Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low- intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements. Keywords: Genetically encoded voltage indicator, GEVI, Photoswitching, Photostability, ASAP Introduction neuronal activities with cell-type specificity [7–9]. Some Understanding neural circuit function requires untangling GEVI mechanisms include fluorescence change upon con- contextual neural activity, ideally by simultaneous moni- formational change of linked voltage-sensing domains toring the activation of all individual neurons in a popula- (VSDs) [10–13], voltage-sensing opsins [8, 14] and their tion. To achieve this goal, various techniques have been eFRET-based compounds [15, 16]. As an example of the developed for large-scale detection of neuronal activity, first mechanism, the recently developed ASAPs are including multi-electrode recording [1, 2] and optical designed as a split voltage-sensing domain (VSD) adapted imaging with calcium indicators [3–7]. Multi-electrode re- from voltage-sensitive phosphatase of Gallus gallus and cording provides ultrahigh temporal resolution but lacks a circular-permuted green fluorescent protein (cpGFP) cellular specificity, while calcium imaging provides cellular [12, 13]. To follow voltage dynamics in neurons by specificity and high spatial accuracy but lacks temporal optical imaging, fast imaging sampling rates and thus resolution. Recent years have seen rapid development of short exposure times are required compared to calcium genetically-encoded voltage indicators (GEVIs), which imaging. This necessitates higher excitation powers, enable high spatial and temporal resolution recording of which can accelerate indicator photobleaching. Photo- bleaching reduces apparent brightness of the indicator over the time, impacting the signal-noise ratio (SNR) * Correspondence: email@example.com; firstname.lastname@example.org for detecting superthreshold spiking or subthreshold Departments of Neurobiology, Bioengineering and Pediatrics, Stanford University, Stanford, CA 94305, USA potential changes. Among GEVIs, ASAPs are unique Hefei National Laboratory for Physical Sciences at the Microscale, CAS for their fast kinetics, high response, and compatibility Center for Excellence in Brain Science and Intelligence Technology, and with in vivo two-photon imaging [9, 13, 17]. However, School of Life Sciences, University of Science and Technology of China, Hefei, China ASAPs, like most GFP-based probes, demonstrate Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Molecular Brain (2018) 11:32 Page 2 of 7 significant fluorescence loss or photobleaching  filter (FF01–535/50, Semrock). Illumination intensity which can be a limitation especially for long-duration was 5 mW/mm in all cases for 470-nm light and imaging experiments. We thus explored the possibility and 0.1~ 2.5 mW/mm for 405-nm light at the speci- of improving voltage imaging of ASAPs by improving men plane. In all experiments, images were acquired their photostability. continuously unless described explicitly, at 200 Hz with a sCMOS camera (Zyla, Andor). The camera and the Materials and methods microscope were connected with a 0.35×-magnification Plasmid construction adapter (Olympus). Synchronization of electrophysiology All plasmids were constructed following standard molecu- and imaging was implemented with a DAQ board lar biology methods and then verified by sequencing of all (PCI-6229, National Instruments) interfaced with Igor Pro cloned fragments. Original pcDNA3.1/Puro-CAG-ASAP1 (Wavemetrics) and Micro-Manager . was obtained from Addgene (Plasmid #52519), and the other variants (ASAP2f, ASAP2f-LE, ASAP2f-V) were Electrophysiology cloned between the NheI and HindIII sites. Whole-cell recordings of neurons were carried out using patch-clamp amplifiers (MultiClamp 700B, Molecular HEK 293 cell culture Devices) at 25 °C. Neurons were perfused in a chamber HEK 293 cells were cultured in Dulbecco’s modified mounted on the microscope stage with the extracellular Eagle’s medium (DMEM, Biowhittaker) supplemented bath solution containing: 150 mM NaCl, 3 mM KCl, with 2 mM L-Glutamine (Gibco), 1 mM Sodium Pyruvate 3 mM CaCl , 2 mM MgCl , 10 mM HEPES, and 5 mM 2 2 (Gibco) and 10% fetal bovine serum (FBS, TBD Science). Glucose. Borosilicate glass pipettes (4~ 6 MΩ open-tip Cells were transfected one day after plated onto cover- resistance) were filled with intracellular solution contain- slips (Φ18 mm; Deckglasser) and imaged 2 days after ing: 130 mM K-gluconate, 6 mM NaCl, 20 mM HEPES, transfection. 