 488 nm power density (Supplementary Note 7), indicating that only the triplet-mediated photobleaching fraction is optically recoverable. We also observed a correlation between the magnitude of recovery and the 488 nm excitation power, with light-induced recovery being more pronounced at higher 488 nm power densities (Fig. 2f). Since RSFPs are off-switched with an near-constant energy dose (power density × illumination time), a higher 488 nm power density shortens the exposure time, thereby maximizing the efficiency of 592 nm-mediated recovery. Regarding the delay between the 488 nm and 592 nm pulses (Fig. 2g), we observed that the magnitude of the recovery decays over a timescale of milliseconds, consistent with the reported lifetime of the triplet state42.

To provide further insights into the photoswitching fatigue mechanism, we fitted our experimental data to the developed kinetic model (Supplementary Note 1), which covers the switching mechanism of rsEGFP2, the photobleaching pathways described in the previous section, as well as the spectroscopic properties of the triplet state, parametrized from the reported data in this manuscript and published studies42. Similarly to recent literature on light-induced RISC in EGFP31 and rsEGFP245, we rationalize the repopulation of the emissive state via RISC from the triplet excited state (T1) to a vibronic state of the singlet manifold (Supplementary Note 1 and 8). All the different wavelengths explored (405, 488, 592, 915 nm) will excite the triplet state42 stimulating both photobleaching and RISC. Upon fluorescence excitation, both T1 and higher triplet states contribute to photobleaching (Fig. 2c), and co-illumination with a 592 nm decreases the probability of photobleaching by reducing the triplet state population. The proposed kinetic model incorporates effective photobleaching quantum yields for ground and excited triplet states, ΦT1 and ΦTn respectively (Fig. 2d). The addition of a photobleaching path from Tn was necessary for reproducing the experimental fatigue curves and not to underestimate the experimental photobleaching. Such a pathway is seemingly relevant since the triplet state of rsEGFP2 presents an absorption peak at 488 nm42, and the triplet state absorption has been identified as a photobleaching promoter in EGFP31.

Overall, the kinetic model is consistent with the data, further supporting that the photoswitching fatigue recovery stems from an optically-induced depopulation of the triplet state. We used the simulation tool to expand the observation over an extended interval of 405, 488 and 592 nm power density (Fig. 2h-i, Supplementary Note 9). In the simulated photoswitching fatigue experiments, the 592 nm illumination time was kept constant at 1 ms and overlapped in time with the 488 nm illumination pulse. Our simulations suggest that the triplet state can be depopulated effectively by co-illuminating with 592 nm (~kW/cm2), with the recovery being more evident at higher 405 nm power densities since a relevant fraction needs to be on-switched to yield enough C- concentration and accumulate triplet state (Fig. 2h). Similarly, the photoswitching fatigue recovery is more pronounced at higher 488 nm excitation power densities. In rsEGFP2 at high 488 nm power densities, the off-switching pulse length can be much shorter than the triplet lifetime (<0.5 ms for 488 nm power densities >600 W/cm2, top half of Fig. 2i). In such conditions, the transient triplet population is effectively depleted during the 592 nm only period, generating the most photoswitching fatigue recovery. The absorption of 592 nm light will not generate additional triplet via ISC but mainly depopulate the transient T1 as shown in the scheme in Fig. 2d. Nonetheless, the photoswitching fatigue recovery reaches a plateau at 488 nm illumination power densities higher than 600 W/cm2 and co-illumination of 592 nm power densities above 10 kW/cm2 which indicates that the triplet state formation and RISC processes reach saturation under the experimental illumination parameters.

Shifting the red co-illumination toward the triplet-state absorption peak at 900 nm enhanced recovery (Fig. 2j). Furthermore, probing across the near-infrared range revealed that recovery efficiency is wavelength-dependent and closely follows the triplet absorption spectrum (Supplementary Note 10). Notably, 810 nm, where the triplet absorption coefficient is similar to that at 592 nm42, resulted in less recovery than 900 nm, comparable to the difference observed between 592 nm and 915 nm. The increased recovery at ~900 nm is attributed to both stronger triplet-state absorption and reduced crosstalk with other photobleaching pathways, such as photobleaching from the on-switching intermediates (Fig. 2g, Supplementary Note 10).

Photoswitching fatigue in other green-emitting RSFPs
We extended our investigation of the fatigue resistance to other RSFPs, focusing on green negative photoswitchers widely used in multiplexing10–13 and super-resolution microscopy28,38,46,47. We observed a correlation between the off-switching speed of the RSFP and the experienced photoswitching fatigue (Fig. 3a, Supplementary Note 11), with slower photoswitchers (in particular, rsEGFP(N205S) and rsGreen1) being more prone to photobleaching upon cycling than their faster switching counterparts (rsEGFP2 and rsGreenF, respectively). The differences in photoswitching fatigue among the screened RSFPs can be rationalized by a less efficient off-switching process, which leads to longer illumination times per cycle and, consequently, a higher probability of triplet-state formation. Assuming the triplet absorption spectrum is similar to that of rsEGFP2, the strong absorption of the T1 state at the excitation wavelength results in an increase of photobleaching (Supplementary Note 11).

When comparing RSFPs, each exhibits a unique switching fatigue profile, therefore we set to explore whether fatigue can serve as an additional multiplexing dimension10–13. Multiplexing strategies generically refer to any method that aim to distinguish RSFPs that emit in the same spectral range by analysing fluorescence signal changes under specific illumination sequences. Methods like LIGHTNING11 and TMI12 resolve the kinetic rates of light-induced transitions, while approaches such as exNEEMO10,13 capture fluorescence intensity changes in response to varying on-switching sequences. Bringing both approaches to high spatiotemporal resolution (e.g., confocal imaging with excitation ~100–500 W/cm2) is difficult, since these techniques rely on the ability to clearly distinguish the off-switching kinetics, either as a fully sampled decay for LIGHTNING and TMI, or as integrated fluorescence for exNEMO (Supplementary Note 12).

To overcome this limitation, we tested photobleaching dynamics as an additional dimension to aid multiplexing strategies, resolving ambiguities under high irradiance. We designed a pulse scheme with four acquisitions per pixel, varying the on-switching energy dose to produce distinct fluorescence responses for each of the four proteins (Fig. 3b). At each pixel, the sample undergoes the pulse sequence in Fig. 3b, with the 4th frame normalizing the recorded intensity of the first three, similar to exNEEMO10,13. To account for photobleaching, 60 frames, 15 unmixing images, were collected for differently labeled bacteria, and the fluorescence loss was calculated. Data for each RSFP were fitted to a Gaussian model in a 4-dimensional space (Fig. 3c), including normalized intensities from the first three image stacks and fluorescence loss. To evaluate the unmixing method, all identified bacteria were reanalyzed using a Gaussian mixture model containing the information of the individual proteins. This pulse scheme is compatible with a parallelized confocal microscope, allowing the acquisition of unmixing images with ~0.5 Hz temporal resolution and over an extended field of view (FOV) ~40×40 μm2, distinguishing four RSFPs with high accuracy (Fig. 3b). This approach demonstrates that tracking the photoswitching fatigue in samples with multiple RSFPs enables accurate identification of individual markers.

We then investigated if the reduction of fatigue by 592 nm co-illumination is a general phenomenon across various RSFP variants. Our data show that rsGreen1 and rsGreenF had a significant recovery of photoswitching fatigue upon co-illumination with 592 nm light (Fig. 3d). We also measured the recovery as a function of the delay between 488 nm and 592 nm illumination pulses (Supplementary Note 11). The recovery magnitude decreased as the delay between the pulses increased, indicating that the effect stems from triplet-state depopulation, consistent with the behavior observed in rsEGFP2. Furthermore, fatigue recovery due to thermal relaxation was reduced in slower photoswitchers (Supplementary Note 11), suggesting that the maximum recoverable fraction is lower than that of faster variants. The differences in photoswitching fatigue behavior are likely due to diminished triplet-state shielding and, consequently, enhanced triplet-mediated photobleaching.

Prolonged time-lapse imaging at high spatiotemporal resolution with RSFPs
Motivated by our findings in a controlled environment, such as PAA gel or bacteria expressing the different RSFPs, we investigated the strength of the phenomenon in living cells to achieve prolonged time-lapse imaging in different modalities.

Taking advantage of our parallelized confocal microscope37,48 we imaged the mitochondrial outer-membrane-protein-25 (OMP-25) labeled with rsEGFP2 in U2OS cells. In this configuration, a spatially patterned 405 nm illumination created by a micro-lens array (MLA) will on-switch fluorophores at multiple focal spots in the xy-plane of the sample while the fluorescence is interrogated with another identical and spatially overlapped pattern of 488 nm light. To form an image, the sample is scanned between consecutive focal spots with a step size tuned depending on the target resolution. Sampling according to the Nyquist criterion, in a confocal image, the average cycling of every protein is less than 10 times. We introduced the 592 nm co-illumination simultaneously with the fluorescence readout step, co-aligning a 592 nm illumination with MLA to trigger the optically-induced depopulation. Our data show a decrease in photobleaching after 500 frames with ~10 kW/cm2 of 592 nm co-illumination (Fig. 4a). As shown in Fig. 4a, the features of the mitochondrial network are better preserved after hundreds of imaging frames when the 592 nm pulse is added. Moreover, we compared photobleaching of timelapses with similar initial signal-to-background ratio (mostly dependent on the transfection efficiency) and observed that photobleaching was slower with the 592 nm co-illumination (Supplementary Note 13). Our results show that optically-induced depopulation of the triplet state can prolong time-lapse optical imaging of living cells.

To explore the benefit of red co-illumination in multiplexing imaging, we imaged bacteria expressing two RSFPs in confocal modality, rsGreen1 and rsGreenF. The nature of the protein can be identified by resolving the off-switching decay of the proteins, like in TMI12 (Fig. 4b), and the global fluorescence is monitored over 50 frames. Without red co-illumination, the weaker RSFPs set the absolute number of frames of acquisition, while the addition of the 592 nm co-illumination extend the absolute observation window for the multiplexing imaging by 20% to 30% (Fig. 4b). Our data show that 592 nm co-illumination can be a powerful aid to extend in time high-resolution single-channel multiplexed imaging with RSFPs in combination with existing unmixing methods.

We further tested the feasibility of the method for super-resolution imaging recordings, specifically in parallelized RESOLFT nanoscopy37,48. In the parallelized confocal geometry, the nanoscale spatial confinement is achieved by off-switching the RSFPs located in the periphery of each focal spot with a grid-like pattern generated by interfering waves. The parallelization of the imaging scheme minimizes the illumination intensities without compromising the overall frame time. As with any other RESOLFT or NL-SIM imaging scheme, photoswitching fatigue represents the ultimate limit to the maximum number of frames which can be recorded.

Similarly to the parallelized confocal imaging experiments, the 592 nm illumination is delivered simultaneously and co-aligned to the fluorescence readout step (Fig. 4c). As the majority of the triplet population will reside in the center of each focal spot, the optically-induced depopulation will be more effective in this position. We observed a decrease in the photobleaching fraction after 20 frames in U2OS cells endogenously expressing vimentin-rsEGFP2 with an increasing 592 nm illumination (Supplementary Note 13) as well as in U2OS cells expressing LifeAct-rsEGFP2. Using the same pipeline, we observed ~20% reduction in the photobleaching fraction after 20 frames (Fig. 4c) by adding the 592 nm co-illumination, which competes with reported chemically-based strategies for reducing photobleaching25,26. As shown in Fig. 4c, co-illumination with 592 nm helped in maintaining the signal-to-noise ratio (SNR) during the time-lapses. Detailed features of the actin network of the cells were still visible after 20 frames (Fig. 4c). Similarly, we transfected U2OS cells with LifeAct tagged to other green negative photoswitchers and evaluated the effect of the 592 nm co-illumination in the same manner (Fig. 4d). Our data in cells correlates well with our findings in the PAA gel for the different RSFPs, showing that 592 nm co-illumination can be a general strategy for reducing photoswitching fatigue in other reversibly switchable fluorescent proteins.

Additionally, we monitored whether the 592 nm co-illumination induced any phototoxicity in the cells. An assay using the DNA-repairing protein XRCC1 (X-Ray cross-complementary factor 1)49 did not report additional DNA photodamage from the 592 nm illumination in the cells (Supplementary Note 14). Similarly, the mobility of mitochondria was assessed as a phototoxicity parameter. Both datasets, with and without 592 nm co-illumination, were inspected for spontaneous events, such as branching, fission, fusion, long stretching and long retraction, and a timestamp of their occurrence was annotated. Our data show the similarity in the cumulative distribution of events with and without 592 nm co-illumination (Supplementary Note 14), suggesting no adverse effects arise from the additional 592 nm illumination regarding mitochondria mobility.

Methods
RSFP characterization in PAA gel
All RSFP characterization experiments were done by embedding the protein of interest in PAA gel. To prepare a sample, a few μl (depending on the RSFP) of purified protein were diluted in phosphate buffer for a total volume of 42.0 μl. The protein solution was mixed with 30.0 μl acrylamide (Rotiphorese Gel 30, Roth, Germany), 0.75 μl 10% ammonium persulfate (Ammonium peroxydisulfate ≥98%, Roth, Germany) and 1 μl 10% TEMED (15524-010, Invitrogen). The final solution was thoroughly mixed by vortex, and a small volume (10–20 μl) was placed on a glass slide (#1.5), and a coverslip was pressed onto the sample to produce a thin layer. After complete polymerization, the sample was sealed with silicon-based glue (Picodent Twinsil, Picodent, Germany) to prevent drying out.

Before each photoswitching fatigue experiment, characterization of the off- and on-switching kinetics of the sample was performed. The off-switching time parametrization was done by averaging 25 off-switching profiles recorded in a small FOV (~1×10 μm2) at different 488 nm illumination (~10–1000 W/cm2). For a given 488 nm power density, the characteristic off-switching time is such that the signal has decayed to 20% of the initial value. To characterize the on-switching response, a 20×20 μm2 FOV is consecutively photoswitched with increasing 405 nm illumination (~10–1000 W/cm2). At each level of 405 nm light, the camera is exposed for the calibrated 488 nm off-switching time, and the measurement is repeated 9 times. The mean of the integrated fluorescence for each cycle, with its standard deviation, is measured and plotted against the 405 nm power density.

To record a photoswitching fatigue experiment, a 20×20 μm2 FOV underwent multiple photoswitching cycles (~1600–2200 depending on the experiment). The camera exposure time was matched to the 488 nm pulse width, and the integrated fluorescence signal was collected for that photoswitching cycle. At the onset of the new cycle, a 1 ms dose of 405 nm light reset the majority of the ensemble population back to the emissive state. Additionally, a 592 nm wavelength was incorporated into the pulse scheme. The typical pulse dwell time was 10 ms, but was adjusted depending on the 488 nm power density.

The nonlinearity of the on-switching process in rsEGFP2 was tested by modifying the delivery of the 405 nm illumination with different combinations of power density and illumination times while keeping a constant total energy of 64 mJ/cm2. The total dose of 64 mJ/cm2 was chosen by previously calibrating the sample to a non-switching ramp with a 1 ms 405 nm illumination time. The same area of 20×20 μm2 was repeatedly exposed to 25 on-off photoswitching cycles for every combination of 405 nm power densities (~3–200 W/cm2) and illumination times (20–0.3 ms). The fluorescence signal was captured by the camera during the 488 nm illumination. For a given experiment, the FOV is initially illuminated with only 488 nm; the fluorescence retrieved in this initial cycle is considered to represent the true concentration of fluorophores in that volume. The fluorescence output for every on-switching condition is normalized to the integrated signal in that initial photoswitching cycle for every FOV. Each data point was built by averaging 20 of the 25 photoswitching cycles for a given condition. The experiment was repeated in 4 different areas of an rsEGFP2-embedded PAA gel.

Moreover, to characterize processes with a higher temporal resolution, a confocal system equipped with an avalanche photodiode (APD) detection system was used. To examine the on-switching mechanism, a burst (5 μs) of high-power 405 nm light (~30 kW/cm2) triggered the on-switching transition, after a variable delay (5 μs–50 ms), the subsequent fluorescence signal was readout with 488 nm illumination at ~40 kW/cm2. The area below the off-switching curve was calculated and plotted against the variable delay. Similarly, we investigated the relaxation of a dark state formed upon 488 nm absorption from the emissive state. In this case, a 5 μs 405 nm burst (~30 kW/cm2) was used as the on-switching light dose, after a 3 ms delay—to ensure full relaxation to the emissive state—a first 488 nm dose (500 μs at ~40 kW/cm2) was delivered to the sample, enough to decrease the observed signal down to 80–90% of the initial value. After that, a second 488 nm pulse with the same illumination dose was delivered. The delay between consecutive 488 nm pulses systematically varied between 5 μs and ~500 ms. The area below the second off-switching curve was compared to a control curve, where no delay existed between pulses, and plotted against the delay time. All the experimental parameters are reported in Supplementary Table 9.

Optical set-ups for characterization experiments
The photoswitching fatigue experiments, as well as the characterization experiments, were carried out on a modified version of the set-up reported here48 (Supplementary Fig. 28). In brief, 3 different wavelengths were combined in a WF microscope configuration with a high numerical aperture (NA) objective (HCX PC APO 100×/1.40 NA oil-immersion; Leica Microsystems). Photoactivation and fluorescence excitation were carried out using modulated laser diodes, 405 nm (200 mW, 06-01, 405 nm; Cobolt, Hübner Photonics) and 488 nm (200 mW, 06-01, 488 nm; Cobolt, Hübner Photonics) respectively. To test the photoswitching fatigue recovery, two longer wavelengths were incorporated, 592 nm (DEMO-2RU-VFL-P-2000-592; MPB Communications) modulated by an acousto-optic tunable filter (AOTF, AOTF-nC-VIS-TN; AA Optoelectronics) and a modulated laser diode 915 nm (250 mW, 06-MLD, 915 nm, Cobolt). The emitted fluorescence was directed to the detection path with a dichroic mirror (ZT405/488//640/775RDC; Chroma) and later to a scientific CMOS (sCMOS) camera (ORCA Fusion C14440-20UP; Hamamatsu). In the microscope, the light doses were digitally modulated by transistor-transistor logic signals with an NIDAQ PCI 6371 (National Instruments) acquisition device. The NIDAQ instrument synchronized the pulse sequence and the camera readout. The length and delay of the laser pulses, the output power of the lasers, the exposure time of the camera, and all other relevant parameters can be controlled through ImSwitch50. For sample screening, we added a wide-field path, which we could alternate with the main WF illumination by flipping a motorized mirror. The power densities of the different beams were calculated by measuring the power at the objective back-aperture, and the area was computed by fitting a Gaussian function to the beam profile imaged on an Alexa-Fluor 488 thin layer, for 405 and 488 nm beams, and Abberior STAR RED, for 592 nm beam.

