to create detailed 
 maps of brain function , one would like to observe 
 the simultaneous activity of thousands or millions of neurons in the 
 intact brain of a behaving animal . large - scale maps of activity at 
 single - neuron and single - spike resolution could give insights into 
 fundamental mechanisms of neural processing . one could map the patterns 
 of activation associated with simple sensory processing tasks or with 
 complex activities such as feeding , locomotion , or social interactions . 
 by correlating the activity of large numbers of single cells , connectomics  have focused on 
 mapping large - scale neural structures using optical and 
 electron microscopies . clever gfp labeling schemes 
 facilitate tracing of neuronal connections in genetically specified 
 cell types . however , connectomic mapping 
 is typically implemented in fixed tissues and thus is incompatible 
 with functional recording . genetically encoded voltage and calcium 
 reporters are now widely 
 used for optical recording of neural activity in vitro and in vivo . these tools are typically used 
 to record from a relatively modest number of cells ( < 1000 ) in a 
 single field of view . a recent technical tour de force demonstrated 
 whole - brain calcium imaging in a live zebrafish , but the imaging bandwidth of 0.8 hz was 1000-fold 
 slower than the duration of a single action potential . one might 
 like to combine large - scale 3d imaging with functional 
 reporters to achieve  functional connectomics first , 
 optical scattering limits imaging in live brain tissue to a depth 
 of 1 mm . to image at greater depth , the brain must be fixed 
 and either chemically clarified or sliced 
 into thin sections . second , existing microscopes 
 can not image large volumes fast enough to resolve simultaneous action 
 potentials ( 1 ms ) or calcium transients ( 200 ms ) in 
 large numbers of cells . for a fast voltage indicator , the signal from 
 a neuronal spike lasts only as long as the spike itself . to image 
 a cubic millimeter of brain with millisecond temporal resolution and 
 micrometer spatial resolution would require a data rate > 10 bits / s , well beyond the bandwidth of existing or conceived 
 microscopes . an alternate strategy is to convert neural activity 
 in a user - defined 
 epoch into a long - lasting ( bio)chemical signal to be read at a later 
 time . in the technique of targeted recombination in active populations 
 ( trap ) , the simultaneous presence of neural activity and a drug ( tamoxifen ) 
 leads to activation of a cre recombinase and subsequent 
 expression of gfp . several 
 proposals have been offered for activity integrators with higher time 
 resolution , but to our knowledge none has 
 been implemented . ( a ) in a real - time voltage 
 reporter , 
 the population in a fluorescent state , f , is a function of membrane 
 voltage , regardless of illumination . ( b ) in a sample and hold voltage 
 sensor , voltage establishes a d1  d2 equilibrium and a write pulse establishes a rapid d2  
 f equilibrium . ( c ) in 
 a light - gated voltage integrator , voltage establishes a d1  d2 equilibrium , and a write pulse drives the 
 unidirectional d2  f transition . the population of 
 f is frozen at the end of the write pulse . in both types of flash 
 memory sensor , the population of f persists in the dark and is later 
 probed via a read pulse that elicits fluorescence . optical gating of an activity recorder is particularly 
 attractive 
 because ( a ) the optical control signal can be gated with nearly arbitrary 
 temporal precision and ( b ) photons used to regulate a photochemical 
 process need not follow a straight - line path from the source to the 
 molecular target . while optical scattering lengths in brain are typically 
 60 m , diffusive transport 
 of photons can easily fill an entire rodent brain with light . thus 
 delivery of an optical control signal is relatively straightforward 
 and does not require sophisticated optics . naturally occurring and 
 engineered rhodopsin proteins have previously been demonstrated to 
 show optical bistability and also to show voltage - dependent 
 switching , but the combination of these two attributes has not , to our knowledge , 
 been demonstrated . here we demonstrate two proof - of - principle 
 approaches to light - gated 
 photochemical recording of membrane voltage . both are based on transmembrane 
 proteins which undergo both voltage- and light - induced conformational 
 changes . figure 1 compares the operation of 
 a standard real - time voltage indicator ( figure 1a ) to the light - gated reporters ( figure 1b , c ) . conventional real - time voltage reporters interconvert between nonfluorescent 