0.2 mM EGTA, 1 mM MgCl , 2 mM MgATP and 0.3 mM Na GTP. Both solution were adjusted to pH 7.3. Primary cultures of rat hippocampal neurons To induce action potentials, 800~ 1600 pA of current Allanimalexperiments were performedfollowing was injected for 1 ms to the neurons. For evaluating the guidelines and with approval from the Animal Experi- voltage responses to single APs, a data set of ≥6 neurons ments Committee of the University of Science and were obtained with ≥50 APs for each neuron. Technology of China. Primary hippocampal neurons were prepared as previously described . Neurons Image analysis were transfected with ASAP variants at 10~ 12 days All image analysis was performed with custom ImageJ in vitro (DIV) and used 2 days after transfection for (NIH) and Matlab (Mathworks) programs. imaging and patch-clamp recording. To avoid errors caused by minor asynchronization of illumination and image acquisition, the initial fluores- Transfection cence was calculated from the fitting curve. The fluores- HEK 293 cells and neurons were transfected with plasmid cence was fitted with a bi- exponential decay function: DNA using a high-efficiency transfection method based on optimized calcium phosphate precipitation . t−t t−t 0 0 − − τ τ 1 2 ftðÞ ¼ A þ A e þ A e ; 0 1 2 Imaging Images of HEK293 cells or neurons were acquired with an inverted microscope (IX71, Olympus) equipped with where A , A , A , t , τ , τ are fitting coefficients and. 0 1 2 0 1 2 a 100×/1.45-NA oil-immersion objective. Cells were f(t )= A + A + A was calculated as the initial fluores- 0 0 1 2 illuminated with high-power light-emitting diodes with cence, and f(+∞)= A was calculated as the residual wavelength of 405 nm (M405LP1, Thorlabs), 450 nm fluorescence. Relative fluorescence was normalized to (M450LP1, Thorlabs) and/or 470 nm (M470 L3, Thorlabs). the calculated initial fluorescence. For evaluating the 405-nm and 470-nm illumination light were filtered at the ASAP response to APs, fluorescence was calibrated by exit of LEDs with band-pass filters centered at 405 nm subtracting the corresponding values of the fitting func- (FBH405–10, Thorlabs) and 475 nm (FF01–475/28, tion and then divided by those values to get a corrected Semrock), respectively. 450 nm illumination was filtered relative ratio (Fig. 1g). For Fig. 1b where decay time con- with a band-pass filters centered at 434 nm to allow stants were evaluated, the fitting curve was simplified as a the 440-nm component passing through. The fluores- mono-exponential function by setting A = 0. For evaluat- cence was collected through a 495-nm dichroic mir- ing the recovery time constants, similar mono-exponential ror (FF495-Di03, Semrock) and a 535/50 emission functions were also used (Fig. 1b and Fig. 4c). Xu et al. Molecular Brain (2018) 11:32 Page 3 of 7 Fig. 1 Photoswitching of ASAPs and light-induced restoration. a An example of fluorescence loss of ASAP1 in the transfected HEK 293 cells. Left, the initial fluorescence; right, fluorescence after 2-s 470-nm light illumination. Scale bar: 5 μm. b Photoswitching and spontaneous recovery kinetics of ASAP1 and ASAP2f fluorescence in transfected HEK 293 cells. Blue bars indicate duration of 470-nm light illumination. Fluorescence intensity was sampled once every 30 s after the initial 2-s continuous imaging. Error bars are SEM (n = 25 for ASAP1; n = 38 for ASAP2f). c The ratio of residual ASAPs fluorescence after photoswitching by 470-nm light at different power density (not significant for any pairs, t-test; n =9 cells). d Reduction and restoration of fluorescence in a cultured hippocampal neuron transfected with ASAP2f. Left, the initial fluorescence; middle, fluorescence after 2-s 470-nm light illumination; right, fluorescence after 0.5-s 405-nm light illumination. e Photoswitching of ASAP2f fluorescence in a neuron excited by 470-nm light (blue bars) illumination was restored by interleaved 405-nm light pulses (purple bars) lasting 1, 10, 100, 1000 ms, respectively. f Photoswitching of ASAPs