Experiments in which higher temporal resolution was required were performed using the confocal microscope configuration shown here8 (Supplementary Fig. 29). In brief, 405 and 488 nm lasers were combined and tightly focused to a diffraction-limited volume with a high NA objective (APO 100×/1.40 NA oil immersion; Leica Microsystems). Photoactivation and fluorescence excitation were carried out using modulated laser diodes, 405 nm (125 mW, 06-01, 405 nm; Cobolt, Hübner Photonics) and 488 nm (70 mW, 06-01, 488 nm; Cobolt, Hübner Photonics) respectively. The 405 nm polarization was controlled with achromatic λ/2 and λ/4 mounted on remotely controlled rotating stages (Thorlabs) and set to circular polarization. To record an experiment, the microscope incorporates a xy galvo scanning system (Cambridge Technology) to scan the light beams across the FOV. The emitted fluorescence is later de-scanned and directed to a pair of APDs (Micro Photon Devices). The counts recorded in both detectors are summed together later in the data treatment. The microscope components were controlled using a field-programmable gate array card (FPGA, PCIe-7852, National Instruments) and custom-designed software in LabVIEW. The power densities of the different beams were calculated by measuring the power at the objective back-aperture, and the area was computed by calculating the FWHM of the beam’s PSF from a gold-bead sample. The schematic of the microscopes, as well as a list of the optical components, is reported in Supplementary Note 15.

The experiments characterizing the photoswitching fatigue recovery effect of near-infrared (NIR) triplet absorption on rsEGFP2 photoswitching fatigue were performed on an epi-illuminated, commercial confocal laser scanning microscope (Olympus FV1200). The setup was integrated by a combination of elements from two previous works51,52 (Supplementary Fig. 30). The PAA-embedded rsEGFP2 was on-switched by a train of 405 nm (LDH-D-C-405, PicoQuant GmbH, Berlin) picosecond pulses extending for 1 ms at a 80 MHz repetition rate and an average power density of ~300 W/cm2 at the sample plane. After a 1 ms delay, the confocal volume was illuminated with a train of picosecond 488 nm (LDH-D-C-485, PicoQuant GmbH, Berlin) pulses spanning 1 ms at the same repetition rate as the 405 nm laser and an average power density of ~14 kW/cm2 at the sample plane. The diode lasers were controlled via a laser driver (Sepia PDL 828) with a repetition rate of 80 MHz. Since the pulse width of the laser was less than 100 ps, a train of pulses constituted a larger pulse. The pulse trains obtained for the final measurements are as mentioned, along with the optical setup schematic in Supplementary Note 15. After a 10 ms period of no illumination, the photoswitching cycle was repeated again for ~5000 cycles.

The NIR co-illumination was provided by a tunable Ti:Sapphire laser (Mira 900, Coherent, pumped by Nd: Vanadate laser (Verdi™ V–10, Coherent)) and was tuned to both 810 and 900 nm for scanning the triplet spectrum. The Ti: Sapphire laser was operated in a continuous mode, and its input was controlled using a mechanical shutter. Contrary to the characterization experiments in the WF configuration, the NIR co-illumination was applied during the whole duration of the photoswitching fatigue experiment. The power of the Ti:Sapphire laser was varied to achieve power densities of ~1–50 kW/cm2 at the sample plane for both wavelengths (810 and 900 nm). The fluorescence was collected back from the same objective (UPlanSApo 60× 1.2 w, Olympus) and then passed through a dichroic mirror (ZT405/488/635rpc-UF2, Chroma), after which the light was focused onto a pinhole of 100 μm. A slightly wider pinhole size was chosen in order to have a larger volume to average the deformities present at every focal plane of the gel. A band pass filter (535/70) was used in the emission path, and finally, the light was collected by two APDs (Tau-SPAD, PicoQuant GmbH, Berlin). The signals were directed to a data acquisition system (Hydraharp 400, Picoquant, Berlin) and time traces of the signals were exported via Symphotime and analysed with a home-built code.

rsEGFP2 photophysical kinetic modeling
To provide an insight into the mechanisms involved in the photoswitching fatigue, we built a photophysics simulation tool. To simulate the response of rsEGFP2, we emulated the experimental framework and calculated the population evolution given a proposed kinetic scheme. The system of kinetic equations is solved in matrix form. Since the photophysical evolution of the RSFP chromophore can be specified as a linear system of ordinary differential equations, it is possible to find an analytical solution to propagate a set of initial populations for each species. The solution involves the diagonalization of the kinetic rate matrix53, which collects all the rates connecting the different species to each other. In general, to simulate a complex pulse scheme, we divide it into time windows—instances in which the laser states are not changing, and thus the kinetic rates are fixed—and solve the rate equations for the length of such a window. For consecutive time windows, the populations of the species at the last simulation point of one window are the initial populations of the next window. A more detailed description of the theory behind the photophysical simulations can be found in Supplementary Note 16.

The proposed photocycle model includes the 4 main states responsible for the photoswitching in rsEGFP2 – TH, CH†, C-, T-†- and an intermediate species, as described in the literature15. Furthermore, to parametrize the observed photoswitching fatigue we included two bleaching pathways: (i), triplet states bleaching, and (ii), photodestruction of the chromophore from the off-state. The complete photocycle scheme with the different transitions between states is shown in Supplementary Note 1. Attaining the different levels of complexity that the photocycle model includes, different pathways were activated or silenced depending on the scope of the simulation. The parameters input in the kinetic model are taken from literature, properly referenced, or by fitting the kinetic model to our data. The parameters used for each of the reported simulations can be found in Supplementary Table 2.

Protein purification
Transformed E. coli cells (TOP10 and BL21 strains, pBAD and pQE expression systems) were grown overnight in LB medium supplemented with ampicillin. The next morning, cells were diluted 1:100 together with ampicillin 1:1000 in LB medium and added to 50-ml tubes so that the tubes were filled to the brim, and the tubes were then sealed and incubated until the optical density (OD) at 600 nm reached 0.6 at 37°C. Then IPTG was added to a final concentration of 1 mM, and the tubes were tightly sealed again to restrict oxygen availability. After overnight incubation, the cells were collected by centrifugation in the same tubes. The resulting bacterial pellet was sonicated and re-concentrated by centrifugation. The remaining supernatant was filtered (MinSart RC Hydrophilic 25 mm 0.2 μm, Sartorius, Germany), and the proteins were purified by Ni-NTA affinity chromatography (His SpinTrap; GE Healthcare) according to the manufacturer’s instructions. The purified proteins were concentrated by ultrafiltration and taken up in phosphate buffer, pH 7.5.

RESOLFT and parallelized confocal microscopy
Live-cell RESOLFT and parallelized confocal imaging were performed on a modified version of the microscope set-up reported here48, the same one used for the photophysical characterization at low illumination power densities (Supplementary Fig. 28). To achieve a spatial resolution below the diffraction limit, the light was spatially patterned in the sample plane by introducing optical elements into the laser paths. Photoactivation was carried out at 405 nm in combination with a micro-lens array (MLA, MLA-150-5C-M; Thorlabs) to create a multi-spot pattern with 625 nm periodicity. Illumination at 488 nm and 592 nm was combined using a dichroic mirror (zt502rdc; Chroma) and focused by an MLA to create a multi-spot pattern at the sample plane with periodicity 625 nm, and that was aligned to the 405 nm illumination. To create the light pattern necessary for fluorescence confinement, the collimated light from the 488 nm digitally modulated diode laser (200 mW, 06-01, 488 nm; Cobolt, Hübner Photonics) was directed to a half-wave plate and polarizing beam splitter (PBS). The half-wave plate was adjusted to equally split the light equally into P and S polarizations. After the polarizing beam splitter, the light was sent through custom-made phase-diffraction gratings of 437-nm-high SiO2 lines with a 25 μm period (Laser Laboratorium Göttingen). After the gratings, the light paths were recombined with another polarizing beam splitter. The ±1 diffraction orders are selected at the back focal plane of the objective to obtain a final off-periodicity of 312.5 nm. A piezo scanning system (BPC303; Thorlabs) was used to apply pulse illuminations across a small region equal to the multi-foci periodicity at precise steps (typically 35 nm but adjusted to the desired optical resolution). The light was digitally modulated by transistor-transistor logic signals with a NIDAQ PCI 6371 (National Instrument) acquisition device. The FOV for MoNaLISA imaging was ~40×40 μm2. The typical imaging pulse scheme consisted of 4 illumination sequences for RESOLFT imaging—on-switching, confinement, readout and recovery, and 3 illumination sequences for parallelized confocal imaging—on-switching, readout and recovery. The NIDAQ instrument synchronized the pulse sequence, the camera readout, and the scanning. The readout laser was synchronized with the sCMOS (ORCA Fusion C14440-20UP; Hamamatsu) camera exposure to detect fluorescence emission. The length and delay of the laser pulses, the output power of the lasers, the exposure time of the camera, and all other relevant parameters were controlled through ImSwitch50. A schematic of the imaging microscope, as well as a list of the optical components, can be found in Supplementary Note 15. The imaging parameters are reported in Supplementary Table 10.

Cell culture and transfection
Wild-type U2OS (ATCC HTB-96) cells and U2OS with endogenously tagged vimentin-rsEGFP254 were cultured in DMEM (Thermo Fisher Scientific; 41966029) supplemented with 10% (vol/vol) FBS (Thermo Fisher Scientific; 10270106) and 1% penicillin-streptomycin (Sigma Aldrich; P4333) and maintained at 37°C and 5% CO2 in a humidified incubator. For transfection, 2×105 cells per well were seeded on coverslips in a six-well plate. After 1 d, cells were transfected using Lipofectamine LTX Reagent with PLUS reagent (Thermo Fisher Scientific; 15338100) according to the manufacturer’s instructions. 24–36 h after transfection, cells were washed in PBS solution, placed with phenol-red-free DMEM or Leibovitz’s L-15 medium (Thermo Fisher Scientific; 21083027) in a chamber, and imaged.

Ratiometric image calculation
We tested experimentally if the fluorescence enhancement we observed in the rsEGFP2-PAA layer system in Fig. 2a was visible in rsEGFP2 labeled live cells. In multiple cells we recorded two images: one we deemed slow image, which consisted of 20 ms illumination at 405 nm and 14 W/cm2 power density followed by a fluorescence readout step with 488 nm, the other, which we named fast image, consisted of a 0.25 ms 405 nm burst at 1.05 kW/cm2 power density followed by the fluorescence readout step with 488 nm illumination. The images were taken sequentially in confocal mode in our WF microscope. To calculate the ratiometric image for every slow/fast image pair, we pre-processed the pair of images by subtracting the background and masked with an intensity threshold using Otsu’s method from the scikit-image Python library. The per-pixel intensity was divided as slow/fast to generate the ratiometric image (Supplementary Fig. 14). A general quantification is shown in Fig. 2b by accounting for all the valid pixels from a pool of 8 ratiometric images. To avoid photobleaching bias from one of the on-switching doses, the first image of each slow/fast images pair was alternated across different cells, i.e., cell 1 was imaged with the slow image pulse scheme first, cell 2 was first imaged with the fast image pulse scheme instead.

Live-cell single channel multiplexing
Multiplexing experiments were carried out on transformed E. coli cells prepared as mentioned above. Bacteria expressing single RSFPs were imaged after centrifugation, and mixed samples were prepared by combining bacteria expressing different proteins at suitable concentrations. Two different methods for unmixing were employed: recording of slow off-switching curves and intra-pixel 405 nm intensity modulation inspired by exNEEMO10,13. For the former, the bacteria in the FOV were off-switched using the 488 nm illumination (~5 μW at the sample plane) of the auxiliary WF shown in Supplementary Fig. 28 with ~100 ms exposure time. Afterwards, the same bacteria were imaged in the parallelized confocal modality in the microscope described above. The scanning step between adjacent multi-foci was set to 90 nm. Since the WF and parallelized confocal images have different coordinate systems, the image stacks were transformed and registered with the MultiStackReg55 plug-in in ImageJ. Following the registration, a custom-written segmentation routine in ImageJ was used to identify single bacteria in both WF and parallelized confocal image stacks and the intensity traces were extracted. The data clustering is performed by a custom-written Matlab script based on a Gaussian mixture model (GMM) that fits the data into a given number of Gaussian components, extracting μ and σ.

On the other hand, the intra-pixel 405 nm intensity modulation method consisted of a series of 4-frame parallelized confocal stacks with modulated 405 nm illumination doses. On every pixel, the sample is consecutively illuminated with 4 different pulse sequences the integrated intensity is collected during the 488 nm excitation and the modulation of the fluorescence levels is given by the on-switching dose. The sample is scanned between adjacent foci with a step of 90 nm. The microscope control software ImSwitch50 was adapted to acquire N-images per pixel. A custom-written Python script splits the acquired frames into 4, and our reconstruction algorithm returns a stack of 4 frames, each with a modulated pixel intensity given by the input illumination sequence, for every acquisition frame. To build a time-lapse recording, this procedure is repeated 15 times, resulting in a total of 60 frames. Following the acquisition, a custom-written script in ImageJ is used for image processing and segmentation of individual bacteria. The intensity traces of the identified bacteria are extracted and processed in a custom-written MATLAB script. Similarly to exNEEMO10,13, the intensity of every 4th frame is used to normalize the intensity of the three previous ones and the average of the normalized 4-frame stack intensity trace across the 60 frames time-lapse is computed. Simultaneously, the fluorescence signal loss due to photobleaching is computed as the difference in average raw intensity between the first 3 normalization frames and the last 3 normalization frames in the stack. The data clustering is performed by a custom-written MATLAB script based on a GMM. The first training step extracts μ and σ for the individual proteins. Afterwards, the whole dataset is reanalyzed using the fitted GMM, and the confusion matrix is created.

