Theoretical prediction of broadband ambient light optogenetic vision restoration with ChRmine and its mutants.

Vision restoration is one of the most promising applications of optogenetics. However, it is limited due to the poor-sensitivity, slow-kinetics and narrow band absorption spectra of opsins. Here, a detailed theoretical study of retinal ganglion neurons (RGNs) expressed with ChRmine, ReaChR, CoChR, CatCh and their mutants, with near monochromatic LEDs, and broadband sunlight, halogen lamp, RGB LED light, and pure white light sources has been presented. All the opsins exhibit improved light sensitivity and larger photocurrent on illuminating with broadband light sources compared to narrow band LEDs. ChRmine allows firing at ambient sunlight (1.5 nW/mm2) and pure white light (1.2 nW/mm2), which is lowest among the opsins considered. The broadband activation spectrum of ChRmine and its mutants is also useful to restore color sensitivity. Although ChRmine exhibits slower turn-off kinetics with broadband light, high-fidelity spikes can be evoked upto 50 Hz. This limit extends upto 80 Hz with the improved hsChRmine mutant although it requires double the irradiance compared to ChRmine. The present study shows that ChRmine and its mutants allow activation of RGNs with ambient light which is useful for goggle-free white light optogenetic retinal prostheses with improved quality of restored vision.

Optogenetically mediated large volume suppression and synchronized excitation of human ventricular cardiomyocytes.

A major challenge in cardiac optogenetics is to have minimally invasive large volume excitation and suppression for effective cardioversion and treatment of tachycardia. It is important to study the effect of light attenuation on the electrical activity of cells in in vivo cardiac optogenetic experiments. In this computational study, we present a detailed analysis of the effect of light attenuation in different channelrhodopsins (ChRs)-expressing human ventricular cardiomyocytes. The study shows that sustained illumination from the myocardium surface used for suppression, simultaneously results in spurious excitation in deeper tissue regions. Tissue depths of suppressed and excited regions have been determined for different opsin expression levels. It is shown that increasing the expression level by 5-fold enhances the depth of suppressed tissue from 2.24 to 3.73 mm with ChR2(H134R) (ChR2 with a single point mutation at position H134), 3.78 to 5.12 mm with GtACR1 (anion-conducting ChR from cryptophyte algae Guillardia theta) and 6.63 to 9.31 mm with ChRmine (a marine opsin gene from Tiarina fusus). Light attenuation also results in desynchrony in action potentials in different tissue regions under pulsed illumination. It is further shown that gradient-opsin expression not only enables suppression up to the same level of tissue depth but also enables synchronized excitation under pulsed illumination. The study is important for the effective treatment of tachycardia and cardiac pacing and for extending the scale of cardiac optogenetics.

Ultra-low power deep sustained optogenetic excitation of human ventricular cardiomyocytes with red-shifted opsins: a computational study.