 and fluorescent states in a voltage - dependent manner ; illumination 
 probes the population in the fluorescent state but does not affect 
 the conformation . formation of a fluorescent product requires 
 simultaneous presence of a depolarizing voltage and illumination . the three - state models shown in figure 1b , c 
 illustrate plausible reaction topologies which could lead to this 
 behavior . in a sample and hold sensor ( figure 1b ) , 
 the population in the fluorescent state tracks the membrane voltage 
 during illumination at a wavelength write ; interconversion 
 ceases the moment the write pulse ends . illumination at a wavelength 
 read at a later time probes the quantity of fluorescent 
 product that existed at the end of the write pulse . these sensors 
 could be used to record snapshots of neural activity at a moment in 
 time . in a light - gated voltage integrator ( figure 1c ) , the population in the fluorescent state accumulates in 
 a voltage - dependent 
 manner during a write pulse . integrators could be used to determine the cumulative 
 level of neuronal activity during a period of illumination . the  sample 
 and hold  and  light - gated integrator  mechanisms 
 are limiting cases of a continuous distribution of light - gated voltage 
 reporters , distinguished by light - dependent kinetics into and out 
 of the fluorescent state during the write pulse . we call the techniques 
 of figure 1b , c  flash memory  
 for their ability to store a record of neural activity upon a flash 
 of light . the three - state reaction scheme of figures 1b , c occurs as a motif within the voltage- and illumination - dependent 
 photocycle of archaerhodopsin - based voltage indicators ( figure s1 ) . while 
 flash memory behavior was not observed in the wild - type protein , we 
 hypothesized that mutants of arch might show kinetics favoring flash 
 memory behavior . we introduce the three - state model here as a conceptual 
 framework for interpreting the data that follows . we give a detailed 
 photophysical characterization of two flash 
 memory sensors , engineered by mutating the real - time voltage reporter 
 archaerhodopsin-3 ( arch ) . the mutant arch(d95h ) approximates a sample 
 and hold sensor , albeit with a slow ( 48 ms ) response to changes in 
 voltage . we used arch(d95h ) to make a photochemical recording of action 
 potentials in a cultured neuron . the mutant arch(d95q ) approximates 
 a light - gated voltage integrator , albeit with poor sensitivity to 
 single spikes . we used arch(d95q ) to count exogenously delivered voltage 
 spikes in a hek cell ( it did not traffic well enough for use in neurons ) . applications in tissue and in vivo will require 
 further technical developments in the protein reporter and in the 
 optical instrumentation and imaging protocols . screens of arch mutants 
 and other microbial rhodopsins may yield reporters with improved sensitivity , 
 kinetics , brightness , and membrane trafficking . raman or 2-photon 
 readout modalities may prevent spurious resetting of proteins by scattered 
 imaging light . for applications where the tissue is fixed and sliced 
 prior to imaging , the robustness of the signal to these procedures 
 must be tested . while whole - brain activity mapping is the ultimate 
 goal , imaging of increasingly large brain subregions will provide 
 useful waypoints . aspartic acid 95 ( analogous to d85 in bacteriorhodopsin ) 
 is the proton acceptor from the schiff base . our lab and others have shown that 
 mutation of this residue can eliminate proton pumping and can modulate 
 photophysical properties of the protein . we generated a library of 
 20 arch(d95x ) mutants and screened for the three attributes of a flash 
 memory sensor : bistability , voltage - sensitivity in the light , and 
 absence of voltage sensitivity in the dark . figure 2a shows the rich colors observed in pellets of e. coli expressing some of these mutants . we tested all 
 arch(d95x ) mutants for bistability , using fluorescence of the retinal 
 chromophore as a readout . we expressed each mutant in e. coli ( materials and methods ) , added carbonyl cyanide m - chlorophenyl hydrazine 
 ( cccp ) to neutralize the membrane potential , and formed a small bacterial 
 pellet for initial spectroscopic characterization . we illuminated 
 each mutant with 16 illumination sequences of the form : ( write , tdark , read ) , with write and read ( 1 s each , 
 10 w / cm ) selected from all pairwise combinations of : 500 , 
 545 , 590 , and 635 nm ( materials and methods ) . we fixed tdark = 5 s. we asked whether 
 the initial fluorescence elicited by read depended 
 on write . such a dependence indicates the presence 
 of at least two states that were stable for at least 5 s in the dark . 