was partially rescued by assistive 405-nm light co-illumination at 0.1~ 2.5 mW/mm . After 0.5-s 405-nm illumination at 0.1~ 2.5 mW/mm alone, fluorescence was partially rescued and followed by photoswitching in 470-nm illumination Results and discussion The spontaneous recovery could be fitted with a ASAPs retain the fluorescent properties of cpGFP, which mono-exponential function, with time constant of ~ 1 min emits green fluorescence when excited by blue light. We (ASAP1, 58.5 ± 1.2 s, n = 25; ASAP2f, 51.8 ± 0.5 s, n = 38), found that ASAPs exhibited fluorescence loss with much larger than the time constant of rapid dimming biphasic kinetics when exposed to blue excitation light following blue light illumination. We observed similar centered at 470-nm (Fig. 1a), with the rapid phase last- effects in HEK293 cells and cultured hippocampal neu- ing less than one second followed by a near-steady state rons transfected with ASAPs. It is noticeable that the with very slow photobleaching (Fig. 1b). At 5 mW/mm , photobleaching curve is different with that in the original a light intensity typically used in voltage detection, ASAP1 study of ASAP2f (Fig. S2 of reference ). A likely ex- and ASAP2f lost ~ 2/3 of their initial fluorescence inten- planation is the ASAP2f bleach curves in the original sities in the rapid phase (ASAP1: 67.2 ± 0.3%, n =25; paper were done with exposure times 4 s apart and the ASAP2f: 67.4 ± 0.5%, n = 38; mean ± SEM, Fig. 1b). fast photoswitching was over before the first image was Altering light intensity from 1.5~ 20 mW/mm did not taken, while in our current study the acquisition was affect the magnitude of fluorescence loss in the rapid synchronized with the illumination and was continuous. phase (Fig. 1c). This fluorescence loss, unlike irreversible The reversible fluorescence loss appeared similar to photobleaching caused by chromophore destruction, was photoswitching events reported in some other GFP vari- reversible and recovered completely in the dark (Fig. 1b). ants such as rsEGFP . Such photoswitching may be Xu et al. Molecular Brain (2018) 11:32 Page 4 of 7 caused by cis-trans isomerization, which enables transi- initial fluorescence (ASAP1: 37.2 ± 0.3%, n = 25; ASAP2f: tion between anionic (~ 470-nm-absorbing) and neutral 37.6 ± 0.5%, n = 23). The result that not all switched fluor- (~ 400-nm-absorbing) states of GFP variants . As escence is recoverable may be due to 405-nm illumination these photoswitching GFP variants show recovery to the also causing some reversible photobleaching, or some anionic state upon 400-nm illumination [22, 23], we chromophores in the trans state being deprotonated and tested whether this was the case with ASAPs. Indeed, thus not absorbing at 405 nm. brief 405-nm illumination restored some of the fluores- Since assistive 405-nm co-illumination significantly in- cence loss induced by 470-nm illumination (Fig. 1d). creased ASAPs fluorescence, we expected that 405-nm The extent of restoration depended on the duration of co-illumination would benefit SNR for measuring voltage 405-nm illumination, with durations up to 100-ms re- transients with ASAP. To test this, we co-illuminated storing increasingly more fluorescence, reaching a max- cultured hippocampal neurons expressing ASAP2f 2 2 imum effect of about half of the lost fluorescence, with 5 mW/mm 470-nm and 0.5 mW/mm 405-nm resulting in a level about 1/3 below the initial fluorescence light (Fig. 2a). Assistive 405-nm light markedly im- (Fig. 1e). Recovered fluorescence could be switched off proved the spike-related ASAP2f signal as represented again by subsequent 470-nm illumination, falling to the by the relative fluorescence change ΔF/F, compared same steady-state intensity as before (Fig. 1e). When to 470-nm illumination alone. While the fluores- applying 405-nm and 470-nm illumination simultan- cence response of ASAP to single action potentials eously, the magnitude of fluorescence restoration (APs) remained unchanged with 405-nm co-illumination depended on the intensity of the 405-nm light. For (without 405-nm light, − 6.1 ± 0.8%, n =6; with 405-nm 5mW/mm 470-nm light, the rescuing effect of light, − 5.9 ± 0.8%, n = 6), the relative brightness and SNR 405-nm co-illumination saturated at ~ 2.5 mW/mm were significantly