The main challenge in cardiac optogenetics is to have low-power, high-fidelity deep excitation of cells with minimal invasiveness and heating. We present a detailed computational study of optogenetic excitation of human ventricular cardiomyocytes (HVCMs) with new ChRmine, bReaChES and CsChrimson red-shifted opsins to overcome the challenge. Action potentials (APs) in ChRmine-expressing HVCMs can be triggered at 6 µW mm-2 (10 ms pulse) and 0.7 µW mm-2 (100 ms pulse) at 585 nm, which is two orders of magnitude lower than ChR2(H134R). This enables safe sustained excitation of deeply situated cardiac cells with ChRmine (7.46 mm) and with bReaChES (6.21 mm) with the light source at the pericardium surface. Deeper excitation up to 10.2 mm can be achieved with ChRmine by illuminating at 650 nm. Photostimulation conditions for minimum charge transfer during APs have been determined, which is important for tissue health under sustained excitation. The AP duration for all the opsins is constant up to 100 ms pulse width but increases thereafter. Interestingly, the AP frequency increases with irradiance under continuous illumination, but APs are suppressed at higher irradiances. The optimal range of irradiance for each opsin to excite HVCMs has been determined. Under optimal photostimulation conditions, each opsin can precisely excite APs up to 2.5 Hz, while latency and power of light pulse for each AP in a sequence remain most stable and an order of magnitude lower, respectively, in ChRmine-expressing HVCMs. The study highlights the importance of ChRmine and bReaChES for resynchronization, termination of ventricular tachycardia and designing optogenetic cardiac pacemakers with enhanced battery life. KEY POINTS: This work is the formulation of accurate theoretical models of optogenetic control of human ventricular cardiomyocytes (HVCMs) expressed with newly discovered opsins (ChRmine, bReaChES and CsChrimson). Under continuous illumination, action potentials in each opsin-expressing HVCMs can only be evoked in a certain range of irradiances. Action potentials in ChRmine-expressing HVCMs can be triggered at ultra-low power (6 µW mm-2 at 10 ms pulse or 0.7 µW mm-2 at 100 ms pulse at 585 nm), which is two to three orders of magnitude lower than reported results. Ongoing action potentials in ChRmine-expressing HVCMs can be suppressed by continuous illumination of 585 nm light at 2 µW mm-2 . ChRmine enables sustained excitation due to its faster recovery from the desensitized state. Optogenetic excitation of deeply situated cardiac cells is possible up to ∼7.46 and 10.2 mm with ChRmine on illuminating the outer surface of pericardium at safe irradiance at 585 nm and 650 nm, respectively. The study opens up prospects for designing energy-efficient light-induced pacemakers, resynchronization and termination of ventricular tachycardia.

Co-expressing fast channelrhodopsin with step-function opsin overcomes spike failure due to photocurrent desensitization in optogenetics: a theoretical study.

Objective.A fundamental challenge in optogenetics is to elicit long-term high-fidelity neuronal spiking with negligible heating. Fast channelrhodopsins (ChRs) require higher irradiances and cause spike failure due to photocurrent desensitization under sustained illumination, whereas, more light-sensitive step-function opsins (SFOs) exhibit prolonged depolarization with insufficient photocurrent and fast response for high-fidelity spiking.Approach.We present a novel method to overcome this fundamental limitation by co-expressing fast ChRs with SFOs. A detailed theoretical analysis of ChETA co-expressed with different SFOs, namely ChR2(C128A), ChR2(C128S), stabilized step-function opsin (SSFO) and step-function opsin with ultra-high light sensitivity (SOUL), expressing hippocampal neurons has been carried out by formulating their accurate theoretical models.Main results.ChETA-SFO-expressing hippocampal neurons shows more stable photocurrent that overcomes spike failure. Spiking fidelity in these neurons can be sustained even at lower irradiances of subsequent pulses (77% of initial pulse intensity in ChETA-ChR2(C128A)-expressing neurons) or by using red-shifted light pulses at appropriate intervals. High-fidelity spiking upto 60 Hz can be evoked in ChETA-ChR2(C128S), ChETA-SSFO and ChETA-SOUL-expressing neurons, which cannot be attained with only SFOs.Significance.The present study provides important insights about photostimulation protocols for bi-stable switching of neurons. This new approach provides a means for sustained low-power, high-frequency and high-fidelity optogenetic switching of neurons, necessary to study various neural functions and neurodegenerative disorders, and enhance the utility of optogenetics for biomedical applications.

Theoretical analysis of optogenetic spiking with ChRmine, bReaChES and CsChrimson-expressing neurons for retinal prostheses.