 in all cases emission its 
 brightness and fluorescence excitation and emission spectra are characterized 
 in figure s3 . fluorescence excited at read = 635 nm was 24% brighter with write = 500 nm than with write = 635 nm . to test whether 
 arch(d95h ) was bistable in mammalian cells , we expressed the protein 
 in hek cells and illuminated the sample with write = 488 or 640 nm , tdark = 1 s , and read = 640 nm ( i = 200 w / cm ) , 
 while using whole - cell voltage clamp to maintain a membrane voltage 
 of 0 mv . illumination at write = 488 nm caused greater 
 initial fluorescence during the read interval than did illumination 
 at write = 640 nm ( figure 2b ) . to illustrate the bistability of arch(d95h ) , we imprinted 
 a photochemical 
 image into a lawn of e. coli expressing 
 arch(d95h ) . a digital micromirror array was used to project an image 
 at write = 488 nm ( 0.7 w / cm ) into the 
 microscope and onto the cells . after tdark = 5 s , the cells were illuminated with homogeneous full - field illumination 
 at read = 640 nm ( 40 w / cm ) , revealing 
 the latent image in the near - infrared fluorescence ( figure 2c ) . this process could be repeated in the same field 
 of view with subsequent patterns written by blue light and read by 
 red light . we varied tdark to measure 
 the lifetime 
 of bistability in arch(d95h ) ( figure 2d ) . a 
 grid pattern of blue light was projected onto the lawn of e. coli . after variable delay , the pattern was probed 
 via wide - field red illumination and near - infrared fluorescence . the 
 contrast remained stable at 10% out to the longest time measured , tdark = 53 min ( figure 2d , inset ) . there was also a slow ( tens of minutes ) increase in the 
 overall brightness of the image , occurring equally in the regions 
 that had and had not been exposed to blue light . the source of this 
 gradual increase in fluorescence is not known , though we speculate 
 that it may have been caused by stray light inducing a gradual buildup 
 of the fluorescent state . ( a ) image of three 
 pellets of e. coli expressing different 
 mutants of arch . ( b ) bistability in arch(d95h ) expressed in hek cells . initial fluorescence under red excitation ( read = 
 640 nm ) was different for write = 640 nm than for 
 write = 488 nm . the write pulse was 500 ms , 200 
 w / cm and the dark interval was tdark = 1 s. ( c ) imprinting of photochemical images in a lawn 
 of e. coli expressing arch(d95h ) . illumination 
 with a pattern of blue light converted arch(d95h ) into a long - lived 
 fluorescent state . after a 5 s delay , the pattern was probed with 
 red excitation and near - infrared fluorescence . a checkerboard pattern was imprinted via 
 blue light and probed via red - induced fluorescence after a variable 
 delay tdark . inset graph shows the difference 
 in fluorescence of the bright and dark squares as a function of tdark . several other mutants , including arch(d95q ) , showed significant 
 bistability in our screen of the arch(d95x ) library . for d95q , fluorescence 
 excited at read = 635 nm was 8% brighter with write = 500 nm than with write = 635 nm ( figure s2 ) . we 
 then expressed all 20 arch(d95x ) mutants in hek cells and characterized 
 their fluorescence ( exc = 640 nm , em = 660760 nm ) as a function of membrane potential ( vm = 150 to + 150 mv ) . all mutants whose 
 fluorescence could be detected showed some degree of voltage sensitivity . at vm = + 150 mv arch(d95h ) was 2-fold 
 brighter than at vm = 150 mv ( figure 3b ) . arch(d95q ) showed the greatest voltage sensitivity , 
 with fluorescence > 7-fold higher at + 150 mv than at 150 
 mv 
 ( figure 3c ) , a consequence of having almost 
 no fluorescence at vm = 150 mv . arch(d95h ) generated a small hyperpolarizing photocurrent ( 5 pa ) under 
 intense illumination at 640 nm ( 500 w / cm ) . wild - type arch typically generated 
 photocurrents > 100 pa , so we deem the small photocurrent of arch(d95h ) 
 to be insignificant . due to the simultaneous presence of optical bistability 
 and voltage - sensitive fluorescence in arch(d95h ) and arch(d95q ) , we 
 further characterized these mutants as prospective flash memory sensors . an acousto - optic tunable filter ( aotf ) 
 on the excitation path dynamically controlled the wavelength and intensity 
 of illumination . a patch clamp amplifier provided control over the 
 membrane potential . a camera recorded fluorescence . a shutter ( not 
 shown ) after the aotf blocked all light from reaching the sample during 
 dark intervals . the aotf , patch clamp apparatus , and camera were synchronized 
 via custom software . ( b ) voltage - sensitive fluorescence of arch(d95h ) 
 expressed in a hek cell under constant illumination at 640 nm . ( c ) fluorescence of arch(d95q ) increased 
 7-fold between 150 and + 150 mv , though most of the sensitivity 
 was at positive voltages , above the physiological range . to test for flash memory behavior , we illuminated 
 hek cells expressing arch(d95h ) or arch(d95q ) with the sequence ( write , tdark , read ) while simultaneously varying the membrane voltage under patch clamp 
 control ( figure 4 ) . each sequence ( write , tdark , read ) was repeated twice , once with vm fixed 
 at 100 mv throughout and once with vm stepped from 100 to + 100 mv during the write interval 
 and then returned to 100 mv for the dark and read intervals . 