improved (relative brightness with (Fig. 1f), resulting again in a level about 1/3 below the 405-nm light, 1.86 ± 0.04; relative SNR with 405-nm light, Fig. 2 Improved photostability and performance of ASAP2f with assistive illumination. a (left) An example image showing a hippocampal neuron expressing ASAP2f driven by current pulses (2 ms, 1600pA, 5 Hz; 22 pulses) delivered via a patch pipette to induce APs. Fluorescence intensity at soma (green ROI) and dendrite (red ROI) was measured separately. Scale bar: 5 μm. (middle) Example traces showing soma fluorescence change in response to APs without (−) or with (+) 405-nm assistive illumination. Spike timings are indicated by short vertical bars. (right) Amplified view of relative fluorescence change in soma and dendrite (dend) without (blue) or with (purple) 405-nm light illumination. Scale bar: − 3%, 0.5 s. b (left to right) Summary of fluorescence changes (ΔF/F), relative brightness and relative SNR in response to single APs, and decay time constant of fluorescence in ASAP2f transfected neurons with (+) and without (−) assistive 405-nm illumination. (***) indicates p < 0.001, paired t-test (n = 6); 2 2 error bars are all SEM. In all experiments, light intensity of 470-nm and 405-nm light was 5 mW/mm and 0.5 mW/mm , respectively. Exposure time was 5 ms for all images Xu et al. Molecular Brain (2018) 11:32 Page 5 of 7 1.58 ± 0.06; n = 6) compared to 470-nm illumination It is known that His-148 of GFP interacts with the alone (Fig. 2b). Even with very weak 405-nm light illumin- deprotonated cis-chromophore of GFP and thus may ation (e.g. 0.2 mW/mm ), significant improvement could stabilize the 470-nm-absorbing state [24, 25]. In GCaMPs be achieved, with ~ 40–50% improvements in brightness that do not exhibit fast photoswitching, residues 144–148 and SNR (Additional file 1:Figure S1).Improved perform- of GFP are deleted and the function of His-148 is replaced ance allowed more reliable voltage detection in subcellular by a water molecule held in place by an Arg residue of areas, such as back-propagating APs in dendrites (Fig. 2a). calmodulin . In contrast, residues 144–148 are retained Additionally, we noticed a shorter decay time before following the circular permutation site in the GFP the fluorescence loss reached its steady state (without of ASAPs. We hypothesized that mutating the 405-nm light, 0.243 ± 0.012 s; with 405-nm light, chromophore-interacting sites could improve the 0.036 ± 0.003 s; n =6) (Fig. 2b). photostability of ASAPs. As a proof of concept, we Studies of photoswitching in other GFP variants mutated positions Ser-147 and His-148 of GFP in suggest that there might be two main states of the ASAPs (Fig. 4a), which correspond to positions 150 ASAPs, a 470-nm-absorbing cis anionic state and a and 151 in the full sequence of ASAP2f, to Leu and 400-nm-absorbing trans neutral state . We hy- Glu. Glu in place of His at 151 (ASAP2f numbering) pothesized that an intermediate wavelength between might be expected to reduce cis-trans isomerization 400-nm and 470-nm may be able to excite both states due to its larger side-chain. Indeed, photostability of and thus mimic the effects of dual illumination with the mutant under 470-nm illumination was signifi- 405-nm and 470-nm light. We indeed verified that cantly improved, with 60% more residual fluores- ASAP2f was also excitable with 458-nm and 488-nm laser cence (ASAP2f-LE: 60.2 ± 0.3% of initial fluorescence; light, producing similar emission spectra (Additional file 1: Fig. 4b). Interestingly, 405-nm co-illumination no Figure S2). We then tested the photostability of ASAP2f longer rescued fluorescence loss, suggesting this mu- upon illumination by a single 440-nm LED. Remarkably, tant did not form a protonated chromophore upon fluorescence loss of ASAP2f was much lower than that 470-nm illumination. Photorecovery in the dark was upon 470-nm illumination alone, reaching steady state also faster than ASAP1 or ASAP2f. (ASAP2f-LE: with 75.6 ± 0.7% of initial fluorescence remaining (n =7; 16.1 ± 0.3 s; Fig. 4c). Although voltage responsivity Fig. 3a). The kinetics was also much faster, similar to that was reduced (ΔF/F -4.1 ± 0.3%, n =6, Fig. 4d), these with 405-nm and 470-nm co-illumination. Interestingly, findings demonstrate that