Objective.Optogenetics has emerged as a promising technique for neural prosthetics, especially retinal prostheses, with unprecedented spatiotemporal resolution. Newly discovered opsins with high light sensitivity and fast temporal kinetics can provide sufficient temporal resolution at safe light powers and overcome the limitations of presently used opsins. It is also important to formulate accurate mathematical models for optogenetic retinal prostheses, which can facilitate optimization of photostimulation factors to improve the performance.Approach.A detailed theoretical analysis of optogenetic excitation of model retinal ganglion neurons (RGNs) and hippocampal neurons expressed with already tested opsins for retinal prostheses, namely, ChR2, ReaChR and ChrimsonR, and also with recently discovered potent opsins CsChrimson, bReaChES and ChRmine, was carried out.Main results.Under continuous illumination, ChRmine-expressing RGNs begin to respond at very low irradiances ∼10-4mW mm-2, and evoke firing upto ∼280 Hz, highest among other opsin-expressing RGNs, at 10-2mW mm-2. Under pulsed illumination at randomized photon fluxes, ChRmine-expressing RGNs respond to changes in pulse to pulse irradiances upto four logs, although very bright pulses >1014photons mm-2s-1block firing in these neurons. The minimum irradiance threshold for ChRmine-expressing RGNs is lower by two orders of magnitude, whereas, the first spike latency in ChRmine-expressing RGNs is shorter by an order of magnitude, alongwith stable latency of subsequest spikes compared to others. Further, a good set of photostimulation parameters were determined to achieve high-frequency control with single spike resolution at minimal power. Although ChrimsonR enables spiking upto 100 Hz in RGNs, it requires very high irradiances. ChRmine provides control at light powers that are two orders of magnitude smaller than that required with experimentally studied opsins, while maintaining single spike temporal resolution upto 40 Hz.Significance.The present study highlights the importance of ChRmine as a potential opsin for optogenetic retinal prostheses.

Theoretical Analysis of Low-power Bidirectional Optogenetic Control of High-frequency Neural Codes with Single Spike Resolution.

Low-power and high-frequency bidirectional control of spatiotemporal patterns of neural spiking is one of the major challenges in optogenetics. A detailed theoretical analysis and optimization with ChR2-NpHR, ChR2(H134R)-eNpHR3.0, Chrimson-GtACR2 and also with prospective opsin pairs namely, Chronos-Jaws, Chronos-eNpHR3.0, CheRiff-Jaws and vf-Chrimson-GtACR2 has been presented. Biophysical circuit models of bidirectional optogenetic control in above opsin pairs expressing hippocampal neurons and fast-spiking neocortical interneurons have been formulated. The models include the important rebound effect of chloride ions and overlapping of absorption spectra. Blue light absorption by red-shifted opsins not only affects the photocurrent, but also its turn-off kinetics. Under continuous illumination, bidirectional control of spiking around 40 Hz in hippocampal neurons requires very low blue and orange light intensities of 0.014 mW/mm2 and 0.8 mW/mm2 with CheRiff-Jaws and 0.04 mW/mm2, and 0.02 mW/mm2 with Chrimson-GtACR2, respectively. Under optimal photostimulation and expression density, high-frequency limit of bidirectional control is 60 Hz and 100 Hz with ChR2-NpHR, 60 Hz and 20 Hz with ChR2(H134R)-eNpHR3.0, 90 Hz and 180 Hz with Chronos-Jaws, and 90 Hz and 250 Hz with Chronos-eNpHR3.0 in neurons and interneurons, respectively. Although, Chrimson-GtACR2 enables bidirectional control at very low-power, vf-Chrimson-GtACR2 provides control with reduced cross-talk. The theoretical analysis highlights the usefulness of computational methods to virtually optimize stimulation protocols for optogenetic tool combinations. The study is useful to generate neural codes with desired spatiotemporal resolution and to design optogenetic neuroprosthetic devices and circuits.

Comparison of low-power, high-frequency and temporally precise optogenetic inhibition of spiking in NpHR, eNpHR3.0 and Jaws-expressing neurons.