Statistics & reproducibility
Data were analyzed using custom-written scripts in MATLAB R2021b, R2022b and R2024b (64 bit) and custom pipelines in Fiji macros and Python scripts for image analysis. The code developed for simulating and fitting the different rsEGFP2 characterization experiments was written in Python. The light doses employed to achieve super-resolution in MoNaLISA were tailored to every experiment day guided by a calibration of the imaging parameters before the experiment. Samples sizes, means, and standard deviations are indicated in the relevant figure legends. The light-induced recovery effect in all samples is reported as the difference between the mean fluorescence loss without (control condition) and with 592/915 nm illumination (experimental condition). The error associated with the mean difference is calculated by propagating the error from the standard error (typically, σ) for both conditions. Detailed information on the experimental parameters is reported in Supplementary Table 9 for the characterization experiments and in Supplementary Table 10 for imaging experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of NCOMMS-25-05441, “All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins,” by Marin-Aguilera et al. This paper develops optimized methods for using light to remove photoswitchable fluorescent proteins from undesirable triplet states that trap population for long time periods. This strategy not only makes the proteins less likely to photobleach, but also makes these molecules available to fluoresce more often. The experimental work in this paper is both thorough and compelling, and the work will be of great value to the community of researchers who use these proteins. The authors demonstrate that this strategy works for other green-emitting fluorescent proteins, and for a number of imaging modalities. I feel that this paper will have a high impact among those in the advanced optical imaging community, and is ultimately worthy of publication in Nature Communications. There is one crucial issue that I feel needs to be addressed first, as well as a range of minor issues. Please do not take the length of the list of the issues I have identified as a negative, I would simply like to see this work have as much impact as possible. To begin with my major concern, the model that the authors present is far from convincing to me; in fact, I am pretty certain that it is flawed, for several reasons. First, along with virtually all of the prior literature in the field of photodeactivation of triplet states, this study makes the unfounded assumption that RISC solely leads to the population of an excited singlet state. In most circumstances, a more likely outcome is the population of a highly vibrationally excited level of the singlet ground state, because the vibrational density of states of S0 at this energy is vastly higher than that of any singlet excited state (this behavior is challenging to prove, but it can be done; see, for instance, Ref. 28 in the paper and dx.doi.org/10.1016/j.isci.2021.103600; as this journal encourages reviewers to reveal their identities, I will say at this point that this publication is one of mine.). This process, which we in the field typically (and, I would argue, incorrectly) call internal conversion (IC) in the case of T1, explains why phosphorescence quantum yields are not typically more than a few tens of percents for organic molecules. Indeed, the typical way to attain a high phosphorescence quantum yield in such molecules is to have the T1 energy be well below that of S1, which not only prevents RISC to S1, but also lowers the probability of IC. The vibrational density of states of S0 grows exponentially with increasing energy, and so the higher the triplet state, the faster RISC to the ground state will be as compared to RISC to an electronic state with an origin of similar energy to the triplet state. That does not mean that no population will undergo RISC to excited singlet states, but in many, in most, organic systems the branching ratio for RISC to excited singlet states is likely to be small. As an example, in Ref. 29 the authors had to invoke photobleaching from the triplet state that also causes repopulation of the singlet manifold to explain the fluorescence intensity does not depend linearly on the intensity of the deactivation wavelength, but rather saturates. This behavior is a natural consequence of RISC going to the S0 state directly, as can be shown by trivial, analytical kinetic modeling. Second, the model, at least as shown schematically, treats all Tn states as having identical behavior. Given what the authors observe, this is manifestly impossible. As mentioned above, the higher the triplet state, the more likely RISC is to occur (and 488-nm light was indeed shown in Ref. 28 to cause RISC to S0, which is missing from the authors’ model), but at the same time it may also be more likely that some sort of photobleaching event occurs. There is absolutely no reason to believe that the branching ratio for these phenomena (not to mention relaxation to lower triplet states) should be the same following triplet absorption with two disparate colors of light. The triplet absorption spectra in Ref. 27 (which predates Ref. 28 and is more relevant to the work presented here) make it quite clear that the different wavelengths used here go to different excited triplet states. Third, although I appreciate that the authors did their best to use literature values for rate constants, I do not trust much of the literature based on the points raised above. Their model contains so many processes and rate constants that it could reproduce almost anything, even though the model is missing the processes discussed in the previous two paragraphs. Indeed, in my view their model is not compatible with the results in Ref. 28. Developing a model that incorporates verifiable photophysics will take a considerable amount of additional experimental work, and I would suggest leaving that for a future paper. Others will likely have a different opinion than mine, but I feel that the modeling work detracts from the paper because, in my opinion, it is at best incomplete, and more likely is inaccurate. Although some of the experiments performed may have been motivated by the model, presenting the interesting results while having those results be motivated by a model that will elicit skepticism in readers like me proves a distraction. I suspect that this opinion will (understandably) not be popular with the authors at first (and maybe afterwards as well), but I urge them to consider my suggestions strongly. It is better to get the modeling right (or to prove the current modeling to be correct) in a subsequent paper (which I feel will also have high impact) than to dilute the impact of impressive experimental work with a model that cannot currently be substantiated. Other, more minor points that need to be addressed, in no particular order of appearance or degree of concern: 1. The authors play fast and loose with the optical spectrum. 405-nm light is violet; there is no such color as “UV-vis.” 592-nm light is orange (and barely so, it’s almost yellow), not red. Calling light “red-shifted” is confusing, and makes it sound as if the authors are looking at a side-band of another laser they used. Please give this light a clear name, such as “light at the triplet deactivation wavelength.” 2. The authors use a lot of vague language in the paper, for instance talking about “several” rounds of photoswitching, and using many unclear antecedents (“it” and “this”) that made the paper hard to read for me in some passages because I had to go back and figure out the antecedents. As I teach my students, this problem can be fixed by avoiding the use of “it” to refer to something prior, and to always follow “this” at the start of a sentence with a noun. 3. Many of the figure panels were difficult for me to parse (e.g., Figure 1d, Figure 1e, Figure 2a, Figure 2h, Figure 2 i,j). Many of the graphs are deceptive, with y-axes not starting at zero, thus exaggerating small effects. At very least, broken scales should be used in such cases. In Figure 2a in the right panel, please add a second x-axis on top that gives the intensity. 90 mJ/cm2 (note that the 2 is not an exponent in the figure) is not an energy. To say that the model reproduces the data in Figures 2e,f is really a stretch. Within your uncertainty, the data in Figure 2f have no dependence on t whatsoever. I also cannot read whatever is in the color bars for the heat maps in Figures 2i,j. 4. There are places in the paper where the authors refer to a spectrum without saying type of spectrum. An example is line 51, “…the…triplet state of PPs has a red-shifted spectrum….” I presume that they mean absorption rather than phosphorescence, but they need to be clear. Another example is in line 180. 5. There are quite a few places in which numbers that should be exponents are not. 6. In the first sentence of the paper, the abbreviation FP is used without definition. There are other similar problems throughout the manuscript (e.g., EGFP, SNR). 7. In the second paragraph of the results section, it would be quite helpful if the authors explained what they mean by an “excitation cycle.” The meaning only becomes clear later on in the paper. 8. There are many “gratuitous” hyphens following words that start with “photo.” For instance, the correct word is “photophysics,” not “photo-physics.” “Infrared” also does not have a hyphen. 9. “Data” is plural, not singular, and Nature correctly follows that usage. 10. Many people who might use the results presented here in their own work have commercial fluorescence microscopes that are not capable of simultaneous exposure at two different wavelengths and are not able to do the sort of multiplexing that the authors demonstrate. Are the authors able to test how much improvement can be achieved if only alternating scans of different colors can be used? If this idea is not easy to pursue, that is not crucial to the publication of this work. To end on a positive note, this work is quite impressive, and I hope to see the paper published in this journal. Please know that all of my comments are meant in an entirely constructive manner; the topic of your work is highly complex, and many of the issues that I have brought up are not well understood or appreciated in the community. I would like to see you publish this work in a manner that is not only exciting, but also “bullet-proof.”", "reviewer_2": "In this manuscript, Marín-Aguilera et al provide an in-depth study of photobleaching mechanisms in reversibly switchable fluorescent proteins (RSFPs) and introduce means of reducing photobleaching under conditions typical of RESOLFT super-resolution microscopy by the addition of red-shifted illumination during switching cycles or possibly dark periods between switching cycles. The paper is a natural follow-up of the recent work of the group of Agathe Espagne (ref 29) who showed that near infrared light is able to induce reversed intersystem crossing from the triplet state of standard green fluorescent proteins such as EGFP, shortcutting photobleaching pathways typically arising downstream this state. Extending the work to the case of RSFPs is important due to the numerous applications that these markers offer in advanced microscopy approaches. The paper is divided into three sections: (i) a thorough experimental investigation of photofatigue decay of purified polyacrylamide-embedded rsEGFP2 as a function of illumination conditions, (ii) an experimental investigation of the level of recovery provided by red-shifted illumination, reaching up to ~15%, and finally (iii) practical realization in the context of biological samples, showing a moderate but significant effect for time-lapse experiments using either parallelized confocal or RESOLFT microscopy. In addition, the authors introduce a novel technique for kinetic-based RSFP multiplexing based on differential photofatigue decays between different variants. All the work is embedded in a conceptual framework based on an elaborated photophysical model of rsEGFP2, allowing the authors, through extensive simulations, to account for the experimental observations made and possibly predict optimized behavior of RSFPs in a variety of imaging experiments. Overall, this represents a large body of interesting and timely work, clearly to the benefit of photo physicists and fluorescent protein developers, and potentially to the benefit of the large community employing RSFPs as labels in their biological experiments. The photophysical aspects are treated in the specific context of intense illuminations, typical of RESOLFT-like experiments, which is rarely addressed in the rich literature concerned with RSFPs photophysics. This makes the paper particularly interesting to photophysicists. Thus, in principle I am supportive of this work. However, I have many interrogations concerning the validity of the proposed model. To my opinion many issues remain to be solved, that may either stem from a certain lack of clarity (the paper is highly complex to read and properly digest) or be related to more serious inconsistencies. All these issues call for a major revision of the manuscript. Major issues concerning the proposed photophysical model of Figure 1e and S1: 1/ In principle the cis anionic and the cis neutral chromophore should be in rapid equilibrium, depending on pH. This equilibrium is not represented in Figure 1e, but should be, as the cis neutral chromophore, even if it is not highly populated at physiological pH, is able to absorb 405 nm light, isomerize to TH, and probably photobleach under such illumination. I guess that in Figure 1e, CH is meant to represent the “cis on like neutral” chromophore of ref 16, not the real “CH”. If this is correct, the “equilibrium” cis neutral chromophore is missing in the model. 2/ According to Figure S1, the triplet state is only accessed from C-, and does not absorb 405 nm light. Yet, the authors make a very strong statement at the beginning of the manuscript (P4-L97: “the data reports the 405 nm light to be the main driving force for increased photo-switching fatigue in rsEGFP2”). If the triplet state is indeed one of the main photobleaching mechanism induced by 405 nm light absorption, either it should be accessible by ISC from a 405 nm absorbing species (a protonated species) or the triplet itself should be able to absorb this light. Otherwise triplet state bleaching must arise from 488 nm light absorption, and in fact this is underlying the discussion along the whole manuscript. This is very confusing. For example, the initial rise in the fatigue fraction seen in Figure 1f relates to 488 nm induced bleaching from an increasingly high C- population due to increasing 405 nm light, which is an indirect effect. In view of this introductory sentence stating that photobleaching relates to 405 nm light, and also figure 1C showing little effect of the 488 nm light, all this needs strong clarification. 3/ In fact, the authors need to justify why T1 (Figure S1) would not absorb 405 nm light. This is by no means suggested by the experimental spectrum in ref 28, which shows at least as much absorption at 405 nm compared to 592 nm. It is in fact likely that T1 absorbs violet light. Have the authors considered the scenario where the off switching phase populates T1 and the immediately following on switching phase leads to reabsorption of 405 nm light by T1 ? Adding a delay between switching cycles would relieve this switching pathway, as observed. 4/ As mentioned by the authors, the data in Figure 1g suggests the presence of one (or several) additional dark state(s) with a lifetime significantly greater than that of T1 (P4-L122). Such additional dark states are indeed expected, and may be radical species. They, too, may absorb 405 nm light to promote either recovery or photobleaching. Can the model of the authors reproduce the data of Figure 1g ? I suspect not, because the long-lived dark states in question are not part of the model, although they are probably crucial to the behavior. The authors might want to consult the following paper: Duan et al, DOI: /10.1021/ja406860e. 5/ The authors, based on Supplementary Note 3, conclude that the Int intermediate absorbs light at 488 nm and is substantially fluorescent. How do they reconcile this finding with the fact that Int has been reported in several publications to be a protonated species, hence with little absorption at 488 nm and, in principle, very low fluorescence ? The authors report an extinction coefficient of Int at 488 nm of 28,000, based on their data and also based on ref 2 (supplementary table 2). This is very unlikely for a fully protonated species, and ref 2 does not support such value (the authors have probably misread table 1 in ref 2, the extinction coefficient of ~28000 of the fully neutral species is at 403 nm, not 488 nm). It is difficult, based on supplementary figure 3A, to understand how supplementary figure 3B is obtained: at early delays, the integrated fluorescence from this figure is about half of the full signal at late delays. But this is not what is suggested by looking at panel A, where the area under the blue curve (5 µs) seems much less than the area under the red curve (51 ms), not even considering the baseline due to residual background. So please explain how supplementary figure 3B is obtained. If a residual substantial amount of the on-state is present at the beginning of the second 488 nm pulse, this will contribute to the fluorescence signal in all curves of panel A, so how do the authors make sure that this amount is negligible ? This being said, I agree that the microsecond rise in the fluorescence could point at Int being fluorescent. All in all, the authors should carefully clarify this point. If they maintain that indeed Int must be fluorescent, they should state it clearly in the main manuscript, as this is a very important finding in view of the available literature, and they should provide a possible mechanistic scenario. Furthermore, if Int is indeed fluorescent, why would it not undergo ISC to the triplet state in the proposed model ? Is there any good reason to neglect this possibility ? 6/ On which basis do the authors assume that C- may convert to Int as a ground state process ? What is “tau_protonation-C” in Supplementary Table 1 ? This parameter is not reported in Supplementary Table 2. Is it used in the actual simulations ? 7/ The authors invoke a direct bleaching pathway from TH, which apparently does not involve the triplet state. The authors should explain why the triplet state is not considered here ? Is this because of the short lifetime of the nonfluorescent TH ? This would make sense, but if the triplet state is not involved, then, photobleaching must arise from the excited singlet state manifold. The authors should provide more details here, notably as RISC is not going to help at all in this case, meaning that, according to the author’s view, light induced recovery by red shifted light would not directly relate to 405 nm induced photobleaching (see point 2 above) … in stark contradiction with the statement that photobleaching mainly arises from excitation at this wavelength. Could it still be that the triplet state and downstream radical species be reached from neutral chromophore species ? The authors might be interested to read: Wulffele et al, DOI 10.1021/acs.jpclett.2c00933. 8/ The authors find that increased power density 488 nm results in higher light induced recovery (figure 2e). This is unexpected in view of the fact that a high-level of 488 nm illumination should by itself promote RISC, decreasing the additional impact of red shifted light. In fact this was noted in refs 28 and 29. I feel that the data shown in figure 2e are misleading, as here the dose of illumination at 488 nm is kept constant, that is, the data at 0.42 kW/cm² are collected with a shorter pulse of 488 nm light than the data at 0.2 kW/cm², while at the same time, if I understand correctly, the 592 nm illumination is kept at 1 ms duration. In this situation, the observation can be easily explained, as, as soon 488 nm illumination terminates, the additional time until the next cycle becomes free for either thermal or light induced relaxation from T1 to S0. These aspects should be clarified to avoid this possible misunderstanding that the more 488 nm illumination the more 592 nm light induced recovery by RISC. 9/ As shown by the authors the RISC effect can as well be obtained by near-infrared light, at 810 or 900 nm. In fact, near infrared light should be the privileged choice, as absorption of the triplet state around 900 nm has been shown to be maximum in green fluorescent proteins. The authors have used 592 nm light probably because this was technically easier (already implemented on their microscope). Interestingly, they found that the achievable effect with 592 nm surpasses that at 810 nm, even with the huge power densities they used in the near infrared. It would be nice to give a precise interpretation of what might be going on. At the end of supplementary note 7, the authors claim that the comparison is difficult based on the fact that the “spatial distribution of light-induced kinetic processes is radically different”. This statement is unclear to me, it would be nice if the authors could elaborate more. In all cases it looks like, from supplementary figure 13, that no matter what intensity is used, the level of light induced recovery at 900 nm cannot be reached at 810 nm. Would the authors have a clue whether this is true and if yes, why ? At the end, using visible red light to promote RISC is nice, as this wavelength is available right away on many microscopes. However, the authors should comment on possible penalties e.g. for 2-color experiments requiring 592 nm light to excite a second red fluorescent marker. 10/ It would be nice to see the experimental data of figure 1F on a linear scale and without the fitted model data superimposed. It looks like these experimental data may follow a simple exponential growth on the used logarithmic scale, meaning a linear growth on a linear scale. Many readers could feel that using such a complicated photophysical model to fit essentially linear data is largely unjustified. Could the authors comment on this ? What are the factors that prevent from only relying on a linear photobleaching effects by the 405 nm light ? 11/ I am puzzled by supplementary figure 4: the data in this figure show the rapid growth of the integrated fluorescence which then levels off without any sign of further decay. On the contrary, the model predicts a decay, as Int is more and more redirected towards TH when the 405 nm power density is increased, and this decay is clearly visible in the fitted data. The experimental data here clearly seem to deviate from the model. Furthermore, the data of figure 1B seem to go in the same direction, as the initial point in the photofatigue decay curves appear to be significantly higher at 0.7 kW/cm² as compared to 0.22 kW/cm², as opposed to what the model would predict. In their experiment of figure S4, could the authors increase further the intensity of the 405 nm illumination to start seeing the expected decay (maybe using their confocal microscope) ? Could it be that Int may also be photoconverted to C- by violet light, so that the relative amount of Int molecules sent to either C- or TH be relatively independent of 405 nm illumination ? It is known that chromophore deprotonation in FPs can be accelerated by light. These aspects should be discussed. 