 remarkably , the initial fluorescence during the read interval , fi , depended on the voltage during the write 
 interval , as required for a flash memory sensor . during the read pulse , 
 the fluorescence gradually relaxed to a steady - state value , ff , determined only by the voltage and illumination 
 during the read pulse . observation of flash memory in arch mutants ( a  c ) 
 arch(d95h ) 
 and ( d  f ) arch(d95q ) . fluorescence of arch(d95h ) at the start of the 
 read pulse was greater for vm = + 100 mv 
 during the write pulse ( purple line ) than for vm = 100 mv during the write pulse ( blue line ) . a voltage 
 pulse in the dark did not influence the fluorescence dynamics during 
 the read interval . the flash memory effect remained unchanged for up to tdark = 2 min . in ( a ( d  f ) same as 
 ( a  c ) for arch(d95q ) . in ( d  f ) write = 532 nm , read = 532 nm . we measured the extent of fluorescence relaxation during 
 the read 
 pulse by the dimensionless quantity in a flash memory sensor , m should 
 be high when v = + 100 mv and low 
 when v = 100 mv . we quantified 
 the flash memory effect bywith v = 100 
 mv in both instances . we tested arch(d95h ) and arch(d95q ) with 
 all combinations of write and read selected from 532 , 594 , and 
 640 nm , keeping tdark fixed ( figures s4 and s5 ) . in arch(d95h ) , the memory 
 effect was maximized with write = 640 nm and read = 594 nm ( figure 4a ) . in arch(d95q ) , 
 the memory effect was maximized with write = 532 
 nm and read = 532 nm ( figure 4d ) . we next asked whether a depolarizing voltage pulse during tdark could overwrite a memory recorded during 
 the write pulse . a 500 ms voltage pulse to + 100 mv in the middle of 
 a 2 s dark interval had no effect on m in 
 either mutant ( figure 4b , e ) . we varied the 
 timing of the voltage pulse in the dark and found no effect on m , except for a small increase in m for arch(d95h ) when the depolarizing voltage pulse ended < 20 
 ms prior to the read pulse ( figure s6 ) . we then varied tdark to measure the 
 persistence of the memory ( figure 4c , f ) . in 
 both mutants the magnitude of m remained constant 
 up to tdark = 2 min . instabilities in 
 the patch clamp connection prevented measurements at larger values 
 of tdark . in arch(d95h ) the memory effect 
 was m = 10% , while in arch(d95q ) the memory 
 effect was m = 20% . we varied the duration of the light pulse during 
 the write interval to measure how fast a photochemical imprint of 
 the voltage could be written . the voltage was held at + 100 mv throughout 
 the write interval ( 300 ms for arch(d95h ) and 800 ms for arch(d95q ) ) , 
 while the duration of the write illumination ( twrite ) was varied between 0 and 200 ms ( figure 5a , b ) . for arch(d95h ) , the value of the memory , m , increased with twrite , following 
 a double - exponential curve with write time constants of fast = 0.14 ms and slow = 12 ms ; the majority 
 of this response ( 57% ) was determined by fast ( figure 5c ) . in contrast , for arch(d95q ) writing took much 
 longer : m also increased with twrite and followed double - exponential kinetics , with time 
 constants fast = 5 ms and slow = 180 ms ; the majority of this response ( 92% ) was determined by 
 slow ( figure 5d ) . kinetics of 
 bright - state formation during the write pulse for ( a , b ) 
 arch(d95h ) and ( c , d ) arch(d95q ) . voltage was held at either + 100 or 
 100 mv during the write interval and at 100 mv during 
 the dark and read intervals . the length of the write flash , twrite , was varied , keeping its end coincident 
 with the step in voltage from + 100 mv to 100 mv . representative 
 fluorescence traces are shown for ( a ) arch(d95h ) and ( c ) arch(d95q ) . ( b , d ) plot of memory effect , m , as a function 
 of twrite . in arch(d95h ) the rise in memory 
 was fit by a double exponential with fast = 0.14 
 ms ( 57% ) and slow = 12 ms ( 43% ) ; a write flash with twrite = 1 ms was sufficient to elicit more than 
 half of the maximal response . ( d ) the dependence of m on twrite in arch(d95q ) was 
 dominated by a slow component . a fit to a double exponential yielded 
 fast = 5 ms ( 8% ) and slow = 180 