A detailed theoretical analysis of low-power, high-frequency and temporally precise optogenetic inhibition of neuronal spiking, with red-shifted opsins namely, NpHR, eNpHR3.0 and Jaws, has been presented. An accurate model for inhibition of spiking in these opsins expressed hippocampal neurons that includes the important rebound activity of chloride ions across the membrane has been formulated. The effect of various parameters including irradiance, pulse width, frequency, opsin-expression density and chloride concentration has been studied in detail. Theoretical simulations are in very good agreement with reported experimental results. The chloride concentration gradient directly affects the photocurrent and inhibition capacity in all three variants. eNpHR3.0 shows smallest inhibitory post-synaptic potential plateau at higher frequencies. The time delay between light stimulus and target spike is crucial to minimize irradiance and expression density thresholds for suppressing individual spike. Good practical values of photostimulation parameters have been obtained empirically for peak photocurrent, time delay and 100% spiking inhibition, at continuous and pulsed illumination. Under continuous illumination, complete inhibition of neural activity in Jaws-expressing neurons takes place at minimum irradiance of 0.2 mW mm-2 and expression density of 0.2 mS cm-2, whereas for pulsed stimulation, it is at minimum irradiance of 0.6 mW mm-2 and 5 ms pulse width, at 10 Hz. It is shown that Jaws and eNpHR3.0 are able to invoke single spike precise inhibition up to 160 and 200 Hz, respectively. The study is useful in designing new experiments, understanding temporal spike coding and bidirectional control, and curing neurological disorders.

Theoretical optimization of high-frequency optogenetic spiking of red-shifted very fast-Chrimson-expressing neurons.

A detailed theoretical analysis and optimization of high-fidelity, high-frequency firing of the red-shifted very-fast-Chrimson (vf-Chrimson) expressing neurons is presented. A four-state model for vf-Chrimson photocycle has been formulated and incorporated in Hodgkin-Huxley and Wang-Buzsaki spiking neuron circuit models. The effect of various parameters that include irradiance, pulse width, frequency, expression level, and membrane capacitance has been studied in detail. Theoretical simulations are in excellent agreement with recently reported experimental results. The analysis and optimization bring out additional interesting features. A minimal pulse width of 1.7 ms at 23 mW / mm 2 induces a peak photocurrent of 1250 pA. Optimal irradiance ( 0.1 mW / mm 2 ) and pulse width ( 50 µ s ) to trigger action potential have been determined. At frequencies beyond 200 Hz, higher values of expression level and irradiance result in spike failure. Singlet and doublet spiking fidelity can be maintained up to 400 and 150 Hz, respectively. The combination of expression level and membrane capacitance is a crucial factor to achieve high-frequency firing above 500 Hz. Its optimization enables 100% spike probability of up to 1 kHz. The study is useful in designing new high-frequency optogenetic neural spiking experiments with desired spatiotemporal resolution, by providing insights into the temporal spike coding, plasticity, and curing neurodegenerative diseases.

Theoretical analysis of low-power fast optogenetic control of firing of Chronos-expressing neurons.

A detailed theoretical analysis of low-power, fast optogenetic control of firing of Chronos-expressing neurons has been presented. A three-state model for the Chronos photocycle has been formulated and incorporated in a fast-spiking interneuron circuit model. The effect of excitation wavelength, pulse irradiance, pulse width, and pulse frequency has been studied in detail and compared with ChR2. Theoretical simulations are in excellent agreement with recently reported experimental results and bring out additional interesting features. At very low irradiances ([Formula: see text]), the plateau current in Chronos exhibits a maximum. At [Formula: see text], the plateau current is 2 orders of magnitude smaller and saturates at longer pulse widths ([Formula: see text]) compared to ChR2 ([Formula: see text]). [Formula: see text] in Chronos saturates at much shorter pulse widths (1775 pA at 1.5 ms and [Formula: see text]) than in ChR2. Spiking fidelity is also higher at lower irradiances and longer pulse widths compared to ChR2. Chronos exhibits an average maximal driven rate of over [Formula: see text] in response to [Formula: see text] stimuli, each of 1-ms pulse-width, in the intensity range 0 to [Formula: see text]. The analysis is important to not only understand the photodynamics of Chronos and Chronos-expressing neurons but also to design opsins with optimized properties and perform precision experiments with required spatiotemporal resolution.