12/ The scheme in Fig1e is difficult to read: the triplet state between C- and B should be shown in clear, notably as the trans states (T) could be confused by some readers with the triplet state (T1). The protonated chromophore state in equilibrium with C- should be shown as CH, and the short-lived intermediates preceding Int along the on switching pathway should be given a different name. Overall, I strongly encourage the authors to revisit their photophysical model in depth. They should also much improve the clarity of their manuscript so that the essential message carried by this model can be captured by the white audience of Nature Communication. At present, what I can extract from the manuscript is that: (i) the effect of 405 nm illumination on photofatigue either relates to the triplet state only via the amount of the on-state population at the beginning of off switching phases, or goes through a singlet state photobleaching mechanism that is nonrecoverable by RISC and (ii) recoverable photobleaching through T1 only occurs upon 488 nm illumination of the on-state. One possible strategy that the authors could consider would be to investigate whether their photophysical model predicts a pH dependence of photofatigue and to carry out corresponding experiments to see whether these predictions are realized. For example, at low pH the fraction of protonated chromophores will increase, likely increasing direct bleaching by 405 nm light. It could also be expected that relaxation of Int to C- slows down, making the initial drop faster. In view of the complexity of the problem, those pH dependent experiments could help to narrow down the photophysical model. Other major points 13/ The authors nicely apply their methodology to both prokaryotic and eukaryotic samples. Figure 4A and 4C suggest that there is a real benefit of applying 592 nm co-illumination. Yet, it is not clear whether the protecting effect shown in the photofatigue decay curves of figure 4A is as pronounced as that observed in vitro. Could the authors comment on this ? Do they see a possible effect of polyacrylamide in augmenting the effect, maybe in relation to oxygen level and/or diffusion, and thus triplet state lifetime, and even maybe propensity for radical formation ? In view of those photofatigue decay curves in figure 4A, which after all predict a relatively faint improvement, how representative are the series of images shown underneath ? The authors may want to show the photofatigue decay of this very sample in the SI. In passing, the first frame of the “with co-illumination” series appears less intense than the first frame of the “without co-illumination” series. Does this correspond to a true, reproducible effect ? If yes, what happens ? The Mona Lisa data are also nice and seem to show a significant gain. Would there be a way to more quantitatively assess the spatial resolution loss along multiframe data collection with and without co-illumination, rather than showing just one profile at a user chosen position (which is the classical old procedure often criticized for potential bias) ? Possibly, a number (~10) of random locations across multiple fibers could be picked and the profiles presented in the SI ? More minor issues 1/ The authors investigated the effect of modulating the on switching light (essentially adding dark times, or reducing power density at constant dose, both resulting in increased data collection time) in the context of their photophysical studies, but they have not implemented such strategies for data collection of biological samples. Thus L19 in the abstract does not really reflect the content of the paper. 2/ P2-L45/46: this sentence calls for references 3/ P3-L64/65: maybe replace “dark state” by “triplet state”: as is, it looks like the authors are not sure of the nature of the state, whereas their photophysical model clearly identifies the triplet state. 4/ P3-L75: the authors might be interested to read: Adam et al, https://doi.org/10.1002/cphc.202200192 It would be interesting to know whether they noticed potential signs of the off-state heterogeneity described in this paper, in view of the ~kHz switching rates that they apply (e.g. non-mono exponential switching kinetics). 5/ P4-L96/97: there seems to be a contradiction between this statement and all the discussion in supplementary note 9 on the different RSFPs tested. In this note, unless I am mistaken, the difference in switching fatigue between the variants is not assigned to 405 nm induced photobleaching, rather to a balance between off switching and ISC induced by 488 nm light. Please explain and improve consistency of the text in this regard. 6/ P4-L122/125: increasing the dark time will not prevent T1-O2 interactions, resulting in ROS production. There are definitely several photobleaching mechanisms at play, please see Duan et al, DOI: /10.1021/ja406860e. 7/ P5-L136: “we investigated how the modulation of the on-switching dose, given a fixed total energy, influences the fluorescence output”: this sentence does not make sense, the authors tend to make a confusion between power density and dose throughout the manuscript. Dose = total energy. 8/ P7-L195/201 (and Table S2): is it reasonable to assume that the quantum yield for photobleaching from the excited triplet is wavelength independent ? As different levels of excited triplet states might be reached as a function of illumination wavelength, the fate could be very different, for example with blue shifted light generating more photobleaching and red shifted light generating more RISC. This is really an open question but the issue should be discussed, and at least the assumptions made clearly stated. A related question is: is it reasonable to assume a much weaker quantum yield for photobleaching from the excited triplet state than from the ground triplet state (supplementary table 2) ? It would be expected indeed that the excited triplet state be more reactive than the ground triplet state. 9/ P9-L276/277: why only dim samples ? 10/ P9-L282: “extends the absolute observation window”: by how much ? Also around 15% ? Please discuss this point. 11/ P10-L294/295: it is not clear to me why co-illumination should only be applied during the readout step. Why not during the switch off step, ideally using a doughnut-shaped red beam ? Isn’t it during this step that most photobleaching occurs ? 12/ P10-L312/313: in fact, a protective effect could be expected, as shown in ref 29. Could such a decrease in phototoxicity be observed by looking at mitochondria mobility ? 13/ P10-L318/321: yes but no experimental demonstration was provided (see point 1 above). The downside of this strategy should also be mentioned (increase in data collection time). 14/ P11-L326: again, “a dark state” rather than the triplet state is invoked here (see point 3 above). This really reads bizarre, as the triplet state is clearly identified throughout the results section. 15/ P11-L346: the authors should recapitulate the benefits of using either visible red light or near infrared light. Have they tried to implement 900 nm illumination on their RESOLFT microscope ? This would be a very nice experiment to do, to check the pros and cons. 16/ The authors have developed the nice rsFusionRed proteins. Readers will naturally wonder whether they have assessed the potential photofatigue enhancement of those red rsFPs with near-IR light. I encourage the authors to answer this question. 17/ It would be nice in the discussion to consider the potential effect of the level of oxygen on the observed light induced recovery. Less oxygen is typically beneficial for photostability because of decreased ROS production, but also increases the triplet state lifetime (this was shown also in FPs). Under anaerobic conditions do the authors think that RISC by red shifted light will still help ? 18/ As RISC by red shifted light increases the singlet state population, not only photostability but also effective brightness should increase. Does this show up in the model and have the authors noticed this experimentally ? Discussing this point would be important. 19/ Using photofatigue as a distinguishing feature for multiplexing is a nice idea, but it should be better explained in what situation it is beneficial to use this technique rather than exNEEMO or TMI. Is this to collect the data faster ? What are the potential consequences in terms of photo/cytotoxicity ? Please discuss these issues. 20/ The provided software appears to work but is based on Python scripts without any manual. It will be very difficult to use unless such manual is made available. Ideally a Python GUI would be desirable to facilitate the use of the software. I am curious to know whether the authors considered using the SMIS simulator (DOI: Bourgeois, 10.1038/s42003-023-04432-x) as an alternative to their software (and then the authors will not have much doubt about the identity of the reviewer …) ? 21/ There are quite a few typos within the manuscript that should be checked … 22/ Figure 2f is very noisy: please perform a statistical test (ANOVA) to check that the fitted decay is significant. 23/ Figure 2h legend: I cannot see any green shaded area. 24/ Figure 3A legend: fluorescence loss after how many cycles ? Why was the chosen off switching 488 nm dose different for each RSFP, while during a real demixing experiment, all RSFPs will be subjected to the same dose. Please explain. “Different multi-foci” -> “different foci” ? Note that some readers may confuse these “foci” with different “focal planes” and have a hard time to realize that they are all in the xy plane. 25/ Figure 4b, lower right panel: why showing the mix with and without 592 nm illumination ? It would be more informative to show the two RSFPs with co-illumination. 26/ Supplementary table 4: bleaching yields are in % ? Please check consistency with table S2. 27/ Supplementary figure 7b: what is the unit on the Y scale ? 28/ Supplementary table 6: bleaching quantum yields from ground and excited triplet seem inverted relative to those in table S2 and S4 ? 29/ I am not sure of some of the arguments presented in supplementary note 9: (i) that photobleaching by slower switching RSFPs is expected to be less recoverable by the RISC effect. If there is more triplet formed relative to off switching, the benefit of RISC should be higher, not lower ? Furthermore, non-switchable Green FPs have been shown to be nicely stabilized by RISC (ref 29); (ii) that the ratio between bleaching from the ground or excited triplet state is the key determinant: the ratio between RISC and photobleaching upon photon absorption by the triplet state is also a key determinant. 30/ supplementary figure 16: do these data support the notion that the long-lived dark state downstream the triplet is possibly also bleached by 405 nm or 488 nm light ? (Remarks on code availability) Below I paste point 20/ in my review: The provided software appears to work but is based on Python scripts without any manual. It will be very difficult to use unless such manual is made available. Ideally a Python GUI would be desirable to facilitate the use of the software. In conclusion for this part, at this stage I consider that the code is not a usable resource for the community.", "reviewer_3": "NCOMMS-25-05441\nPoint-by-point responses to reviewers of the manuscript NCOMMS-25-05441:\nAll-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent\nProteins\nMarin-Aguilera et al\nWe appreciate the comments and the suggestions of the Reviewers. All the inputs helped to improve both\nthe structure and clarity of the manuscript.\nIn the revised version we integrated the comments of both reviewers to strengthen and clarify the model.\nThis brought us to uniform the model with previous literature on triplet and fast photoswitching studies of\nrsEGFP2. Through simulation and experimental work, we have validated the drawn photocycle for\nrsEGFP2 as well as tested its expansion to incorporate additional states and the different pathways\nhighlighted by the reviewers. In the text we have now clarified the limit and strength of the model, that is\nintended to be an effective model rather than a comprehensive theoretical description.\nBelow we provide a point-by-point response to the comments of each Reviewers. The comments of the\nReviewers are written in blue and our responses in black.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nReview of NCOMMS-25-05441, “All-Optical Strategies to Minimize Photo-Bleaching in Reversibly\nSwitchable Fluorescent Proteins,” by Marin-Aguilera et al.\nThis paper develops optimized methods for using light to remove photoswitchable fluorescent proteins from\nundesirable triplet states that trap population for long time periods. This strategy not only makes the proteins\nless likely to photobleach, but also makes these molecules available to fluoresce more often. The\nexperimental work in this paper is both thorough and compelling, and the work will be of great value to the\ncommunity of researchers who use these proteins. The authors demonstrate that this strategy works for\nother green-emitting fluorescent proteins, and for a number of imaging modalities. I feel that this paper will\nhave a high impact among those in the advanced optical imaging community, and is ultimately worthy of\npublication in Nature Communications.\nWe appreciate the reviewer’s positive evaluation of our work.\nSince the concerns raised all pertain to the model, it seems best to first clarify its intended role within our\nstudy. Based on our experimental findings, we constructed a minimal, effective kinetic model to provide a\nmechanistic explanation. In bleaching photophysics, kinetic models are often simplified, because\nfluorophore bleaching typically arises from complex, oxidative reactions involving many environmental\nfactors and chemical species. Adding too many free parameters would weaken the model’s predictive\npower; conversely, reporting only the experimental trends would leave readers without a framework for\ninterpreting these inherently complex photophysical processes. Hence, we developed a minimal kinetic\nmodel: we began with the established core-state scheme for rsEGFP2 and added only those extra states and\ntransitions required to capture our experimental observations. We made simplifications and added effective\npathways, and we thank the reviewer for helping us clarify our assumptions.\nThere is one crucial issue that I feel needs to be addressed first, as well as a range of minor issues. Please\ndo not take the length of the list of the issues I have identified as a negative, I would simply like to see this\nwork have as much impact as possible.\nTo begin with my major concern, the model that the authors present is far from convincing to me; in fact, I\nam pretty certain that it is flawed, for several reasons. First, along with virtually all of the prior literature in\nthe field of photodeactivation of triplet states, this study makes the unfounded assumption that RISC solely\nleads to the population of an excited singlet state. In most circumstances, a more likely outcome is the\npopulation of a highly vibrationally excited level of the singlet ground state, because the vibrational density\nof states of S0 at this energy is vastly higher than that of any singlet excited state (this behavior is\nchallenging to prove, but it can be done; see, for instance, Ref. 28 in the paper and\ndx.doi.org/10.1016/j.isci.2021.103600; as this journal encourages reviewers to reveal their identities, I will\nsay at this point that this publication is one of mine.). This process, which we in the field typically (and, I\nwould argue, incorrectly) call internal conversion (IC) in the case of T1, explains why phosphorescence\nquantum yields are not typically more than a few tens of percents for organic molecules. Indeed, the typical\nway to attain a high phosphorescence quantum yield in such molecules is to have the T1 energy be well\nbelow that of S1, which not only prevents RISC to S1, but also lowers the probability of IC. The vibrational\ndensity of states of S0 grows exponentially with increasing energy, and so the higher the triplet state, the\nfaster RISC to the ground state will be as compared to RISC to an electronic state with an origin of similar\nenergy to the triplet state. That does not mean that no population will undergo RISC to excited singlet states,\nbut in many, if not most, organic systems the branching ratio for RISC to excited singlet states is likely to\nbe small. As an example, in Ref. 29 the authors had to invoke photobleaching from the triplet state that also\ncauses repopulation of the singlet manifold to explain the fluorescence intensity does not depend linearly\non the intensity of the deactivation wavelength, but rather saturates. This behavior is a natural consequence\nof RISC going to the S0 state directly, as can be shown by trivial, analytical kinetic modeling.\nWe acknowledge the literature complexity of the triplet to singlet transition. In outlining the used model,\nwe have built on the cited literature, nevertheless we took the reviewer’s comment as an inspiration to\nvalidate and compare the different pathways in the context of our data as well as to better ground our\ndecision. Our intention is to have a solid effective model that is able to reproduce the experimental data of\nphotobleaching.\nThe extended spectroscopical studies recently reported on rsEGFP2 (among particularly insightful for this\ncontext is Rane et al. 2023, J. Phys. Chem. B ) allow us to have an indication of where the triplet states are\nin the energy ladder (Byrdin et al., 2018, J. Am. Chem. Soc) or which are their absorption properties (Rane\net al. 2023, J. Phys. Chem. B, Byrdin et al., 2018, J. Am. Chem. Soc) (Fig. R1). However, they lack\nFigure R1. Schematic of energy ladder of singlet and triplet states for rsEGFP2 in eV. The position of\nS1 was determined from the maximum in the emission spectra (Grotjohann et al., 2012, eLife). The\nposition of T1 was determined from the maximum in phosphorescence emission spectra of EGFP (Byrdin\net al., 2018, J. Am. Chem. Soc) as an approximation of the energy level of the first triplet state of\nrsEGFP2. The energy levels of the subsequent excited triplet states were calculated as E(λ) = 1.69 +\n1.24/λ, with λ in μm. The colored arrows indicate which wavelength would populate each of the energy\nlevels: 3.06 eV, 900nm; 3.78 eV, 592 nm; 4.24 eV, 488 nm; 4.75 eV, 405 nm.\nconclusive evidence for which is the precise mechanism that leads to the repopulation of the singlet form\nvia RISC. In the literature, the effects of light-induced RISC in EGFP (Ludvíková et al., 2023, Nat.\nBiotechnol.) and rsEGFP2 (Byrdin, M., et al., 2025, Biol. Cell.) mainly assume that any conversion of\npopulation from the triplet to the singlet will occur via excited states. The existence of such pathway in\nfluorescent proteins has been proven and observed by means of optically-activated delayed fluorescence\n(OADF) (Peng, B., 2021, J. Phys. Chem. B., Lu, Y-H., 2023, J. Phys. Chem. B.) where a fluorescent photons\nare measured after illuminating the sample with red-shifted light that is delayed in time with respect to\nfluorescence excitation. This phenomenon is possible if the RISC mechanism involves the excited states\nfrom the singlet manifold, but as the reviewer points out this mechanism might not be the dominant RISC\npathways in typical organic dyes.\nAs the reviewer suggests, RISC could happen from excited triplet states directly into the vibronic manifold\nof S followed by intramolecular vibrational relaxation (IVR), this would be supported by the fact that the\n0\ndensity of vibronic states increases exponentially with the excitation energy above a state. We recognize\nthat, since light-induced RISC events are rare compared to the overall fluorescence emissions in a typical\nfatigue experiment, the final influence of landing to S or S after a RISC/IVR should not produce\n1 0\nappreciable change in our observable. If light-induced RISC populates S , in a few nanoseconds the great\n1\nmajority of the events will lead anyway to S , with a very small chance of ISC (Φ 10-3).\nWe explored whether channelling RISC T S or RISC T S would have a sensible impact on the\nn 1 n 0\nnumerical results of the kinetic simulation of the fatigue experiments. This was verified by simulating the\nlight-induced fatigue recovery in two identical simulation settings, with all the parameters as already\ndescribed in the main text and Supporting Information Note 1. The only change in the two conditions is the\nconnection of the transition from the excited triplet state to the singlet states. In one case T converts to S\nn 1\n(as in all the remaining simulations in the manuscript) and in the second case T converts to S . The dashed\nn 0\narrows in Fig. R2 represents RISC/IVR, thus including the vibrational relaxation. There are no notable\nchanges in the computed light-induced recovery.\nFigure R2. Example of light-induced photoswitching fatigue recovery computed as a function of the 592\nnm power density at a fixed 488 nm power density (300 W/cm2). (a) Condition with RISC mechanism\nT S . (b) Condition with RISC mechanism T S . All the simulations were carried out at 1 ms 405\nn 1 n 0\nnm and 100 W/cm2 power density.\nIn the main manuscript, we decided to keep the original RISC T S mechanism, because we believe it is\nn 1\nmore consistent with the cited literature and does not produce relevant changes for the results of our work\ncompared to RISC T S . Nevertheless, in order to make the reader aware of this discussion, we included\nn 0\nthe presented discussion and reference in Supplementary Note 8, properly referring to it in the main text.\nSecond, the model, at least as shown schematically, treats all Tn states as having identical behavior.\nGiven what the authors observe, this is manifestly impossible. As mentioned above, the higher the triplet state,\nthe more likely RISC is to occur (and 488-nm light was indeed shown in Ref. 28 to cause RISC to S0,\nwhich is missing from the authors’ model), but at the same time it may also be more likely that some sort of\nphotobleaching event occurs. There is absolutely no reason to believe that the branching ratio for these\nphenomena (not to mention relaxation to lower triplet states) should be the same following triplet absorption\nwith two disparate colors of light. The triplet absorption spectra in Ref. 27 (which predates Ref. 28 and is\nmore relevant to the work presented here) make it quite clear that the different wavelengths used here go to\ndifferent excited triplet states.\nAs the reviewer rightfully points out, it is uncommon that different wavelengths share the same RISC\nefficiency. Indeed, the different wavelengths are parameterized separately in our model, but notably we\naccount only for the different absorption cross-section as reported in Rane et al. J. Phys. Chem. B 2023, but\nthey share the effective RISC quantum yield. Such effective quantum yield is retrieved by fitting of the\ndata.