Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators
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Abstract
Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent changes in opsin absorption to modulate the fluorescence of an atached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and 2. We found that the voltage sensitivity came from a photocycle intermediate, not from the opsin ground state. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo .
Teaser
Voltage sensitivity in FRET-opsin indicators comes from a photocycle intermediate, reachable via optimized 2P excitation.
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Possibly photobleaching, and possibly excitation of voltage-insensitive background fluorescence.
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Oops, yes, those are low and high-power regimes.
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I don't know.
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We explored interleaving 1P and 2P excitation to sensitize the 2P signal. This looked promising, but we didn't push it far enough to be sure.
Yes, Fig. 2d,e tell you about the illumination and "rest" timescales that will leave the protein in the sensitized state.
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he complex photophysics of the FRET-opsin GEVIs suggest that future protein engineering efforts shouldbe accompanied, at a minimum, by a quan�fica�on of intensity-dependent voltage sensi�vity. Aninteres�ng avenue for future explora�ons would be to determine the photocycle basis for the intensity-dependent changes in voltage sensi�vity and voltage step-response waveforms shown in Figs. 1 and 2..CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeThe copyright holder for this preprint (whichthis version posted April 2, 2024.;https://doi.org/10.1101/2024.04.01.587540doi:bioRxiv preprint
Is it conceptually possible to engineer out the S2 excited state or change its excitation wavelength?
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A beter strategy would be to interleave epochs of intense (> 10 mW/mm2) illumina�on withepochs of darkness. Similarly, for voltage imaging of large samples (e.g. an en�re mouse heart), theexcita�on intensi�es may be low, leading to a loss of voltage sensi�vity.
That's a very valuable insight! Do you think there would be any benefit in simultaneous 1P and 2P illumination to increase SNR?
For interleaving stimulation, is the idea to use the kinetics from Fig 2d,e to determine the min period that can be used to illuminate the same spot?
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This observa�on shows thatstatements of FRET-opsin voltage sensi�vity are only meaningful if illumina�on intensity is specified
That's very good to know!
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Figure 2
Maybe I missed it but the "I" and "II" labels in a,b,d,e are not explained in the legend. I assume I = low-power light regime and II = high-power?
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b) Fluorescence traces (le� axis, detrended ΔF/F; right axis, F) of from 1P (top) and 2P(botom) epochs of a single recording
In the 2P panel it looks like the spike amplitude paradoxically decreases as you increase laser power -- is that due to photobleaching?
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