All-optical ultrafast XOR/XNOR logic gates, binary counter, and double-bit comparator with silicon microring resonators.

We present designs of all-optical ultrafast YES/NOT, XOR/XNOR logic gates, binary counter, and double-bit comparator based on all-optical switching by two-photon absorption induced free-carrier injection in silicon 2 × 2 add-drop microring resonators. The proposed circuits have been theoretically analyzed using time-domain coupled-mode theory based on reported experimental values to realize low power (∼ 28 mW) ultrafast (∼ 22 ps) operation with high modulation (80%) and bit rate (45 Gb/s). The designs are complementary metal-oxide semiconductor compatible and provide advantages of high Q-factor, tunability, compactness, cascadibility, scalability, reconfigurability, simplicity, and minimal number of switches and inputs for realization of the desired logic. Although a two-bit counter has been shown, the scheme can easily be extended to N-bit counter through cascading.

Optoelectronic logic gates based on photovoltaic response of bacteriorhodopsin polymer composite thin films.

We present designs of optoelectronic OR, AND, NOR, and NAND logic gates with multiple pulsed pump laser beams based on the photovoltaic response of bacteriorhodopsin (BR) molecules embedded in a polyvinyl matrix coated on ITO. A detailed experimental study of the photovoltaic response reveals that continuous pulsed exposure to 532 nm and 405 nm laser light results in a large photocurrent/photovoltage, due to rapid reprotonation and chromophore reisomerization, taking BR to the ground state in hundreds of nanoseconds. It also helps in sustaining the photovoltage at higher frequencies and in maintaining the shape of the photovoltage. It is shown experimentally that for a pulsed laser beam at 532 nm with peak pump intensity of 1.19 W/cm (2), a photovoltage of 50 mV is generated. A detailed numerical simulation of the photovoltaic response of BR has been carried out taking into account all the six states (B, K, L, M, N, and O) in the BR photocycle to ascertain the effect of various parameters such as lifetime of the M-state, the pump pulse-width, pump intensity, lifetime of excited protons, and rate constant of excited protons. Experimental results are in good agreement with theoretical simulations. The present study opens up new prospects for protein-based optoelectronic computing.

Design of all-optical reconfigurable logic unit with bacteriorhodopsin protein coated microcavity switches.

We present a theoretical design of an all-optical reconfigurable logic unit based on optically controlled microcavity switches, for realization of all-optical computing circuits. It can execute different logic and arithmetic operations such as half and full adder or subtractor, by only changing the control inputs on the same circuit. Theoretical designs considering bacteriorhodopsin (BR) protein coated microcavities in tree architecture have been presented. The combined advantages of high Q-factor, tunability, compactness, switching of near-IR signals at telecom wavelengths (1310/1550 nm) with low-power control signals, and flexibility of cascading switches to form circuits, makes the designs promising for practical applications. They combine the ultrahigh sensitivity of both BR and microresonators to define a novel paradigm of all-optical computing based on hybrid nanobiophotonic integration.

Switching light with light in chlorophyll-A molecules based on excited-state absorption.