\nAt this point it is important to consider the nature of our experimental data: the loss in fluorescence after\nconsecutive on/off cycling. This is not information rich, as data coming from more conventional time\nresolved spectroscopy methods. Considering the limited predicting capability of such data, we have fixed\nmost of the parameters from literature and helped where possible with ad-hoc experiments. The RISC\nparameters are instead directly fitted. Overall, we believe that a finer description of RISC would not add\nsubstantial mechanistic insight, which is the primary aim of our modeling. Our RISC pathways are to be\nintended as effective ones and could be refined in the future if additional experiments and work will be\navailable regarding the details of RISC in rsEFGP2.\nWe made clear in the main text and Supporting Note 8 that RISC from different T states in general has\nn\ndifferent efficiencies, and that we are working with a minimal effective model still able to reproduce\nsatisfactorily the data.\n“Similarly to recent literature on light-induced RISC in EGFP32 and rsEGFP245, we rationalize the\nrepopulation of the emissive state via RISC from the triplet excited state (T ) to a vibronic state of the singlet\nn\nmanifold (Supplementary Note 1 and 8). All the different laser lines of the experiment (405, 488, 592, 915\nnm) will excite the triplet state31, stimulating both bleaching and RISC.”\nThird, although I appreciate that the authors did their best to use literature values for rate constants, I do not\ntrust much of the literature based on the points raised above. Their model contains so many processes and\nrate constants that it could reproduce almost anything, even though the model is missing the processes\ndiscussed in the previous two paragraphs. Indeed, in my view their model is not compatible with the results\nin Ref. 28. Developing a model that incorporates verifiable photophysics will take a considerable amount\nof additional experimental work, and I would suggest leaving that for a future paper. Others will likely have\na different opinion than mine, but I feel that the modeling work detracts from the paper because, in my\nopinion, it is at best incomplete, and more likely is inaccurate. Although some of the experiments performed\nmay have been motivated by the model, presenting the interesting results while having those results be\nmotivated by a model that will elicit skepticism in readers like me proves a distraction. I suspect that this\nopinion will (understandably) not be popular with the authors at first (and maybe afterwards as well), but I\nurge them to consider my suggestions strongly. It is better to get the modeling right (or to prove the current\nmodeling to be correct) in a subsequent paper (which I feel will also have high impact) than to dilute the\nimpact of impressive experimental work with a model that cannot currently be substantiated.\nWe understand the reviewer perspective and agree on the importance of grounding the kinetic model in\nverifiable photophysics. In the revised main manuscript, we refocused the kinetic model’s role and\ndrastically reduced its overall importance.\nThe model is now presented strictly as a minimal, descriptive framework that helps readers follow the\nexperimental trends. We have flipped the order of some paragraphs in the first two sections, first we report\nthe data and then we follow with the comparison with the kinetic model. Furthermore, all forward-looking\nor predictive sections have been moved to the Supplementary Information, meaning former Fig. 2h and its\ndiscussion is now moved to Supplementary Note 9 where we expand and clarify the limitations.\nWe agree with the reviewer that the experimental part of the manuscript could be self-consistent. At the\nsame time we believe that without a simple kinetic model the reader would not understand in depth the\nexperimental results. This is primarily because rsFPs have a complex photophysical switching behavior,\nalready without considering bleaching pathways and triplet states. A simplified effective kinetic model,\nalthough with limitations and simplifications, is a great tool for the reader to navigate the experimental\nresults.\nWe have now first stated the intention of the model at the very beginning of the manuscript:\n“Using this information, we built a minimal effective kinetic model for rsEGFP2 that could aid the\nmechanistic explanation of the observed photoswitching fatigue behavior (Supplementary Note 1)”\nMore clearly framed the simplification and assumption of it, this reflects both throughout the main text and\nin the extended and revised Supplementary Information. Also following the comments of Reviewer #2,\ndirected to the model, we now have added the following Supplementary Note related to the model:\n- Supplementary Note 5. Thermal relaxation of long lived dark in rsEGFP2\n- Supplementary Note 8. Higher excited triplet state and risk mechanisms.\nFinally, we conclude putting in prospective future expansions of the model to bring it outside of the live\ncell imaging context.\n“Our results showed that a deeper understanding of the species involved in the photocycle of RSFPs as well\nas the mechanisms governing photobleaching can lead to longer and higher quality live-cell imaging\nexperiments. Further expansion of the model, for example, by incorporating additional long-lived dark\nstates (e.g., radicals) or extended pH equilibria, would generalize it to a wider range of experimental\nschemes beyond live cell imaging.”\nWe believe these changes keep the valuable mechanistic guidance the kinetic model provides, while\neliminating the risk of over-interpretation, without weakening the experimental narrative.\nOther, more minor points that need to be addressed, in no particular order of appearance or degree of\nconcern:\n1. The authors play fast and loose with the optical spectrum. 405-nm light is violet; there is no such color\nas “UV-vis.” 592-nm light is orange (and barely so, it’s almost yellow), not red. Calling light “red-shifted”\nis confusing, and makes it sound as if the authors are looking at a side-band of another laser they used.\nPlease give this light a clear name, such as “light at the triplet deactivation wavelength.”\nWe understand the confusion that the term “red-shifted” may have caused to the reviewer. We clarified in\nthe main text the precise meaning of the term, i.e. light of a wavelength that is shifted towards the red side\nof the visible spectrum compared to the primary fluorescence excitation wavelength. We also remark that\n“red-shifted” is a commonly used term in spectroscopic literature, that does not necessary mean that the\nlaser light we used is of color red, but that is shifted to longer wavelengths or lower energies. In general,\nwe have improved the main text and tried to avoid confusion regarding the term.\n2. The authors use a lot of vague language in the paper, for instance talking about “several” rounds of\nphotoswitching, and using many unclear antecedents (“it” and “this”) that made the paper hard to read for\nme in some passages because I had to go back and figure out the antecedents. As I teach my students, this\nproblem can be fixed by avoiding the use of “it” to refer to something prior, and to always follow “this” at\nthe start of a sentence with a noun.\nWe improved the main manuscript by reducing the instances of antecedents. We agree that in this way the\nreadability of the text is improved.\n3. Many of the figure panels were difficult for me to parse (e.g., Figure 1d, Figure 1e, Figure 2a, Figure 2h,\nFigure 2 i,j). Many of the graphs are deceptive, with y-axes not starting at zero, thus exaggerating small\neffects. At very least, broken scales should be used in such cases. In Figure 2a in the right panel, please add\na second x-axis on top that gives the intensity. 90 mJ/cm2 (note that the 2 is not an exponent in the figure)\nis not an energy. To say that the model reproduces the data in Figures 2e,f is really a stretch. Within your\nuncertainty, the data in Figure 2f have no dependence on t whatsoever. I also cannot read whatever is in\nthe color bars for the heat maps in Figures 2i,j.\nWe have updated the mentioned figures to enhance their clarity.\n- Broken axes have been added to more clearly reflect the magnitude of the change.\n- Figure 2a now reports both illumination time and power density of 405 nm, and the exponent has\nbeen fixed.\n- For old Figure 2f (now Figure 2g) we collected a new dataset. The updates in the model and finer\nsampling defined a better accordance between experimental data and simulation. Previously, the\n488 nm illumination time (0.9 ms) was shorter than the 592 nm pulse (1 ms) while now both 488\nnm and 592 nm pulses are 1 ms. With these experimental parameters, we can now better reproduce\nthe data trends, especially the increase in light-induced recovery observed between 0 ms delay and\n1 ms delay.\n- The labels on the color map are now in black for clarity or outside the bar.\n4. There are places in the paper where the authors refer to a spectrum without saying type of spectrum. An\nexample is line 51, “…the…triplet state of PPs has a red-shifted spectrum….” I presume that they mean\nabsorption rather than phosphorescence, but they need to be clear. Another example is in line 180.\nWe have now corrected the mistake in the paper.\n5. There are quite a few places in which numbers that should be exponents are not.\nWe have now addressed it.\n6. In the first sentence of the paper, the abbreviation FP is used without definition. There are other similar\nproblems throughout the manuscript (e.g., EGFP, SNR).\nWe have updated accordingly.\n7. In the second paragraph of the results section, it would be quite helpful if the authors explained what\nthey mean by an “excitation cycle.” The meaning only becomes clear later on in the paper.\nWe have rephrased the sentence to solve its ambiguity; the text is now:\nWe homogeneously illuminated a region (20 × 20 μm2) of a thin layer of rsEGFP2 embedded in\npolyacrylamide (PAA) gel with consecutive on-and-off photoswitching cycles. The emitted fluorescent\nsignal during each off-switching phase was recorded until a decrease of 80%, mimicking the imaging\nconditions, and the photoswitching fatigue curve was built by repeating the photocycle hundreds of times\n(Fig. 1a).\n8. There are many “gratuitous” hyphens following words that start with “photo.” For instance, the correct\nword is “photophysics,” not “photo-physics.” “Infrared” also does not have a hyphen.\nWe have made corrections throughout the text.\n9. “Data” is plural, not singular, and Nature correctly follows that usage.\nWe corrected to the plural form of “data”.\n10. Many people who might use the results presented here in their own work have commercial fluorescence\nmicroscopes that are not capable of simultaneous exposure at two different wavelengths and are not able to\ndo the sort of multiplexing that the authors demonstrate. Are the authors able to test how much improvement\ncan be achieved if only alternating scans of different colors can be used? If this idea is not easy to pursue,\nthat is not crucial to the publication of this work.\nOur results indicate that the fatigue‐resistance benefit of >592 nm co‐illumination remains apparent in a\nsequential scheme, provided the interval between the 405 nm + 488 nm excitation and the >592 nm recovery\npulse is under 2 ms. In a scanning microscope, this could be implemented by interleaving the 592 nm\nillumination with each line or pixel scan, or by reducing the field of view to shorten the overall frame‐\nacquisition time.\nTo end on a positive note, this work is quite impressive, and I hope to see the paper published in this journal.\nPlease know that all of my comments are meant in an entirely constructive manner; the topic of your work\nis highly complex, and many of the issues that I have brought up are not well understood or appreciated in\nthe community. I would like to see you publish this work in a manner that is not only exciting, but also\n“bullet-proof.”\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Marín-Aguilera et al provide an in-depth study of photobleaching mechanisms in\nreversibly switchable fluorescent proteins (RSFPs) and introduce means of reducing photobleaching under\nconditions typical of RESOLFT super-resolution microscopy by the addition of red-shifted illumination\nduring switching cycles or possibly dark periods between switching cycles. The paper is a natural follow-\nup of the recent work of the group of Agathe Espagne (ref 29) who showed that near infrared light is able\nto induce reversed intersystem crossing from the triplet state of standard green fluorescent proteins such as\nEGFP, shortcutting photobleaching pathways typically arising downstream this state. Extending the work\nto the case of RSFPs is important due to the numerous applications that these markers offer in advanced\nmicroscopy approaches.\nThe paper is divided into three sections: (i) a thorough experimental investigation of photofatigue decay of\npurified polyacrylamide-embedded rsEGFP2 as a function of illumination conditions, (ii) an experimental\ninvestigation of the level of recovery provided by red-shifted illumination, reaching up to ~15%, and finally\n(iii) practical realization in the context of biological samples, showing a moderate but significant effect for\ntime-lapse experiments using either parallelized confocal or RESOLFT microscopy.\nIn addition, the authors introduce a novel technique for kinetic-based RSFP multiplexing based on differential photofatigue\ndecays between different variants.\nAll the work is embedded in a conceptual framework based on an elaborated photophysical model of\nrsEGFP2, allowing the authors, through extensive simulations, to account for the experimental observations\nmade and possibly predict optimized behavior of RSFPs in a variety of imaging experiments.\nOverall, this represents a large body of interesting and timely work, clearly to the benefit of photo physicists\nand fluorescent protein developers, and potentially to the benefit of the large community employing RSFPs\nas labels in their biological experiments. The photophysical aspects are treated in the specific context of\nintense illuminations, typical of RESOLFT-like experiments, which is rarely addressed in the rich literature\nconcerned with RSFPs photophysics. This makes the paper particularly interesting to photophysicists. Thus,\nin principle I am supportive of this work. However, I have many interrogations concerning the validity of\nthe proposed model. To my opinion many issues remain to be solved, that may either stem from a certain\nlack of clarity (the paper is highly complex to read and properly digest) or be related to more serious\ninconsistencies. All these issues call for a major revision of the manuscript.\nWe thank the reviewer for the positive comments and the thorough revision that made us clarify and expand\nthe investigation.\nMajor issues concerning the proposed photophysical model of Figure 1e and S1:\n1/ In principle the cis anionic and the cis neutral chromophore should be in rapid equilibrium, depending\non pH. This equilibrium is not represented in Figure 1e, but should be, as the cis neutral chromophore,\neven if it is not highly populated at physiological pH, is able to absorb 405 nm light, isomerize to TH,\nand probably photobleach under such illumination. I guess that in Figure 1e, CH is meant to represent the “cis\non like neutral” chromophore of ref 16, not the real “CH”. If this is correct, the “equilibrium” cis neutral\nchromophore is missing in the model.\nWe apologized for the confusion on the nomenclature. We have now clarified the terminology of the states\ninvolved in photoswitching of rsEGFP2 in Supplementary Note 1 and throughout the manuscript. We have\naligned the terminology to the one of Uriarte, et al. (J. Chem. Phys. Lett, 2022), CH† in our scheme is a\ntransient state corresponding to “Cis On-like neutral”. Fig. R3 shows the states of the model mapped over\nScheme 2 of Uriarte et al. J Phys Chem Lett 2022.\nFigure R3. Scheme of the proposed off-to-on photoswitching mechanism of rsEGFP2, adapted from\nScheme 2 of Uriarte et al. J Phys Chem Lett 2022. On the top we clarify the labels implemented in the\nrevised work.\nCH† is formed from TH by an excited state isomerization reaction upon 405 nm illumination and evolves\nfurther to C- by consecutive relaxation steps. Given the time resolution of our experimental investigation,\nall the fast relaxation steps happening in the ps – ns timescale are indistinguishable, and we have collected\nthem over CH†. The last step of this chain relaxes in the μs and therefore it is relevant and observable in\nour data (Woodhouse et al Nat Comm 2020). CH relaxes to the on-state (C-) with a much slower timescale\nint\nof ~ 1 ms.\nThe reviewer suggests that the CH state, in fast pH equilibrium with C-, could be relevant because of its\nability to absorb 405 nm light and interconvert to TH and bleach. In the kinetic model used throughout the\nmanuscript we neglected the pH equilibrium based on the following assumptions: (i) pKa=5.9 and at\nexperimental pH of 7.5 the equilibrium concentration ratio is [CH]/[C-] ~ 0.025; (ii) our experiments use\ncyclic illumination schemes that induces cyclic modulation of the populations of the states of rsFPs (Fig.\nR4b). Light at 405 nm is shined on the sample mainly on the off-state. After a waiting time of 1 ms,\nlight at 488 nm is shined on the sample mainly on the on-state. This is a rather general properties of all our\nsequences, and 405 nm light is never shined on the sample mainly in the on-state C- when also its protonated\npartner CH has the chance to accumulate from the fast pH equilibrium.\nWe decided to test these assumptions by setting up an extended version of the kinetic model including CH\nand the connected pathways, we will refer to this kinetic model as “with pH equilibrium” (Fig. R4a). The\nparametrization of the kinetic constants connected to the equilibrium C-/CH is set up using pH-pK and the\na\ndeprotonation time of CH of 35 μs from Woodhouse et al., Nat. Comm 2020. All the remaining\nspectroscopical parameters of CH are borrowed from CH†, including the bleaching quantum yield. To\nexamine if the kinetic model with pH equilibrium would predict notable changes with respect to the original\nkinetic model, we reproduced Figure 1d,f with using both models (Fig. R4c). We observe only minimal\nchanges of the predicted fatigue fraction. We believe that the simplified kinetic model without the pH\nequilibrium is reasonable in the context of our cyclic experiments with rsFPs, we also acknowledge that\nthis simplification might not be valid in other conditions, i.e. not cyclic experiments or if relevant 405 nm\nlight doses are shined on the system with substantial on-state population.\n2/ According to Figure S1, the triplet state is only accessed from C-, and does not absorb 405 nm light.\nYet, the authors make a very strong statement at the beginning of the manuscript (P4-L97: “the data reports the\n405 nm light to be the main driving force for increased photo-switching fatigue in rsEGFP2”). If the triplet\nstate is indeed one of the main photobleaching mechanism induced by 405 nm light absorption, either it\nshould be accessible by ISC from a 405 nm absorbing species (a protonated species) or the triplet itself\nshould be able to absorb this light. Otherwise triplet state bleaching must arise from 488 nm light absorption,\nand in fact this is underlying the discussion along the whole manuscript. This is very confusing. For\nexample, the initial rise in the fatigue fraction seen in Figure 1f relates to 488 nm induced bleaching from\nan increasingly high C- population due to increasing 405 nm light, which is an indirect effect. In view of\nthis introductory sentence stating that photobleaching relates to 405 nm light, and also figure 1C showing\nlittle effect of the 488 nm light, all this needs strong clarification.\nFirstly, we apologize for the lack of clarity: 405 nm is indeed one of the wavelengths that promote the\nexcitation of the first triplet state of C- to a higher triplet state in our kinetic model. We have updated the\nFigure R4 (a) Proposed kinetic models with C-/CH pH equilibrium for the photophysics rsEGFP2, the\nkinetic model without pH equilibrium can be found in Supplementary Figure 1b. In the kinetic model\nwith pH equilibrium we have included CH as an additional state that can be photoswitched to TH or\nbleached upon 405 and 488 nm illumination. The colored arrows describe processes that are light-driven\nand the color of the arrows indicate the wavelengths in the pulse scheme (405 nm, violet; 488 nm, cyan;\n592 nm, orange). (b) Representation of the time evolution of TH, CH and C- over the sequential\nint\nillumination profile generally used in the experiment, i.e. 488 and 405 nm interleaved with a delay\nwithout any illumination (c) Photobleaching dependencies to 405 nm power densities. The addition of\nCH did not significantly change the magnitude of the fatigue fraction.\nillustration of Supplementary Figure 1 to clearly show the wavelengths at which each state is optically\naccessible using a color legend.\nThe statement regarding 405 nm light being the main drive force for increased photo-switching fatigue in\nrsEGFP2 emerges from the experimental data. An increase of 405 nm power in our typical experiments has\na higher impact on the fatigue fraction compared to an increase of 488 nm light. As the reviewer comments,\nthere are two modes in which 405 nm light contributes to bleaching. At low power density in Fig. R5 (cyan\nbox) there is an indirect effect: the increasingly high population of the on-state makes the sample more\ncapable of absorbing 488 nm light stimulating the bleaching pathways from the triplet states. At high power\ndensity (Fig. R5, violet box), when the on-switching process is saturated, direct bleaching from absorption\nof 405 nm light becomes increasingly relevant. We clarified this aspect on the main text.\nEven if there are not explicit triplet states connected to the off-states in our simplified kinetic model,\nwe cannot rule out that some of the bleaching involves them, and the direct bleaching pathways from 405 nm\nabsorption are to be intended as effective ones, modeling in a lumped way all the bleaching pathways.\nNevertheless, the short fluorescence lifetime of the off-state should reduce the ISC quantum yields, making\nthe population of triplet-states less probable, and overall it should not be as relevant for the overall photo-\nphysics.\nFigure R5. The fatigue fraction at increasing 405 nm power density is shown in relation of the on-\nswitching fraction of the 405 nm light.\n3/ In fact, the authors need to justify why T1 (Figure S1) would not absorb 405 nm light. This is by no\nmeans suggested by the experimental spectrum in ref 28, which shows at least as much absorption at 405\nnm compared to 592 nm. It is in fact likely that T1 absorbs violet light. Have the authors considered the\nscenario where the off switching phase populates T1 and the immediately following on switching phase\nleads to reabsorption of 405 nm light by T1 ? Adding a delay between switching cycles would relieve this\nswitching pathway, as observed.\nAs in the previous reply, the triplet state 3C- is indeed optically accessible by 405, 488, and 592 nm light\nwith the cross-section for each wavelength as in Rane et al. 2023, J. Phys. Chem. B (Fig. R1 and new Figure\n2d in the main manuscript). Therefore, the effect that a 405 nm pulse can play on the triplet population\ncreated by the previous off-switching is included in the kinetic modeling. In the following main point 4 of\nthe peer review, we go more in depth on the discussion about interpreting data with a delay between\nswitching cycles, that also lets the complete relaxation of the triplet state population before shining a 405\nnm light dose in the next photocycle.