 ms ( 92% ) . we also performed the complementary 
 experiment of changing the 
 duration of the voltage pulse during the write interval while keeping 
 the duration of the light pulse fixed at 1000 ms . the memory effect 
 in arch(d95h ) saturated with a time constant for the voltage pulse 
 of 48 ms . for arch(d95q ) the corresponding time constant was 146 ms 
 ( figure s7 ) . we tested whether arch(d95h ) could function as 
 a flash memory sensor for recording neuronal action potentials . the 
 submillisecond response of the protein to a flash of light at constant 
 voltage ( figure 5 ) indicated that the light - driven 
 transition into the fluorescent state was fast compared to the duration 
 of an action potential . however , the 48 ms response to a step in voltage 
 under constant illumination ( figure s7 ) 
 implied that the voltage - dependent transition was slow : the rate of 
 conformational change would low - pass filter the underlying voltage 
 dynamics of the neuron . despite this limitation , we tested whether 
 arch(d95h ) could record an imprint of a single neuronal action potential . we fused the c - terminus of arch(d95h ) to an endoplasmic reticulum 
 export motif , followed by an eyfp expression marker and a trafficking 
 sequence , as described in ref ( 23 ) ( materials and methods ) . we cloned 
 this construct into a lentiviral mammalian expression vector under 
 the camkii promoter . hippocampal neurons and glia were dissociated 
 from postnatal day 0 ( p0 ) rats and cultured on poly - d - lysine 
 coated glass - bottomed dishes ( materials and methods ) . at 4 days in vitro ( div ) we transfected the cells with 
 arch(d95h)-eyfp at 7 div using calcium phosphate , and we measured 
 activity at 1215 div . at the time of measurement , our construct 
 had trafficked to the plasma membranes of the soma and processes , 
 although considerable protein remained internalized in intracellular 
 membranes ( figure 6a ) . injection of current 
 pulses ( 500 pa for 4 ms ) via whole - cell patch 
 clamp reliably induced single action potentials . we paired single 
 action potentials with a 2 ms flash at write = 594 
 nm ( i = 200 w / cm ) . the flash was delivered 
 either before ( t < 0 ms ) , during ( 0 ms 
 < t < 10 ms ) , or after ( t > 10 ms ) the action potential . after a dark interval 
 of tdark = 1 s , we used write = read = 594 nm , on the logic that in a neuroscience 
 application 
 it might be most convenient to use light of a single wavelength . our 
 signal - to - noise ratio in these measurements was not adequate to detect 
 a signature of the action potential in the read fluorescence . we attributed 
 this negative result to the slow response of arch(d95h ) to a step 
 in voltage (  = 48 ms , figure s7 ) . we performed numerical simulations of the three - state model of 
 figure 1b with different approaches to pairing 
 flashes of light with action potentials ( figure 
 s8 ) . these simulations showed that repeated trains of action 
 potentials paired with brief flashes of light could build up population 
 in the fluorescent state . in the simulations , the fluorescence during 
 the read pulse reflected the temporal overlap of the voltage and light 
 in the write pulses . paired action potentials and flashes of orange 
 light led to increased formation of a fluorescent product only when 
 the action potentials and orange flashes coincided in time . the fluorescent 
 product was probed at tdark = 1 s after 
 the last action potential . ( b ) memory effect , m , recorded 
 during the read interval ( circles ) overlaid on the electrical recording 
 of the action potential acquired during the write interval ( green ) . we thus modified our illumination protocol to pair a train 
 of 10 
 action potentials with a train of 10 light flashes . action potentials 
 were induced at 50 ms intervals , and each was paired with a 2 ms write 
 flash at write = 594 nm ( i = 200 
 w / cm ) . for each set of 10 action potentials , the write 
 flashes were delivered either before ( t < 
 0 ms ) , during ( 0 ms < t < 10 ms ) , or 
 after ( t > 10 ms ) the corresponding action 
 potentials . figure 6a shows the revised protocol . 