We analyze all-optical switching in chlorophyll-A (Chl-A) molecules for different combinations of pump-probe wavelengths, based on nonlinear intensity-induced excited-state absorption. It is shown that for a pulsed pump beam at 672 nm with peak pump intensity of 5 kW/cm(2) and Chl-A concentration of 1.5 mM, the transmission of a continuous-wave probe beam at 476 nm can be completely switched off (100% modulation) with switch on-off time of 0.58 and 0.18 micros, respectively. It is also shown that the switching characteristics can be inverted by changing the probe beam wavelength. The effect of various parameters, such as concentration, pump beam intensity, pump pulse width, absorption cross section of the ground state, and lifetimes of different states, on the switching characteristics has been analyzed in detail. It is shown that there exists an optimum value of concentration of Chl-A for maximum switching contrast, for the case when the ground state also absorbs the probe beam. The switching characteristics of Chl-A have also been compared with Chl-B and Bchl. Experimental results for all-optical switching in Chl-A with a train of pulses are in good agreement with theoretical results. It is shown that higher contrast and faster switching can be achieved as opposed to what was reported recently in other biomolecules such as archael rhodopsin and phototropin proteins. The results have also been used to design switches and logic gates.

All-optical switching in plant blue light photoreceptor phototropin.

We theoretically analyze all-optical switching in the recently characterized LOV2 domain from Avena sativa (oat) phot1 phototropin, a blue-light plant photoreceptor, based on nonlinear intensity-induced excited-state absorption. The transmission of a cw probe laser beam at 660 nm corresponding to the peak absorption of the first excited L-state, through the LOV2 sample, is switched by a pulsed pump laser beam at 442 nm that corresponds to the maximum initial D state absorption. The switching characteristics have been analyzed using the rate equation approach, considering all the three intermediate states and transitions in the LOV2 photocycle. It is shown that for a given pump pulse intensity, there is an optimum pump pulsewidth for which the switching contrast is maximum. It is shown that the probe laser beam can be completely switched off (100% modulation) by the pump laser beam at 50 kW/cm2 for a concentration of 1 mM with sample thickness of 5.5 mm. The switching characteristics are sensitive to various parameters such as concentration, rate constant of L-state, peak pump intensity and pump pulse width. At typical values, the switch-off and switch-on time is 1.6 and 22.3 micros, respectively. The switching characteristics have also been used to design all-optical NOT and the universal NOR and NAND logic gates.

All-optical switching in Pharaonis phoborhodopsin protein molecules.

Low-power all-optical switching with pharaonis phoborhodopsin (ppR) protein is demonstrated based on nonlinear excited-state absorption at different wavelengths. A modulating pulsed 532-nm laser beam is shown to switch the transmission of a continuous-wave signal light beam at: 1) 390 nm; 2) 500 nm; 3) 560 nm; and 4) 600 nm, respectively. Simulations based on the rate equation approach considering all seven states in the ppR photocycle are in good agreement with experimental results. It is shown that the switching characteristics at 560 and 600 nm, respectively, can exhibit negative to positive switching. The switching characteristics at 500 nm can be inverted by increasing the signal beam intensity. The profile of switched signal beam is also sensitive to the modulating pulse frequency and signal beam intensity and wavelength. The switching characteristics are also shown to be sensitive to the lifetimes of ppR(M) and ppR(O) intermediates. The results show the applicability of ppR as a low-power wavelength tunable all-optical switch.

All-optical biomolecular parallel logic gates with bacteriorhodopsin.

All-optical two input parallel logic gates with bacteriorhodopsin (BR) protein have been designed based on nonlinear intensity-induced excited-state absorption. Amplitude modulation of a continuous wave (CW) probe laser beam transmission at 640 nm corresponding to the peak absorption of O intermediate state through BR, by a modulating CW pump laser beam at 570 nm corresponding to the peak absorption of initial BR state has been analyzed considering all six intermediate states in its photocycle using the rate equation approach. The transmission characteristics have been shown to exhibit a dip, which is sensitive to normalized small-signal absorption coefficient (beta), rate constants of O and N intermediate states and absorption of the O state at 570 nm. There is an optimum value of beta for a given pump intensity range for which maximum modulation can be achieved. It is shown that 100% modulation can be achieved if the initial state of BR does not absorb the probe beam. The results have been used to design low-power all-optical parallel NOT, AND, OR, XNOR, and the universal NAND and NOR logic gates for two cases: 1) only changing the output threshold and 2) considering a common threshold with different beta values.