\n4/ As mentioned by the authors, the data in Figure 1g suggests the presence of one (or several) additional\ndark state(s) with a lifetime significantly greater than that of T1 (P4-L122). Such additional dark states are\nindeed expected, and may be radical species. They, too, may absorb 405 nm light to promote either recovery\nor photobleaching. Can the model of the authors reproduce the data of Figure 1g? I suspect not, because the\nlong-lived dark states in question are not part of the model, although they are probably crucial to the\nbehavior. The authors might want to consult the following paper: Duan et al", "reviewer_τ": "Φ and Φ ). (b)\nR• T1-to-radical Bleach-radical\nExperimental data and simulation of photoswitching fatigue thermal recovery. For this experiment, the\n405 nm dose was 1 ms and 240 W/cm2 while the 488 nm dose was 5 ms and 40 W/cm2. The dotted lines\nrepresent the simulation while the shaded areas are ± σ of the experimental data. Below, are the residuals\nof each experimental curve as data - simulation. (c) R-• at the end of each photoswitching cycle pulse\nscheme. For a total dwell time of 10 ms (blue curve) the population of radical species continuously\nincreases, while for longer total dwell (25 ms – orange, 50 ms – yellow and 100 ms – magenta) times the\nradical state opulation reaches a saturation level.\nevolve further into a bleached state. The kinetic model fitted on the experimental data suggests a radical\nstate lifetime of ~ 17 seconds (Fig. R6b), in agreement with previous reports of reversible photobleaching\nin both organic dyes and fluorescent proteins (Ha, 2012; Schuster et al., 2005; Sinnecker et al., 2005;\nBerardozzi et al., 2016).\nFrom the kinetic model, the concentration of the radical state shows a continuous build-up (Fig. R6c). With\nlonger waiting time the radical state has more time to relax, and a lower steady state level is reached. Under\nthe main characterization condition of the presented work, the radical lifetime (~16 s) is in the same order\nof magnitude of the experimental time (~ 20 s). The radical bleaching and recovery will progress slowly\nduring the experiments, on average the radical species will be a passive player during the experiment\nmimicking a bleaching species. Accordingly, in the minimal kinetic model presented here, the radical\npathway is effectively lumped with irreversible photobleaching. Overall, this will result on a reasonable\nrepresentation of the overall kinetic behavior with some small biases on the specific kinetic constant related\nto the triplet branch. It is important to note that co‐illumination with light above 592 nm completely\nbypasses radical formation by directly accessing the triplet state, the precursor of any long‐lived dark state.\nBecause of the strong correlation of the triplet and radical parameters, attempting to fit the expanded kinetic\nmodel simultaneously will converge difficulty, weakening the presented effective kinetic model.\nIn this regard, the lifetime of the radical that we have estimated could be deduced only once all the other\nparameters\nhave been fixed.\nConsidering the radical lifetime could provide practical guidelines in imaging experiments. In widefield\nmicroscopy, slower acquisition speeds (< 0.05 Hz) will minimize the build-up of substantial steady-state\nradical population, whereas faster speeds (> 0.05 Hz) will maximize it. In confocal imaging, the scanning\ninherently introduces dark intervals of 20–100 ms or more, with parallelized confocal and RESOLFT\nsystems providing shorter dark times than point‐scanning approaches. However, the radical lifetime in\nbiological samples could be shorter than our experiments in PAA and difficult to predict a priori. Moreover,\nin live‐cell experiments, acquisition timing is dictated by the dynamics of the biological process rather than\nby RSFP photophysics.\nThis investigation has been now added in a new Supplementary Note 5 and referenced accordingly in the\nmain text.\n5/ The authors, based on Supplementary Note 3, conclude that the Int intermediate absorbs light at 488 nm\nand is substantially fluorescent. How do they reconcile this finding with the fact that Int has been reported\nin several publications to be a protonated species, hence with little absorption at 488 nm and, in principle,\nvery low fluorescence? The authors report an extinction coefficient of Int at 488 nm of 28,000, based on\ntheir data and also based on ref 2 (supplementary table 2). This is very unlikely for a fully protonated\nspecies, and ref 2 does not support such value (the authors have probably misread table 1 in ref 2, the\nextinction coefficient of ~28000 of the fully neutral species is at 403 nm, not 488 nm).\nWe thank the reviewer for pointing out the miss reference of the extinction coefficient value from reference\n2 in the old Supplementary Table 2. We removed the reference in the updated version of the Supporting\nInformation. The value of the extinction coefficient of CH was deduced primarily and in a self-consistent\nInt\nmanner from our experimental data according to our updated Supplementary Note 3. The nature of CH is\nInt\nassigned from the best knowledge available in literature, i.e. from Uriarte et al. J Phys Chem Lett 2022.\nThe extinction coefficient of 28000 M-1L-1 best reproduces our transient fluorescence experiments in Figure\n2c of the main manuscript. The only assumption was to assign the same quantum yield of fluorescence to\nCH and C-, and assign the difference in fluorescence signal only to a difference in extinction coefficient.\nInt\nWe clarified these assumptions in Supplementary Note 3. A proof that CH is substantially fluorescence\nInt\ncomes from the experiments with impulsive on-switching light at 405 nm in Supplementary Figure 4, which\nshow a rise of fluorescence emission with approximately 5 microseconds time constant. The non-negligible\nextinction coefficient value at 488 nm of CH is also supported by fast-spectroscopy data (Woodhouse et\nInt\nal Nat Comm 2020, Figure 2), that shows the initial shift and growth of the main transition band of the on-\nspecies in a time-scales compatible with the lifetime of CH .\nInt\nIt is difficult, based on supplementary figure 3A, to understand how supplementary figure 3B is obtained:\nat early delays, the integrated fluorescence from this figure is about half of the full signal at late delays. But\nthis is not what is suggested by looking at panel A, where the area under the blue curve (5 µs) seems much\nless than the area under the red curve (51 ms), not even considering the baseline due to residual background.\nSo please explain how supplementary figure 3B is obtained. If a residual substantial amount of the on-state\nis present at the beginning of the second 488 nm pulse, this will contribute to the fluorescence signal in all\ncurves of panel A, so how do the authors make sure that this amount is negligible ?\nThe experiment in Supplementary Figure 3a consists of a first long 488 nm pulse that will trigger the\ncomplete off-switching transition. Given that there is a non-zero probability of on-switching at 488 nm,\nthere will be a residual signal at the end of that illumination pulse. Immediately after, a burst of 405 nm\nwill induce the on-switching transition and after a variable delay a 488 nm illumination dose is used for\nfluorescence read-out and to bring most of the molecules back to the off-state. If there were no 405 nm\nillumination, the signal at the 2nd 488 nm pulse would remain constant at equilibrium value of the end of\nthe 1st 488 nm pulse (assuming no significant relaxation processes in the first few μs). The background level\nat the end of the first 488 nm pulse is subtracted from the signal of the overall time trace. Therefore, we\nestablished that any signal that we observe after the 405 nm burst will come from the on-switching transition\nand the dynamics we observe in the integrated signal of Supplementary Fig. 3a,b stem from the relaxation\nof a ms-lived state that appears after on-switching.\nThe computation of the values shown in Supplementary\nFigure 3b is done with a trapezoidal numerical integration method.\nWe enriched and clarified the explanation in the relative Supplementary Note 3.\nThis being said, I agree that the microsecond rise in the fluorescence could point at Int being fluorescent.\nAll in all, the authors should carefully clarify this point. If they maintain that indeed Int must be fluorescent,\nthey should state it clearly in the main manuscript, as this is a very important finding in view of the available\nliterature, and they should provide a possible mechanistic scenario.\nWe clarified in the main manuscript that the argument supporting the fluorescent nature of CH comes\nInt\nfrom the microsecond rise of the fluorescence in experiment with impulsive 405 nm light on-switching.\nThe nature of state CH is interpreted as described above according to recent spectroscopic literature. We\nInt\nthank the reviewer for stressing that this is an interesting finding that deserves to be highlighted more in the\nmain text.\nFurthermore, if Int is indeed fluorescent, why would it not undergo ISC to the triplet state in the proposed\nmodel ? Is there any good reason to neglect this possibility?\nWe have not included the formation of triplet state from CH in our simplified kinetic model. We agree\nInt\nwith the reviewer that if a lot of fluorescence would be emitted by CH , the formation of its triplet state\nInt\nwould become a relevant pathway. We believe that the simplification in the proposed kinetic model holds\nfor the description of our bleaching and imaging experiments because we always wait 1 ms between 405\nnm and 488 pulses. During the 1 ms waiting time, most of the CH fraction is converted into C-,\nInt\nsignificantly reducing the fluorescence signal generated from CH and the formation of its associated\nInt\ntriplet population. This aspect is represented more clearly in the sketch of Figure R4b and Supplementary\nNote 1.\n6/ On which basis do the authors assume that C- may convert to Int as a ground state process ? What is\n“tau_protonation-C” in Supplementary Table 1 ? This parameter is not reported in Supplementary Table 2.\nIs it used in the actual simulations ?\nFollowing the interpretation of the states according to Uriarte et al. J Phys Chem Lett 2022 in Fig. R3a and\nthe refined state model as in Supplementary Figure 2 of the updated Supporting Information, we corrected\nthe kinetic model. CH relaxes to C- as maturation of the on-state. Conversely C- does not convert to CH .\nInt Int\nC- could interconvert to CH through a ground state pH-equilibrium, that is not included in the simplified\nkinetic model used throughout the manuscript. We have now made this clarification in Supporting\nInformation. Accordingly, the parameters table is now updated removing the parameter connected to the\nkinetics C-  CH. This discussion was expanded above in major point 1, where we argument on the\nrelevance of the pH equilibrium for our experimental conditions.\n7/ The authors invoke a direct bleaching pathway from TH, which apparently does not involve the triplet\nstate. The authors should explain why the triplet state is not considered here ? Is this because of the short\nlifetime of the nonfluorescent TH ? This would make sense, but if the triplet state is not involved, then,\nphotobleaching must arise from the excited singlet state manifold. The authors should provide more details\nhere, notably as RISC is not going to help at all in this case, meaning that, according to the author’s view,\nlight induced recovery by red shifted light would not directly relate to 405 nm induced photobleaching\n(see point 2 above) … in stark contradiction with the statement that photobleaching mainly arises from\nexcitation at this wavelength. Could it still be that the triplet state and downstream radical species be reached from\nneutral chromophore species ? The authors might be interested to read: Wulffele et al, DOI\n10.1021/acs.jpclett.2c00933.\nAccording to ultrafast spectroscopic data, the lifetime of the off-state is a few picoseconds, e.g. from Uriarte\net al, J Phys Chem Lett, 2022. As the reviewer suggests, this implies a lower chance of populating the triplet\nstates through ISC from the off-state. In our simplified kinetic model we have a pathway for direct photo-\nbleaching from the off-states, mainly driven by 405 nm light. This is an effective pathway necessarily to\nreproduce the experimental data and account for the direct photobleaching with 405 nm light that happens\nwhen the on-switching process is saturated, i.e. when the population is driven almost fully to the on-state\nat every cycle. We cannot rule out the complete absence of a small fraction of triplet state from the off-\nstates, nor the formation of very long-lived dark states with lifetime much slower than our experiments.\nNevertheless, bleaching from the off-states is to be taken as a lumped process that models in a simplified\nway the direct bleaching.\nAs discussed above in major point 2, increasing the 405 nm power density when below saturation will\nindirectly increase the population of C- at every cycle, and it will stimulate the formation of an increasingly\nhigh amount of triplet state population and the connected pathways (RISC/bleach). Thus, even if we notice\nan increase in fatigue with increasing 405 nm power density, in the regime below saturation, triplet mediated\nbleaching is still the most relevant channel and rescuable with RISC. We clarified in the main text that our\nstatement regarding the trend of the fatigue fraction with 405 nm increasing power density does not mean\nthat the bleaching only arises from the direct excitation with this wavelength. The experimental data\nsuggests that direct bleaching from excitation of the off-states is not effectively rescuable by the red-shifted\nlight. It is reasonable to assume that the possible intermediates mediating bleaching are not absorbing\nsubstantially at the red-shifted wavelengths used for rescuing, or that rescue mechanisms from those states\nare not very probable.\n8/ The authors find that increased power density 488 nm results in higher light induced recovery (figure\n2e). This is unexpected in view of the fact that a high-level of 488 nm illumination should by itself promote\nRISC, decreasing the additional impact of red shifted light. In fact this was noted in refs 28 and 29. I feel\nthat the data shown in figure 2e are misleading, as here the dose of illumination at 488 nm is kept constant,\nthat is, the data at 0.42 kW/cm² are collected with a shorter pulse of 488 nm light than the data at 0.2\nkW/cm², while at the same time, if I understand correctly, the 592 nm illumination is kept at 1 ms duration.\nIn this situation, the observation can be easily explained, as, as soon 488 nm illumination terminates,\nthe additional time until the next cycle becomes free for either thermal or light induced relaxation from\nT1 to S0. These aspects should be clarified to avoid this possible misunderstanding that the more 488 nm\nillumination the more 592 nm light induced recovery by RISC.\nWe are aware of the opposite behavior of the studied RSFPs to 488 nm power ramp respects to what reported\nin the mentioned reference. This is due to the photoswitchable nature of the RSFPs respect to the fluorescent\nproteins under investigation by Ludvikova et al., 2023, Nat Biotechnol. Our aim is to investigate negative\nRSFPs in conditions close to imaging settings, and this demands working at constant 488 nm energy dose.\nThe user would only deliver the necessary light dose to the sample, meaning that to reach the same off-\nswitched fraction at higher power densities, one would illuminate for a shorter period. This constraint does\nnot apply to conventional microscopy, where more illumination does bring to a steady increase of\nfluorescence. In super-resolution microscopy with negative RSFPs, the optimal spatial confinement is\nobtained at a fixed energy dose, i.e. when the illumination time roughly matches the off-switching time. In\nthis scenario, the shorter the 488 nm light the lower the chance for the population in the triplet state to do\nRISC by 488 nm illumination. This defines the opposite behavior with respect to a conventional fluorescent\nprotein where the 488 nm can always be increased to obtain a higher signal, and indeed this was the\nillumination condition explored by Ludvikova et al., 2023, Nat. Biotechnol.\nWe have now clarified this aspect in the main text.\n9/ As shown by the authors the RISC effect can as well be obtained by near-infrared light, at 810 or 900\nnm. In fact, near infrared light should be the privileged choice, as absorption of the triplet state around 900\nnm has been shown to be maximum in green fluorescent proteins. The authors have used 592 nm light\nprobably because this was technically easier (already implemented on their microscope). Interestingly, they\nfound that the achievable effect with 592 nm surpasses that at 810 nm, even with the huge power densities\nthey used in the near infrared. It would be nice to give a precise interpretation of what might be going on.\nAt the end of supplementary note 7, the authors claim that the comparison is difficult based on the fact that\nthe “spatial distribution of light-induced kinetic processes is radically different”. This statement is unclear\nto me, it would be nice if the authors could elaborate more.\nIn all cases it looks like, from supplementary figure 13, that no matter what intensity is used, the level of\nlight induced recovery at 900 nm cannot be reached at 810 nm. Would the authors have a clue whether this\nis true and if yes, why ?\nAt the end, using visible red light to promote RISC is nice, as this wavelength is available right away on\nmany microscopes. However, the authors should comment on possible penalties e.g. for 2-color\nexperiments requiring 592 nm light to excite a second red fluorescent marker.\nThe difficulty in comparing 810 nm and 592 nm stands from the different experimental setups used for\nthose two measurements: point-scanning with pulsed lasers on one side and widefield (~30 µm in diameter)\nwith continuous wave lasers on the other. For this reason, in the work, we have decided to compare 810 nm\nwith 900 nm, using the 810 nm as the control wavelength where we can assume a cross section for T1\nsimilar to the one at 592 nm.\nWe agreed that it would have been valuable for the reader to have a direct comparison, therefore we have\nperformed a new experiment where we directly compare 592 nm and 915 nm (as it is laser line available in\ncommercial CW laser) on the widefield microscope. As can be seen in Fig. R7 at 915 nm the recovery is\nhigher than at 592 nm. This aligned with Rane et al., J Phys Chem B, 2023, where the triplet state absorbs\nmore efficiently 915 nm than 592 nm light. At the same time, it is counterintuitive with the expectation of\nhigher RISC probability for higher excitation energies (see also major point 1 of reviewer #1).\nSimilarly to what observed in the confocal data comparison, there is higher recovery when using 900/915\nnm light. At the same wavelengths the triplet state absorbs more efficiently according to Rane et al, J Phys\nChem B, 2023. The absorption cross-section of the triplet seems to be the dominant effect, compared to\nRISC efficiency and other pathways addressed by the red shifted light, following the trends reported in\nLudvikova et al, Nat Biotechnol 2023, where the higher RP effect matches the peak of the triplet absorption\nspectrum of EGFP.\nThe new data is now available in Figure 2j and Supplementary Note 10.\nFigure R7. Light-induced recovery as a function of the 592 nm and 915 nm power density in the\nwidefield microscope. We observed a more efficient recovery as well as a higher maximum recovery\nvalue with 915 nm co-illumination than at 592 nm. The dashed lines correspond to an exponential\nfunction ((cid:1) = (cid:3)(1−exp ((cid:11)/(cid:13)))) fit performed on the data with k = 0.42 kW/cm2 and k = 1.33\n915 592\nkW/cm2.\n10/ It would be nice to see the experimental data of figure 1F on a linear scale and without the fitted model\ndata superimposed. It looks like these experimental data may follow a simple exponential growth on the\nused logarithmic scale, meaning a linear growth on a linear scale. Many readers could feel that using such\na complicated photophysical model to fit essentially linear data is largely unjustified. Could the authors\ncomment on this ? What are the factors that prevent from only relying on a linear photobleaching effects\nby the 405 nm light ?\nWe changed the plot of Figure 1f to linear scale, and changed the label to make it more clear.\nWe apologize for the lack of clarity in the main figure. Figure 1f of the main manuscript shows a section of\nthe experimental data (dark shaded data of Figure 1d) together with the expected trends computed from the\nkinetic model. Supplementary Note 4 explains the procedure to fit the bleaching quantum yields of the\nkinetic model. Briefly we fit only two parameters for the kinetic model with only bleaching from the triplet\nstates (Φ , Φ ) and three parameters for the kinetic model with also bleaching from\nBleach-triplet Bleach-triplet-excited\nthe off-states (Φ , Φ , Φ ). For the actual fitting we use a subset of the\nBleach-triplet Bleach-triplet-excited Bleach-trans-neutral\nexperimental data as shown in Supplementary Figure 7b of the revised Supplementary Information. Thus,\nFigure 1f of the main manuscript shows the extrapolated trend predicted from the kinetic model for a range\nof 405 nm power density. The computed fatigue fraction contains all the bleaching contributions included\nin the kinetic model.\n11/ I am puzzled by supplementary figure 4: the data in this figure show the rapid growth of the integrated\nfluorescence which then levels off without any sign of further decay. On the contrary, the model predicts a\ndecay, as Int is more and more redirected towards TH when the 405 nm power density is increased, and this\ndecay is clearly visible in the fitted data. The experimental data here clearly seem to deviate from the model.\nFurthermore, the data of figure 1B seem to go in the same direction, as the initial point in the photofatigue\ndecay curves appear to be significantly higher at 0.7 kW/cm² as compared to 0.22 kW/cm², as opposed to\nwhat the model would predict. In their experiment of figure S4, could the authors increase further the\nintensity of the 405 nm illumination to start seeing the expected decay (maybe using their confocal\nmicroscope) ? Could it be that Int may also be photoconverted to C- by violet light, so that the relative\namount of Int molecules sent to either C- or TH be relatively independent of 405 nm illumination ? It is\nknown that chromophore deprotonation in FPs can be accelerated by light. These aspects should be\ndiscussed.