engineering of ASAPs to 405-nm co-illumination no longer improved photo- improve their photostability is possible. stability of ASAP fluorescence when imaged with In summary, we found that ASAP-family GEVIs, 440-nm light. Compared to 470-nm, 440-nm illumin- including ASAP1 and ASAP2f are photoswitchable, exhi- ation also did not alter voltage responsivity of ASAP biting rapid decay to a lower-intensity steady state upon (Fig. 3b). Thus, 440-nm illumination is a simple and continuous 470-nm illumination. We also noticed that convenient method for improving the photostability of ASAP2s (), and newer mutants show biphasic photo- ASAPs during voltage imaging. switching of similar extents (data not shown). A full Fig. 3 Improved photostability of ASAP2f with single 440-nm illumination. a Unlike 470-nm illumination, 440-nm light elicited much less photoswitching of ASAP2f in transfected neurons and was not further improved by assistive 405-nm co-illumination (indicated by a purple bar). b Fluorescence response of ASAP2f to single APs remained unchanged under 440-nm illumination (440-nm: - 5.7 ± 0.6%, n = 7; 470-nm: - 6.1 ± 0.8%, n = 6; n.s.: p > 0.05, t-test) Xu et al. Molecular Brain (2018) 11:32 Page 6 of 7 Fig. 4 Mutations at linker sites improved ASAPs photostability. a Schematic diagram showing the constructs of ASAP2f and its mutant ASAP2f-LE. b ASAP2f-LE mutant expressed in neurons showed less photoconversion compared to original ASAP1 and ASAP2f. The fluorescence loss of ASAP2f-LE was no longer recoverable by 405-nm co-illumination. c Spontaneous recovery from photoswitching of the mutant in the dark. All imaging conditions were similar to that in Fig. 1b. d ASAP2f-LE showed lower voltage sensitivity in response to single APs. p = 0.04, t-test; n =6 for ASAP2f, n = 6 for ASAP2f-LE characterization will be performed in future studies. performance and perhaps new properties. For instance, We demonstrated that 405-nm co-illumination with given the precedence of irreversibly photoactivatable 470-nm illumination, or a single excitation wavelength GFP-based probes such as PA-GCaMP , it may be of 440 nm, reduces photoswitching and greatly im- possible to create irreversibly photoactivatible ASAP proves the performance of ASAPs while preserving variants, which may be beneficial in applications require their voltage responsivity. This insight yields a simple high contrast. and effective approach to improve brightness and thus SNR of ASAPs in voltage imaging. We think that simi- Additional file lar mechanisms would work in longer-term experi- ments although this must be test in future studies. One Additional file 1: Supporting Figures. Figure S1. Weak 405-nm light likely explanation for the photoswitching effect is the illumination improved ASAPs performance on AP detection. Figure S2. transition between the two states that previously char- Emission spectrum of ASAP2f excited by 458-nm and 488-nm acterized for GFP-like proteins: a 470-nm-absorbing cis illumination. (DOCX 192 kb) anionic state and a 405-nm-absorbing trans neutral state. In addition, we cannot rule out the possibility of a non-fluorescent anionic state that is induced by Abbreviations cpGFP: Circularly permuted GFP; DIV: Day(s) in vitro; eFRET: Electrochromic 470-nm light. The presence of this state would explain Förster resonance energy transfer; GEVI: Genetically encoded voltage the inability of 405-nm light to fully restore ASAP indicator; GFP: Green fluorescent protein; SNR: Signal-to-noise ratio; fluorescence. It may also explain why the Glu-151 mu- VSD: Voltage sensing domain tant of ASAP2f still shows some loss of fluorescence upon 470-nm light, but without the ability to be re- Acknowledgements stored by 405-nm light. Our findings may be instructive We thank B. Zhang for hippocampal cultures, L. Qi, C. Xia and other lab for future engineering of ASAP probes with improved members for help and discussion. Xu et al. Molecular Brain (2018) 11:32 Page 7 of 7 Funding 12. St-Pierre F, Marshall JD, Yang Y, Gong Y, Schnitzer MJ, Lin MZ. High-fidelity This study was supported by grants from the CAS (XDB02050000) and NSFC optical reporting of neuronal electrical activity with an ultrafast fluorescent (31630030). voltage sensor. 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