 a plot of the memory effect , m , during the 
 read interval as a function of t during the 
 write interval reproduced the underlying waveform of the action potential 
 ( figure 6b ) . this measurement demonstrates 
 that arch(d95h ) can record a photochemical imprint of action potentials 
 in a neuron , though an improved signal - to - noise ratio will be needed 
 for application in neuroscience . finally , we explored whether 
 arch(d95q ) could function as a light - gated 
 voltage integrator . for a true integrator , the memory signal due to 
 a voltage pulse should not depend on when in the write interval the 
 pulse occurs . that is , population transferred to the bright state 
 during an action potential must not revert to the dark state during 
 a subsequent hyperpolarization . thus there must be a negligible rate 
 from bright state to the dark state during the write pulse ( figure 1c ) . after a search of wavelengths and intensities 
 for the write pulse , we found that iwrite = 1 w / cm and write = 532 nm caused 
 arch(d95q ) to function as a light - gated voltage integrator ( figure s9 ) . ( a ) top : sequence of illumination and voltage pulses to 
 test the function of arch(d95q ) as a light - gated voltage integrator . a series of n voltage pulses ( 100 to + 100 
 mv , 1 ms ) was paired with dim green illumination ( 1 w / cm ) to produce fluorescent product at a voltage- and light - dependent 
 rate . after a delay of tdark = 0.5 s , 
 the fluorescence was probed by a green read pulse ( 200 w / cm ) . ( b ) memory effect , m , probed 
 in the read interval as a function of number of spikes in the write 
 interval . in the presence of the write pulse , when the write pulse was omitted , spikes 
 did not induce a memory effect ( black ) . error bars are the sample 
 standard deviation calculated from six repetitions of the experimental 
 pulse sequence . arch(d95q ) did not traffic 
 efficiently to the plasma membrane of 
 neurons , so we tested its ability to count imposed voltage spikes 
 in hek cells instead , using the protocol shown in figure 7a . a cell expressing arch(d95q ) was held under voltage 
 clamp conditions via a patch pipet , initially at a resting voltage 
 of 100 mv . a reset pulse ( reset = 635 nm , treset = 0.5 s , ireset = 300 w / cm ) drove the population into the nonfluorescent 
 state . during the write period , a dim green pulse ( write = 532 nm , twrite = 0.4 s , iwrite = 1 w / cm ) was paired with a variable 
 number of voltage spikes ( 100 mv to + 100 mv , 1 ms in duration ) . after a dark interval tdark = 0.5 s , the 
 fluorescence was probed by a green pulse ( read = 
 532 nm , tread = 0.5 s , iread = 200 w / cm ) . we compared the value of 
 the memory effect , m , in the presence of n voltage spikes to its value in the absence of voltage 
 spikes . figure 7a shows representative 
 raw fluorescence 
 traces with and without n = 100 voltage spikes . figure 7b shows that the memory effect ( m ) increased with the number of voltage spikes during the write interval . although the voltage spikes in this experiment were not action potentials , 
 this preliminary result shows the feasibility of using an arch - based 
 sensor to count voltage spikes in a light - gated manner . what is 
 the molecular basis of flash memory in archaerhodopsin mutants ? while 
 a complete characterization of the photocycles of arch(d95h ) and d95q 
 is beyond the scope of this paper , here we show that a simple three - state 
 model reproduces the main qualitative features of the data . varying 
 the illumination parameters can tune the behavior of the model continuously 
 between sample and hold and light - gated integrator behavior . figure 8a shows the model and figure 8b , c shows numerical simulation results . a voltage - dependent 
 equilibrium exists between two nonfluorescent states , d1 and d2 . the fluorescent 
 state , f , is connected to d2 by a light - driven process ( presumably retinal isomerization ) . the action spectra of the transitions into and out of state f are different ; blue light drives the transition into the 
 fluorescent state ( d2  f ) , red light drives the reverse reaction ( f  d2 ) , and orange light drives 
 both reactions . red light also excites fluorescence of f. to use the protein as a sample and hold sensor ( figure 8b ) , one illuminates with a wavelength write that simultaneously drives both the d2  f and f  d2 transitions . during the write interval the 
 ratio of [ f ] to [ d2 ] 
 is determined by write and the forward and reverse 
 action spectra . voltage sets the ratio of [ d1 ] to [ d2 ] and thereby sets the 
 population of f. the moment the light turns off , 
 the population in f is trapped , decoupled from voltage - dependent 
 dynamics in the d manifold . during the read pulse , 
 light at read excites fluorescence from f but at the same time re - establishes equilibrium between f and the d manifold . numerical simulation 
 of three - state kinetic model of flash memory 
 effect in arch mutants . ( a ) reaction scheme in which illumination 
 wavelength tunes the d2  f equilibrium . ( b ) in a sample - and - hold sensor , the population 
 of f follows the voltage - dependent d1  d2 equilibrium 
 until the end of the write pulse . the solid red trace on the right 
 is a numerical simulation of the population in f. 