\nWe investigated the apparent drop in the simulated integrated fluorescence curve by extending the\nexperimental investigation at higher 405 nm photoactivation dose. We increased the 405 nm illumination\ntime as we were limited in the power density we can deliver at the sample plane with the widefield\nconfiguration of the microscope. As shown in Figure R8a, increasing the illumination time yields a faster\nrise of the fluorescence signal at lower 405 nm, however, there is a decrease of the fluorescence at higher\ndoses, and that drop is more significant for longer illumination times. Similarly, the normalized\nfluorescence in Figure Rb decreases at > 1 J/cm2. Taking this into consideration, we rationalize the\ndiscrepancy between the simulated and experimental curve in the previous Supplementary Figure 4 as an\noverestimation of the delivered power density to the sample.\nWhen we calculate the power density for a given condition, we assume that all the power measured at the\nback aperture of the objective will be delivered in the sample plane across an area with a diameter equal to\nFWHM of the beam profile (~ 30 - 40 μm). We choose our experimental region of interest (ROI) small\nenough so that the power delivered in the ROI is constant. We estimate that the discrepancies observed\nbetween simulations and the data stem from these considerations: i) there might be a loss of laser power\nbetween the point of measurement and the sample plane, ii) the extension of the beam profile that is relevant\nto the calculation of the power density in the sample plane is larger than FWHM, iii) the power across the\nexperimental ROI is not constant which will lead to a reduction the photoactivation efficiency away from\nthe center of the beam.\nFigure R8. (a) Normalized fluorescence as function of the 405 nm energy. The data are coming from\nthree dataset acquired over the same interval of power densities but increasing illumination time (1, 2\nand 3 ms). (b) On-switching curve data and fit. Each data point is an average of 9 complete photocycle\nrepetitions. The simulation consisted of a 5-state model without triplet and bleaching pathways. The\nparameter α scales the 405 nm power density such as the simulation matches the experimental data points.\nTo quantify the discrepancy between the experimental observations and the simulation output, we\nrecompute the on-switching curve with an additional correction factor α (0.642 ± 0.06) that scales the power\ndensity such as the simulation output best matches the experimental values. We deemed that the best way\nto account for this source of experimental error is to study the correlation between the different parameters\nusing the simulation tool. The resulting Φ and εINT from the model fitting in Supplementary Figure\nINT→TH 405\n5 are to be understood in the context of a family of solutions where certain experimental parameters that\ngreatly influence the model fit have an implicit uncertainty and correlation among each other.\nThis correction in power density is now applied in Figure 1a, 1b, 1d, 1f and 2a as well as Supplementary\nFigure 5 and 7 where we studied the dependency of the 405 nm power density and updated the experimental\n405 nm power density values in Supplementary Table 10. The correction is introduced in Supplementary\nNote 3.\nConcerning the question about a light driven pathway connecting CH and C-, we have ourself faced this\nint\nquestion when building the kinetic model. In all the recent spectroscopic literature on negative RSFPs that\nwe have consulted, the light always induces the cis/trans isomerization through a conical intersection\nrelaxation. CH is a protonated species (Woodhouse et al Nat Comm 2020) thus we have assumed a\nint\ncis/trans isomerization toward TH or a similar species. Of course, we would be happy to consult the\nliterature that the reviewer is suggesting that could help the interpretation of the photoinduced processes of\nCH .\nint\n12/ The scheme in Fig1e is difficult to read: the triplet state between C- and B should be shown in clear,\nnotably as the trans states (T) could be confused by some readers with the triplet state (T1). The protonated\nchromophore state in equilibrium with C- should be shown as CH, and the short-lived intermediates\npreceding Int along the on switching pathway should be given a different name.\nWe have updated Figure 1e with a simplified scheme that primarily reports on the two bleaching pathways\nidentified in the study: triplet-mediated and off-state-mediated bleaching, with the wavelengths that mainly\ndrive these pathways. An updated full scheme is now presented in Supplementary Figure 1b, where we\nhave clarified and corrected the nomenclature and identities of the different states considered for building\nthe kinetic model.\nOverall, I strongly encourage the authors to revisit their photophysical model in depth. They should also\nmuch improve the clarity of their manuscript so that the essential message carried by this model can be\ncaptured by the white audience of Nature Communication. At present, what I can extract from the\nmanuscript is that: (i) the effect of 405 nm illumination on photofatigue either relates to the triplet state\nonly via the amount of the on-state population at the beginning of off switching phases, or goes through a\nsinglet state photobleaching mechanism that is nonrecoverable by RISC and (ii) recoverable photobleaching\nthrough T1 only occurs upon 488 nm illumination of the on-state.\nThe comments of both reviewer #1 and #2 helped strengthen the model and define its working conditions.\nAs we mentioned also at the beginning of this point-to-point rebuttal letter, we conceive the model as a\ndescriptive tool rather than a predictive one. Overall, the model acts in support of the main observations of\nthe work that are exactly what the reviewer highlights.\nOne possible strategy that the authors could consider would be to investigate whether their photophysical\nmodel predicts a pH dependence of photofatigue and to carry out corresponding experiments to see whether\nthese predictions are realized. For example, at low pH the fraction of protonated chromophores will\nincrease, likely increasing direct bleaching by 405 nm light. It could also be expected that relaxation of Int\nto C- slows down, making the initial drop faster. In view of the complexity of the problem, those pH\ndependent experiments could help to narrow down the photophysical model.\nFollowing the reviewer suggestion, we performed a series of photoswitching fatigue experiments at\ndifferent pHs (6, 6.5, 7.5 and 8) and with varying 405 nm power densities. Across the 2-dimensional space\nof parameters we monitored the fatigue fraction observed in our data and compared it to the prediction of\nthe kinetic model. Overall, we see a very good agreement between our experimental data and the simulated\nfatigue fraction, especially notable at low 405 nm power densities. At higher 405 nm power densities, our\ndata shows a less pronounced pH dependency of the fatigue fraction than what is predicted by the kinetic\nmodel. To check whether the pH of the rsEGFP2-PAA embedded gel was correct we monitored the mean\ninitial fluorescence intensity as function of the pH and we see a sigmoidal shape as would be expected.\nOur photophysical investigation aims at moving close to live cell imaging conditions and therefore guides\noptimized imaging. For this reason, the majority of our experiments were carried out at the cell’s\nphysiological pH of 7.4, and we see a good agreement between experimental data and the model within a\nunit pH around the physiological pH.\nFigure R9. (a) Fatigue fraction as a function of the pH and the 405 nm power density. Overall, the\nsimulated photobleaching matches the experimental data across the different explored pH. At higher 405\nnm power densities, our data shows a less pronounced pH dependency than the prediction by the kinetic\nmodel. (b) Fatigue fraction data and simulation at different pHs. At pH = 6.0 our data shows a lower\nfatigue fraction than predicted by the simulation.\nOther major points\n13/ The authors nicely apply their methodology to both prokaryotic and eukaryotic samples. Figure 4A and\n4C suggest that there is a real benefit of applying 592 nm co-illumination. Yet, it is not clear whether the\nprotecting effect shown in the photofatigue decay curves of figure 4A is as pronounced as that observed in\nvitro. Could the authors comment on this ? Do they see a possible effect of polyacrylamide in augmenting\nthe effect, maybe about oxygen level and/or diffusion, and thus triplet state lifetime, and even maybe\npropensity for radical formation ? In view of those photofatigue decay curves in figure 4A, which after all\npredict a relatively faint improvement, how representative are the series of images shown underneath ? The\nauthors may want to show the photofatigue decay of this very sample in the SI. In passing, the first frame\nof the “with co-illumination” series appears less intense than the first frame of the “without co-illumination”\nseries. Does this correspond to a true, reproducible effect ? If yes, what happens ?\nTo help the transversal comparison among the different systems studied, from synthetic PAA layers of the\nRSFPs to cells, we have compared the fatigue in each sample under identical illumination conditions and\nthe recovered fraction under 592 nm co-illumination (Fig. R10).\nWe set the imaging conditions to maximize the temporal resolution of the acquisition with 4 ms total dwell\ntime and 60 nm scanning steps and monitored the fluorescence over 50 imaging frames. As shown in Figure\n10a, different live-cell samples (bacteria, vimentin, actin and mitochondria) have their characteristic\nbleaching curve. Additionally, we incorporated a sample of rsEGFP2 embedded in a PAA matrix at the\nsame concentration as in the characterization experiments. We observed photobleaching between 20 to 40\n% in eukaryotic cells, with actin labelled rsEGFP2 showing the higher bleaching resistance (29%\nnormalized fluorescence at the last frame in magenta) compared to rsEGFP2 tagged to vimentin and\nmitochondria (15% and 16% normalized fluorescence at the last frame, in yellow and green, respectively).\nIn prokaryotic cells, we observed the highest photobleaching resistance (52% normalized fluorescence at\nthe last frame in orange) which we attributed to the specific geometry of the sample, i.e. a think pellet of\nbacterial cell rather than a semi-2D sample like for the other samples. Similarly, we measured the\nphotobleaching with 592 nm co-illumination (15 kW/cm2 and 0.5 ms) added in the pulse scheme and we\nobserved a decrease in photobleaching in all samples, specifically higher in bacteria (21% recovery in\norange) and in the PAA layer (13% in blue). An interpretation of these results can be linked to a slower\ndiffusivity of molecular oxygen both in biofilms (De Beer, D., et al, 1994, Biotechnol. Bioeng., Stewart,\nP.S., 2003, J. Bacteriol.) and in the PAA matrix (Hepworth, S,J,, et al, 1999, Phys. Med. Biol., Ju, L.-K., et\nal, 1986, Chem. Eng. Sci., Marek, P., et al., 2014, J. Sens. Sens. Syst.), which can prolong the lifetime of\nthe triplet and reduce its reactivity downstream to generate irreversible photobleaching. In this context,\ngiven that live-cells are imaged in aqueous media, is plausible that the reactivity of the triplet state of\nrsEGFP2 generates more photoproducts that can lead into photobleaching and the observed fluorescence\nloss.\nRegarding the difference in the first frame of Fig. 4a, the reason lies in the variability of expression levels\nassociated with transient transfection, explicitly shown in Supplementary Figure 25c as the bleaching\nconstant against the initial signal-to-background ratio. The average raw and normalized bleaching curves\nhave now been added to the same Supplementary Figure 25 for completeness.\nThe presented comparative study is now reported in Supplementary Note 14.\nFigure R10. (a) Intensity traces for a variety of samples containing rsEGFP2 imaged in parallelized\nconfocal mode. In blue, a rsEGFP2-embedded polyacrylamide layer; in orange, a biofilm of\nEscherichia Coli expressing rsEGFP2; in yellow, rsEGFP2-vimentin in U2OS cells; in magenta,\nrsEGFP2-ActinChromobody in U2OS cells, and in green, rsEGFP2-OMP2 in U2OS cells. The lines\nrepresent the mean intensity traces, and the shaded area shows ± σ from the mean. (b) Light-induced\nphotobleaching recovery with 592 nm co-illumination in the aforementioned samples. The 592 nm\nillumination dose were 0.5 ms at 15 kW/cm2. The bars represent the mean light-induced recovery\nand the error bars shows ± σ from the mean.\nThe Mona Lisa data are also nice and seem to show a significant gain. Would there be a way to more\nquantitatively assess the spatial resolution loss along multiframe data collection with and without co-\nillumination, rather than showing just one profile at a user chosen position (which is the classical old\nprocedure often criticized for potential bias) ? Possibly, a number (~10) of random locations across multiple\nfibers could be picked and the profiles presented in the SI ?\nFollowing the suggestion of the reviewer we have extended the characterization of the SNR and spatial\nresolution at different time points within MoNaLISA timelapse recording of U2OS cells tagged with\nLifeAct-rsEGFP2 (Figure 4c in the main text). We traced ~ 20 – 40 line profiles of actin filaments per image\nand performed a Lorentzian fitting with a custom-written MATLAB script. We calculated the SNR as well\nas the FWHM of the Lorentzian peak of each line profile in the first and last frame of each timelapse\nrecording. As shown in Figure R11a, the SNR with or without 592 nm co-illumination is higher in the initial\nframe (median values 14.01 with 592 nm, 10.88 without), however we observed a greater reduction in SNR\nwithout 592 nm co-illumination (~ 40 % decrease in SNR after 20 RESOLFT imaging frames) than when\nthe 592 nm co-illumination is applied (~ 27 % decrease in SNR after 20 RESOLFT imaging frames). Along\nwith the intensity traces shown in Figure 4c of the main text, the SNR quantification highlights the benefits\nof incorporating the 592 nm co-illumination for prolonged timelapse imaging in intensity-demanding\ntechniques such as MoNaLISA. At the same time, we quantified the FWHM measured on > 100 actin\nfilaments (over 4 – 5 cells) per condition in Figure R11b. Considering that our determination depends on a\nfitted parameter, what we see as a result of the reduced SNR is a broadening of the FWHM distribution\n(Figure R11b) and an increase on failed fitting score (Figure R11c) at the 20th frame without co-\nillumination.\nThis further quantification is now reported in detail in Supplementary Note 14 and Supplementary Figure\n26.\nFigure R11. (a) SNR of U2OS-LifeAct-rsEGFP2 measured from > 100 actin filaments per condition.\nThe size of the box represents the 25th-75th percentiles of the data, and the lines represent the median\nvalue. The whiskers extend to 1.5 x the interquartile distance below or above the 25th-75th percentiles.\nThe reduction in SNR at the 20th frame is less significant when 592 nm co-illumination is applied (27\n%) than when is not (40 %). (b) FWHM from Lorentzian fit of U2OS-LifeAct-rsEGFP2 measured\nfrom > 100 actin filaments per condition. The size of the box represents the 25th-75th percentiles of the\ndata, and the lines represent the median value. The whiskers extend to 1.5 x the interquartile distance\nbelow or above the 25th-75th percentiles. The distribution of sizes of the measured filaments in the 20th\nframe without 592 nm co-illumination is skewed towards broader filaments indicating a loss of\nresolution. (c) Percentage of fits that lie within 3σ of the median. Applying the 592 nm co-illumination\nincreases the number of representatives fits at 20th frame.\nMore minor issues\n1/ The authors investigated the effect of modulating the on switching light (essentially adding dark times,\nor reducing power density at constant dose, both resulting in increased data collection time) in the context\nof their photophysical studies, but they have not implemented such strategies for data collection of\nbiological samples. Thus L19 in the abstract does not really reflect the content of the paper.\nFollowing the reviewer’s suggestion, we verified the direct influence of on-switching time modulation on\nimaging. In Figure R12 (now Supplementary Figure 14 and Figure 2b), actin filaments labeled with\nrsEGFP2 were imaged using on-switching times of 20 ms and 0.25 ms for a power density of 0.14 and 1.05\nkW/cm2, respectively; the displayed image represents the pixel-wise ratio of these two acquisitions. Because\nfluorescence increased in the image recorded with a longer 405 nm illumination duration at lower intensity,\nthe histogram of the resulting ratio is shifted above 1.\nFigure R12. (a) Slow image pulse scheme. The 405 nm illumination dose was set to 20 ms and 14 W/cm2,\nthe fluorescence was captured with a 488 nm read-out pulse (500 W/cm2 and 1 ms). (b) Fast image pulse\nscheme. The 405 nm illumination dose was set to 0.25 ms and 1.05 kW/cm2, the fluorescence was\ncaptured with a 488 nm read-out pulse (500 W/cm2 and 1 ms). (c) Ratiometric image (Slow image /\nFast\nimage). The pseudocolor of each pixel in the actin fibres shows the value of the intensity ratio. (d)\nHistogram of the pixel values in (c). The colour bar on top represents the pseudocolor in associated to\neach bin in the histogram.\n2/ P2-L45/46: this sentence calls for references\nWe have now added a reference to a review article in the above-mentioned position.\n3/ P3-L64/65: maybe replace “dark state” by “triplet state”: as is, it looks like the authors are not sure of\nthe nature of the state, whereas their photophysical model clearly identifies the triplet state.\nWe have now updated the text accordingly, clearly addressing the triplet state since the beginning of the\ntext.\n4/ P3-L75: the authors might be interested to read: Adam et al", "reviewer_author_response": "NCOMMS-25-05441\nPoint-by-point responses to reviewers of the manuscript NCOMMS-25-05441:\nAll-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent\nProteins\nMarin-Aguilera et al\nWe appreciate the comments and the suggestions of the Reviewers. All the inputs helped to improve both\nthe structure and clarity of the manuscript.\nIn the revised version we integrated the comments of both reviewers to strengthen and clarify the model.\nThis brought us to uniform the model with previous literature on triplet and fast photoswitching studies of\nrsEGFP2. Through simulation and experimental work, we have validated the drawn photocycle for\nrsEGFP2 as well as tested its expansion to incorporate additional states and the different pathways\nhighlighted by the reviewers. In the text we have now clarified the limit and strength of the model, that is\nintended to be an effective model rather than a comprehensive theoretical description.\nBelow we provide a point-by-point response to the comments of each Reviewers. The comments of the\nReviewers are written in blue and our responses in black.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nReview of NCOMMS-25-05441, “All-Optical Strategies to Minimize Photo-Bleaching in Reversibly\nSwitchable Fluorescent Proteins,” by Marin-Aguilera et al.\nThis paper develops optimized methods for using light to remove photoswitchable fluorescent proteins from\nundesirable triplet states that trap population for long time periods. This strategy not only makes the proteins\nless likely to photobleach, but also makes these molecules available to fluoresce more often. The\nexperimental work in this paper is both thorough and compelling, and the work will be of great value to the\ncommunity of researchers who use these proteins. The authors demonstrate that this strategy works for\nother green-emitting fluorescent proteins, and for a number of imaging modalities. I feel that this paper will\nhave a high impact among those in the advanced optical imaging community, and is ultimately worthy of\npublication in Nature Communications.\nWe appreciate the reviewer’s positive evaluation of our work.\nSince the concerns raised all pertain to the model, it seems best to first clarify its intended role within our\nstudy. Based on our experimental findings, we constructed a minimal, effective kinetic model to provide a\nmechanistic explanation. In bleaching photophysics, kinetic models are often simplified, because\nfluorophore bleaching typically arises from complex, oxidative reactions involving many environmental\nfactors and chemical species. Adding too many free parameters would weaken the model’s predictive\npower; conversely, reporting only the experimental trends would leave readers without a framework for\ninterpreting these inherently complex photophysical processes. Hence, we developed a minimal kinetic\nmodel: we began with the established core-state scheme for rsEGFP2 and added only those extra states and\ntransitions required to capture our experimental observations. We made simplifications and added effective\npathways, and we thank the reviewer for helping us clarify our assumptions.\nThere is one crucial issue that I feel needs to be addressed first, as well as a range of minor issues. Please\ndo not take the length of the list of the issues I have identified as a negative, I would simply like to see this\nwork have as much impact as possible.\nTo begin with my major concern, the model that the authors present is far from convincing to me; in fact, I\nam pretty certain that it is flawed, for several reasons. First, along with virtually all of the prior literature in\nthe field of photodeactivation of triplet states, this study makes the unfounded assumption that RISC solely\nleads to the population of an excited singlet state. In most circumstances, a more likely outcome is the\npopulation of a highly vibrationally excited level of the singlet ground state, because the vibrational density\nof states of S0 at this energy is vastly higher than that of any singlet excited state (this behavior is\nchallenging to prove, but it can be done; see, for instance, Ref. 28 in the paper and\ndx.doi.org/10.1016/j.isci.2021.103600; as this journal encourages reviewers to reveal their identities, I will\nsay at this point that this publication is one of mine.). This process, which we in the field typically (and, I\nwould argue, incorrectly) call internal conversion (IC) in the case of T1, explains why phosphorescence\nquantum yields are not typically more than a few tens of percents for organic molecules. Indeed, the typical\nway to attain a high phosphorescence quantum yield in such molecules is to have the T1 energy be well\nbelow that of S1, which not only prevents RISC to S1, but also lowers the probability of IC. The vibrational\ndensity of states of S0 grows exponentially with increasing energy, and so the higher the triplet state, the\nfaster RISC to the ground state will be as compared to RISC to an electronic state with an origin of similar\nenergy to the triplet state. That does not mean that no population will undergo RISC to excited singlet states,\nbut in many, if not most, organic systems the branching ratio for RISC to excited singlet states is likely to\nbe small. As an example, in Ref. 29 the authors had to invoke photobleaching from the triplet state that also\ncauses repopulation of the singlet manifold to explain the fluorescence intensity does not depend linearly\non the intensity of the deactivation wavelength, but rather saturates. This behavior is a natural consequence\nof RISC going to the S0 state directly, as can be shown by trivial, analytical kinetic modeling.