 ( c ) in a light - gated integrator , blue light converts population from d2 to f but does not allow the 
 reverse process . population in f accumulates in a 
 voltage - dependent manner during the write pulse . the reset 
 pulse is given at a wavelength reset sufficiently 
 far red that it drives f  d2 but not d2  f , thereby initializing the population in the dark d manifold . the write pulse is chosen with write sufficiently blue that it can drive d2  f but not f  d2 . thus , when the voltage is high enough to 
 populate d2 and the write pulse is on , 
 molecules take a one - way trip from d2 to f. this model predicts that by tuning the intensity and 
 wavelength of the write pulse , one can adjust the dynamic range of 
 the integrator . a large kdf increases 
 sensitivity to single spikes but causes the integrator to saturate 
 at a smaller number of spikes , while a small kdf has the opposite effect . our simple analysis suggests that 
 additional control over the state of the system could be obtained 
 by illuminating with two wavelengths simultaneously during the write 
 interval . by choosing a blue and a red wavelength , one could independently 
 control the rates into and out of the fluorescent state . while 
 this model is sufficient to explain the main features we 
 observed in flash memory proteins , these molecules likely have more 
 than three significant states . if one were to map the simple model 
 of figure 8a onto a canonical proton pump photocycle , 
 the dark manifold would likely correspond to the set of states that 
 interconvert in a voltage - dependent way in the main photocycle ( m and n intermediates ) , and the state f would correspond to the off - pathway photogenerated fluorescent 
 state called q in ref ( 21 ) . we have introduced the 
 concept of flash memory as a technique to 
 record light - gated photochemical imprints of membrane voltage . two 
 mutants of the fluorescent voltage indicator arch can be used as flash 
 memory sensors , albeit with small signal amplitudes that limit immediate 
 practical utility . this protein could store a photochemical record of action potentials 
 in a rat neuron . this protein could report the number of electrical spikes that had 
 occurred in a hek cell during a user - selected recording epoch . many aspects of flash memory sensors need further improvement . 
 these include : plasma membrane trafficking in neurons , overall brightness , 
 and contrast between the  high - voltage  and  low - voltage  
 states . ideally , the protein would switch fully within the physiological 
 range of 70 to + 30 mv . for readouts that involve fixing and 
 slicing the tissue , one must also test whether the memory effect is 
 robust to fixation procedures and whether it lasts for days , not just 
 minutes . in view of the very limited search for flash memory 
 proteins undertaken 
 here , we are optimistic that superior performance may be found among 
 other rhodopsin - like proteins . one should not restrict the search 
 to mutants of arch . among the vast number of natively bistable rhodopsin - like 
 proteins , there may be some that are 
 fluorescent and voltage sensitive . a more detailed structural analysis 
 of arch(d95h ) and d95q would help guide this search . in particular , 
 it may be helpful to identify the isomerization state of the retinal 
 in the fluorescent state as well as the voltage - induced shifts in 
 structure and protonation . if one is content 
 to image the optically accessible 
 region of the brain , then the readout could be performed in the live 
 animal . this approach has the advantage that the protein can be reset 
 and the measurement repeated multiple times , thereby averaging out 
 uncorrelated baseline activity . if one wishes to image a larger or 
 deeper region of the brain than is optically accessible , then one 
 could fix the brain and either clarify or slice the tissue . while we have focused on fluorescence 
 as a readout , other modalities 
 may also be feasible . particularly attractive are multiphoton techniques 
 such as two - photon fluorescence and stimulated raman scattering , as 
 these techniques have greater depth penetration than the visible light 
 used in one - photon imaging . two - photon fluorescence provides a very 
 localized excitation volume , avoiding the problem of unintentional 
 resetting of proteins from scattered imaging illumination . nonresonant 
 raman or infrared absorption techniques may be able to determine the 
 isomerization state of the retinal without inducing changes in this 
 state . these techniques could integrate signal for longer times than 
 fluorescent readouts , thereby increasing sensitivity . there 
 are several ways in which one might use flash memory proteins 
 in neuroscience experiments . sample and hold proteins are probably 
 most useful when the neural activity is linked to a repeatable stimulus , 
 e.g. , in a sensory processing experiment . one could then repeat the 
 stimulus multiple times , interleaved with trials without the stimulus . 
 by varying the interval between stimulus and  write flash , one may determine the precise sequence in which the stimulus 
 activates neurons . light - gated integrators may be more useful in identifying 
 brain regions that show enhanced activity during spontaneously generated 
 behaviors . one could deliver a flash of light to the brain upon observing 
 the desired behavior and then fix and image the brain region of interest . see the supporting information for detailed 
 materials and methods , summarized below . a library of arch(d95x ) mutants was 
 generated by performing saturation mutagenesis of residue asp95 in 
 arch in the pet-28b vector . to allow for expression in hek-293 t cells , 
 the arch(d95x ) library was cloned into a lentiviral mammalian expression 
 vector ( addgene plasmid 22051 cut with the restriction enzymes bamhi and agei ) . the final 
 library consisted of arch(d95x ) fused to c - terminal egfp , under a 
 ubiquitin promoter . for neuronal expression , the ( d95h ) point mutation 
 was made on addgene plasmid 35514 ( plenti - camkiia - earch 3.0-eyfp ) . arch(d95x ) mutants were expressed 
 in e. 
 coli ( strain bl21 ) as previously described . carbonyl cyanide m - chlorophenyl 
 hydrazine ( cccp , 50 g / ml ) was added to neutralize membrane 
 potential , and the cells were spread on a glass coverslip for imaging . white light emission from a supercontinuum laser ( fianium sc-450 - 6 ) 
 was spectrally selected using an acousto - optic tunable filter . a digital 
 micromirror device ( texas instruments lightcrafter ) fluorescence 
 imaging of arch mutants in mammalian cells ( hek-293 t and neurons ) 
 was performed on a home - built , inverted wide - field microscope . patch - clamp 
 experiments were performed at room temperature ( 25 c ) using 
 an axopatch 200b amplifier ( molecular devices ) . all patch - clamp data 
 in hek cells were acquired in voltage - clamp mode ; all patch - clamp 
 data in neurons were acquired in current - clamp mode . in each combined 
 fluorescence and patch - clamp experiment , we illuminated the sample 
 with a series of laser pulses ( i = 250 w / cm unless otherwise specified ) while varying the voltage or current 
 across the cell membrane . the experimental sequence was repeated multiple 
 times to ensure that observed effects were not due to photobleaching . numerical 
 simulation of a three - state model of light - gated voltage integration 
 was implemented in matlab . a system of ordinary differential equations 
 was defined with states d1 , d2 , 
 and f , and rates kd1d2 , kd2d1 , kd2f , and kfd2 . rates kd1d2 and kd2d1 were assumed to depend on membrane voltage , while rates kd2f and kfd2 were assumed to depend on illumination 
 wavelength and intensity .
we developed a technique , 
 flash 
 memory  , to record 
 a photochemical imprint of the activity state  firing or not 
 firing  of a neuron at a user - selected moment in time . 
 the key 
 element is an engineered microbial rhodopsin protein with three states . 
 two nonfluorescent states , d1 and d2 , exist 
 in a voltage - dependent equilibrium . 
 a stable fluorescent state , f , 
 is reached by a photochemical conversion from d2 . 
 when 
 exposed to light of a wavelength write , population 
 transfers from d2 to f , at a rate determined by the d1  d2 equilibrium . 
 the population of f maintains 
 a record of membrane voltage which persists in the dark . 
 illumination 
 at a later time at a wavelength read excites fluorescence 
 of f , probing this record . 
 an optional third flash at a wavelength 
 reset converts f back to d2 , for a subsequent 
 write  read cycle . 
 the flash memory method offers the promise 
 to decouple the recording of neural activity from its readout . in 
 principle 
 , the technique may enable one to generate snapshots of neural 
 activity in a large volume of neural tissue , e.g. , a complete mouse 
 brain , by circumventing the challenge of imaging a large volume with 
 simultaneous high spatial and high temporal resolution . 
 the proof - of - principle 
 flash memory sensors presented here will need improvements in sensitivity , 
 speed , brightness , and membrane trafficking before this goal can be 
 realized .