\nWe acknowledge the literature complexity of the triplet to singlet transition. In outlining the used model,\nwe have built on the cited literature, nevertheless we took the reviewer’s comment as an inspiration to\nvalidate and compare the different pathways in the context of our data as well as to better ground our\ndecision. Our intention is to have a solid effective model that is able to reproduce the experimental data of\nphotobleaching.\nThe extended spectroscopical studies recently reported on rsEGFP2 (among particularly insightful for this\ncontext is Rane et al. 2023, J. Phys. Chem. B ) allow us to have an indication of where the triplet states are\nin the energy ladder (Byrdin et al., 2018, J. Am. Chem. Soc) or which are their absorption properties (Rane\net al. 2023, J. Phys. Chem. B, Byrdin et al., 2018, J. Am. Chem. Soc) (Fig. R1). However, they lack\nFigure R1. Schematic of energy ladder of singlet and triplet states for rsEGFP2 in eV. The position of\nS1 was determined from the maximum in the emission spectra (Grotjohann et al., 2012, eLife). The\nposition of T1 was determined from the maximum in phosphorescence emission spectra of EGFP (Byrdin\net al., 2018, J. Am. Chem. Soc) as an approximation of the energy level of the first triplet state of\nrsEGFP2. The energy levels of the subsequent excited triplet states were calculated as E(λ) = 1.69 +\n1.24/λ, with λ in μm. The colored arrows indicate which wavelength would populate each of the energy\nlevels: 3.06 eV, 900nm; 3.78 eV, 592 nm; 4.24 eV, 488 nm; 4.75 eV, 405 nm.\nconclusive evidence for which is the precise mechanism that leads to the repopulation of the singlet form\nvia RISC. In the literature, the effects of light-induced RISC in EGFP (Ludvíková et al., 2023, Nat.\nBiotechnol.) and rsEGFP2 (Byrdin, M., et al., 2025, Biol. Cell.) mainly assume that any conversion of\npopulation from the triplet to the singlet will occur via excited states. The existence of such pathway in\nfluorescent proteins has been proven and observed by means of optically-activated delayed fluorescence\n(OADF) (Peng, B., 2021, J. Phys. Chem. B., Lu, Y-H., 2023, J. Phys. Chem. B.) where a fluorescent photons\nare measured after illuminating the sample with red-shifted light that is delayed in time with respect to\nfluorescence excitation. This phenomenon is possible if the RISC mechanism involves the excited states\nfrom the singlet manifold, but as the reviewer points out this mechanism might not be the dominant RISC\npathways in typical organic dyes.\nAs the reviewer suggests, RISC could happen from excited triplet states directly into the vibronic manifold\nof S followed by intramolecular vibrational relaxation (IVR), this would be supported by the fact that the\n0\ndensity of vibronic states increases exponentially with the excitation energy above a state. We recognize\nthat, since light-induced RISC events are rare compared to the overall fluorescence emissions in a typical\nfatigue experiment, the final influence of landing to S or S after a RISC/IVR should not produce\n1 0\nappreciable change in our observable. If light-induced RISC populates S , in a few nanoseconds the great\n1\nmajority of the events will lead anyway to S , with a very small chance of ISC (Φ 10-3).\nWe explored whether channelling RISC T S or RISC T S would have a sensible impact on the\nn 1 n 0\nnumerical results of the kinetic simulation of the fatigue experiments. This was verified by simulating the\nlight-induced fatigue recovery in two identical simulation settings, with all the parameters as already\ndescribed in the main text and Supporting Information Note 1. The only change in the two conditions is the\nconnection of the transition from the excited triplet state to the singlet states. In one case T converts to S\nn 1\n(as in all the remaining simulations in the manuscript) and in the second case T converts to S . The dashed\nn 0\narrows in Fig. R2 represents RISC/IVR, thus including the vibrational relaxation. There are no notable\nchanges in the computed light-induced recovery.\nFigure R2. Example of light-induced photoswitching fatigue recovery computed as a function of the 592\nnm power density at a fixed 488 nm power density (300 W/cm2). (a) Condition with RISC mechanism\nT S . (b) Condition with RISC mechanism T S . All the simulations were carried out at 1 ms 405\nn 1 n 0\nnm and 100 W/cm2 power density.\nIn the main manuscript, we decided to keep the original RISC T S mechanism, because we believe it is\nn 1\nmore consistent with the cited literature and does not produce relevant changes for the results of our work\ncompared to RISC T S . Nevertheless, in order to make the reader aware of this discussion, we included\nn 0\nthe presented discussion and reference in Supplementary Note 8, properly referring to it in the main text.\nSecond, the model, at least as shown schematically, treats all Tn states as having identical behavior.\nGiven what the authors observe, this is manifestly impossible. As mentioned above, the higher the triplet state,\nthe more likely RISC is to occur (and 488-nm light was indeed shown in Ref. 28 to cause RISC to S0,\nwhich is missing from the authors’ model), but at the same time it may also be more likely that some sort of\nphotobleaching event occurs. There is absolutely no reason to believe that the branching ratio for these\nphenomena (not to mention relaxation to lower triplet states) should be the same following triplet absorption\nwith two disparate colors of light. The triplet absorption spectra in Ref. 27 (which predates Ref. 28 and is\nmore relevant to the work presented here) make it quite clear that the different wavelengths used here go to\ndifferent excited triplet states.\nAs the reviewer rightfully points out, it is uncommon that different wavelengths share the same RISC\nefficiency. Indeed, the different wavelengths are parameterized separately in our model, but notably we\naccount only for the different absorption cross-section as reported in Rane et al. J. Phys. Chem. B 2023, but\nthey share the effective RISC quantum yield. Such effective quantum yield is retrieved by fitting of the\ndata.\nAt this point it is important to consider the nature of our experimental data: the loss in fluorescence after\nconsecutive on/off cycling. This is not information rich, as data coming from more conventional time\nresolved spectroscopy methods. Considering the limited predicting capability of such data, we have fixed\nmost of the parameters from literature and helped where possible with ad-hoc experiments. The RISC\nparameters are instead directly fitted. Overall, we believe that a finer description of RISC would not add\nsubstantial mechanistic insight, which is the primary aim of our modeling. Our RISC pathways are to be\nintended as effective ones and could be refined in the future if additional experiments and work will be\navailable regarding the details of RISC in rsEFGP2.\nWe made clear in the main text and Supporting Note 8 that RISC from different T states in general has\nn\ndifferent efficiencies, and that we are working with a minimal effective model still able to reproduce\nsatisfactorily the data.\n“Similarly to recent literature on light-induced RISC in EGFP32 and rsEGFP245, we rationalize the\nrepopulation of the emissive state via RISC from the triplet excited state (T ) to a vibronic state of the singlet\nn\nmanifold (Supplementary Note 1 and 8). All the different laser lines of the experiment (405, 488, 592, 915\nnm) will excite the triplet state31, stimulating both bleaching and RISC.”\nThird, although I appreciate that the authors did their best to use literature values for rate constants, I do not\ntrust much of the literature based on the points raised above. Their model contains so many processes and\nrate constants that it could reproduce almost anything, even though the model is missing the processes\ndiscussed in the previous two paragraphs. Indeed, in my view their model is not compatible with the results\nin Ref. 28. Developing a model that incorporates verifiable photophysics will take a considerable amount\nof additional experimental work, and I would suggest leaving that for a future paper. Others will likely have\na different opinion than mine, but I feel that the modeling work detracts from the paper because, in my\nopinion, it is at best incomplete, and more likely is inaccurate. Although some of the experiments performed\nmay have been motivated by the model, presenting the interesting results while having those results be\nmotivated by a model that will elicit skepticism in readers like me proves a distraction. I suspect that this\nopinion will (understandably) not be popular with the authors at first (and maybe afterwards as well), but I\nurge them to consider my suggestions strongly. It is better to get the modeling right (or to prove the current\nmodeling to be correct) in a subsequent paper (which I feel will also have high impact) than to dilute the\nimpact of impressive experimental work with a model that cannot currently be substantiated.\nWe understand the reviewer perspective and agree on the importance of grounding the kinetic model in\nverifiable photophysics. In the revised main manuscript, we refocused the kinetic model’s role and\ndrastically reduced its overall importance.\nThe model is now presented strictly as a minimal, descriptive framework that helps readers follow the\nexperimental trends. We have flipped the order of some paragraphs in the first two sections, first we report\nthe data and then we follow with the comparison with the kinetic model. Furthermore, all forward-looking\nor predictive sections have been moved to the Supplementary Information, meaning former Fig. 2h and its\ndiscussion is now moved to Supplementary Note 9 where we expand and clarify the limitations.\nWe agree with the reviewer that the experimental part of the manuscript could be self-consistent. At the\nsame time we believe that without a simple kinetic model the reader would not understand in depth the\nexperimental results. This is primarily because rsFPs have a complex photophysical switching behavior,\nalready without considering bleaching pathways and triplet states. A simplified effective kinetic model,\nalthough with limitations and simplifications, is a great tool for the reader to navigate the experimental\nresults.\nWe have now first stated the intention of the model at the very beginning of the manuscript:\n“Using this information, we built a minimal effective kinetic model for rsEGFP2 that could aid the\nmechanistic explanation of the observed photoswitching fatigue behavior (Supplementary Note 1)”\nMore clearly framed the simplification and assumption of it, this reflects both throughout the main text and\nin the extended and revised Supplementary Information. Also following the comments of Reviewer #2,\ndirected to the model, we now have added the following Supplementary Note related to the model:\n- Supplementary Note 5. Thermal relaxation of long lived dark in rsEGFP2\n- Supplementary Note 8. Higher excited triplet state and risk mechanisms.\nFinally, we conclude putting in prospective future expansions of the model to bring it outside of the live\ncell imaging context.\n“Our results showed that a deeper understanding of the species involved in the photocycle of RSFPs as well\nas the mechanisms governing photobleaching can lead to longer and higher quality live-cell imaging\nexperiments. Further expansion of the model, for example, by incorporating additional long-lived dark\nstates (e.g., radicals) or extended pH equilibria, would generalize it to a wider range of experimental\nschemes beyond live cell imaging.”\nWe believe these changes keep the valuable mechanistic guidance the kinetic model provides, while\neliminating the risk of over-interpretation, without weakening the experimental narrative.\nOther, more minor points that need to be addressed, in no particular order of appearance or degree of\nconcern:\n1. The authors play fast and loose with the optical spectrum. 405-nm light is violet; there is no such color\nas “UV-vis.” 592-nm light is orange (and barely so, it’s almost yellow), not red. Calling light “red-shifted”\nis confusing, and makes it sound as if the authors are looking at a side-band of another laser they used.\nPlease give this light a clear name, such as “light at the triplet deactivation wavelength.”\nWe understand the confusion that the term “red-shifted” may have caused to the reviewer. We clarified in\nthe main text the precise meaning of the term, i.e. light of a wavelength that is shifted towards the red side\nof the visible spectrum compared to the primary fluorescence excitation wavelength. We also remark that\n“red-shifted” is a commonly used term in spectroscopic literature, that does not necessary mean that the\nlaser light we used is of color red, but that is shifted to longer wavelengths or lower energies. In general,\nwe have improved the main text and tried to avoid confusion regarding the term.\n2. The authors use a lot of vague language in the paper, for instance talking about “several” rounds of\nphotoswitching, and using many unclear antecedents (“it” and “this”) that made the paper hard to read for\nme in some passages because I had to go back and figure out the antecedents. As I teach my students, this\nproblem can be fixed by avoiding the use of “it” to refer to something prior, and to always follow “this” at\nthe start of a sentence with a noun.\nWe improved the main manuscript by reducing the instances of antecedents. We agree that in this way the\nreadability of the text is improved.\n3. Many of the figure panels were difficult for me to parse (e.g., Figure 1d, Figure 1e, Figure 2a, Figure 2h,\nFigure 2 i,j). Many of the graphs are deceptive, with y-axes not starting at zero, thus exaggerating small\neffects. At very least, broken scales should be used in such cases. In Figure 2a in the right panel, please add\na second x-axis on top that gives the intensity. 90 mJ/cm2 (note that the 2 is not an exponent in the figure)\nis not an energy. To say that the model reproduces the data in Figures 2e,f is really a stretch. Within your\nuncertainty, the data in Figure 2f have no dependence on t whatsoever. I also cannot read whatever is in\nthe color bars for the heat maps in Figures 2i,j.\nWe have updated the mentioned figures to enhance their clarity.\n- Broken axes have been added to more clearly reflect the magnitude of the change.\n- Figure 2a now reports both illumination time and power density of 405 nm, and the exponent has\nbeen fixed.\n- For old Figure 2f (now Figure 2g) we collected a new dataset. The updates in the model and finer\nsampling defined a better accordance between experimental data and simulation. Previously, the\n488 nm illumination time (0.9 ms) was shorter than the 592 nm pulse (1 ms) while now both 488\nnm and 592 nm pulses are 1 ms. With these experimental parameters, we can now better reproduce\nthe data trends, especially the increase in light-induced recovery observed between 0 ms delay and\n1 ms delay.\n- The labels on the color map are now in black for clarity or outside the bar.\n4. There are places in the paper where the authors refer to a spectrum without saying type of spectrum. An\nexample is line 51, “…the…triplet state of PPs has a red-shifted spectrum….” I presume that they mean\nabsorption rather than phosphorescence, but they need to be clear. Another example is in line 180.\nWe have now corrected the mistake in the paper.\n5. There are quite a few places in which numbers that should be exponents are not.\nWe have now addressed it.\n6. In the first sentence of the paper, the abbreviation FP is used without definition. There are other similar\nproblems throughout the manuscript (e.g., EGFP, SNR).\nWe have updated accordingly.\n7. In the second paragraph of the results section, it would be quite helpful if the authors explained what\nthey mean by an “excitation cycle.” The meaning only becomes clear later on in the paper.\nWe have rephrased the sentence to solve its ambiguity; the text is now:\nWe homogeneously illuminated a region (20 × 20 μm2) of a thin layer of rsEGFP2 embedded in\npolyacrylamide (PAA) gel with consecutive on-and-off photoswitching cycles. The emitted fluorescent\nsignal during each off-switching phase was recorded until a decrease of 80%, mimicking the imaging\nconditions, and the photoswitching fatigue curve was built by repeating the photocycle hundreds of times\n(Fig. 1a).\n8. There are many “gratuitous” hyphens following words that start with “photo.” For instance, the correct\nword is “photophysics,” not “photo-physics.” “Infrared” also does not have a hyphen.\nWe have made corrections throughout the text.\n9. “Data” is plural, not singular, and Nature correctly follows that usage.\nWe corrected to the plural form of “data”.\n10. Many people who might use the results presented here in their own work have commercial fluorescence\nmicroscopes that are not capable of simultaneous exposure at two different wavelengths and are not able to\ndo the sort of multiplexing that the authors demonstrate. Are the authors able to test how much improvement\ncan be achieved if only alternating scans of different colors can be used? If this idea is not easy to pursue,\nthat is not crucial to the publication of this work.\nOur results indicate that the fatigue‐resistance benefit of >592 nm co‐illumination remains apparent in a\nsequential scheme, provided the interval between the 405 nm + 488 nm excitation and the >592 nm recovery\npulse is under 2 ms. In a scanning microscope, this could be implemented by interleaving the 592 nm\nillumination with each line or pixel scan, or by reducing the field of view to shorten the overall frame‐\nacquisition time.\nTo end on a positive note, this work is quite impressive, and I hope to see the paper published in this journal.\nPlease know that all of my comments are meant in an entirely constructive manner; the topic of your work\nis highly complex, and many of the issues that I have brought up are not well understood or appreciated in\nthe community. I would like to see you publish this work in a manner that is not only exciting, but also\n“bullet-proof.”\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Marín-Aguilera et al provide an in-depth study of photobleaching mechanisms in\nreversibly switchable fluorescent proteins (RSFPs) and introduce means of reducing photobleaching under\nconditions typical of RESOLFT super-resolution microscopy by the addition of red-shifted illumination\nduring switching cycles or possibly dark periods between switching cycles. The paper is a natural follow-\nup of the recent work of the group of Agathe Espagne (ref 29) who showed that near infrared light is able\nto induce reversed intersystem crossing from the triplet state of standard green fluorescent proteins such as\nEGFP, shortcutting photobleaching pathways typically arising downstream this state. Extending the work\nto the case of RSFPs is important due to the numerous applications that these markers offer in advanced\nmicroscopy approaches.\nThe paper is divided into three sections: (i) a thorough experimental investigation of photofatigue decay of\npurified polyacrylamide-embedded rsEGFP2 as a function of illumination conditions, (ii) an experimental\ninvestigation of the level of recovery provided by red-shifted illumination, reaching up to ~15%, and finally\n(iii) practical realization in the context of biological samples, showing a moderate but significant effect for\ntime-lapse experiments using either parallelized confocal or RESOLFT microscopy.\nIn addition, the authors introduce a novel technique for kinetic-based RSFP multiplexing based on differential photofatigue\ndecays between different variants.\nAll the work is embedded in a conceptual framework based on an elaborated photophysical model of\nrsEGFP2, allowing the authors, through extensive simulations, to account for the experimental observations\nmade and possibly predict optimized behavior of RSFPs in a variety of imaging experiments.\nOverall, this represents a large body of interesting and timely work, clearly to the benefit of photo physicists\nand fluorescent protein developers, and potentially to the benefit of the large community employing RSFPs\nas labels in their biological experiments. The photophysical aspects are treated in the specific context of\nintense illuminations, typical of RESOLFT-like experiments, which is rarely addressed in the rich literature\nconcerned with RSFPs photophysics. This makes the paper particularly interesting to photophysicists. Thus,\nin principle I am supportive of this work. However, I have many interrogations concerning the validity of\nthe proposed model. To my opinion many issues remain to be solved, that may either stem from a certain\nlack of clarity (the paper is highly complex to read and properly digest) or be related to more serious\ninconsistencies. All these issues call for a major revision of the manuscript.\nWe thank the reviewer for the positive comments and the thorough revision that made us clarify and expand\nthe investigation.\nMajor issues concerning the proposed photophysical model of Figure 1e and S1:\n1/ In principle the cis anionic and the cis neutral chromophore should be in rapid equilibrium, depending\non pH. This equilibrium is not represented in Figure 1e, but should be, as the cis neutral chromophore,\neven if it is not highly populated at physiological pH, is able to absorb 405 nm light, isomerize to TH,\nand probably photobleach under such illumination. I guess that in Figure 1e, CH is meant to represent the “cis\non like neutral” chromophore of ref 16, not the real “CH”. If this is correct, the “equilibrium” cis neutral\nchromophore is missing in the model.\nWe apologized for the confusion on the nomenclature. We have now clarified the terminology of the states\ninvolved in photoswitching of rsEGFP2 in Supplementary Note 1 and throughout the manuscript. We have\naligned the terminology to the one of Uriarte, et al. (J. Chem. Phys. Lett, 2022), CH† in our scheme is a\ntransient state corresponding to “Cis On-like neutral”. Fig. R3 shows the states of the model mapped over\nScheme 2 of Uriarte et al. J Phys Chem Lett 2022.\nFigure R3. Scheme of the proposed off-to-on photoswitching mechanism of rsEGFP2, adapted from\nScheme 2 of Uriarte et al. J Phys Chem Lett 2022. On the top we clarify the labels implemented in the\nrevised work.\nCH† is formed from TH by an excited state isomerization reaction upon 405 nm illumination and evolves\nfurther to C- by consecutive relaxation steps. Given the time resolution of our experimental investigation,\nall the fast relaxation steps happening in the ps – ns timescale are indistinguishable, and we have collected\nthem over CH†. The last step of this chain relaxes in the μs and therefore it is relevant and observable in\nour data (Woodhouse et al Nat Comm 2020). CH relaxes to the on-state (C-) with a much slower timescale\nint\nof ~ 1 ms.\nThe reviewer suggests that the CH state, in fast pH equilibrium with C-, could be relevant because of its\nability to absorb 405 nm light and interconvert to TH and bleach. In the kinetic model used throughout the\nmanuscript we neglected the pH equilibrium based on the following assumptions: (i) pKa=5.9 and at\nexperimental pH of 7.5 the equilibrium concentration ratio is [CH]/[C-"}, "author_response": ""}]

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