Downloaded by guest on October 1, 2021 a iiiy ope ihtefc httelftm fteexcited the of lifetime the that fact the sen- with high most This coupled TPLSM. than sitivity, in larger used be commonly to molecules two-photon out CR fluorescent turns the of which estimate cross-section, an provide absorption to used single- is a state. analysis ground by Our CR find described the of we are depletion TPLSM, incorporating photocurrents model for CR photocycle of used ms) characterization intensities (<25 light brief biophysical typical that initial At CR. an of provide TPE to cells tured by possible currently tar- for illumination. is precision (blue-light) than single-photon spatial using of photostimulation degree neuronal to higher method geted much analogous a (TPE) of excitation confer an two-photon plane would using that by the hypothesized photocurrents around CR We activate section 11). optical (10, tis- intrinsic focus scattering an in providing deep achieve sue, excitation to which fluorescence by choice localized of method spatially the is beam IR focused in for circuits. favored neural typically of geometry studies a vivo 9), with ref. see pattern (but illumination of spatial single projected a to con- or be fined cannot excitation beam Additionally, (8). depth focused scattering and tissue increasing definition a optically spatial of an the reducing is intensity light, example, defocuses single-photon that using for medium by tissue, tissue biological Brain thick excitation. in achieve to ficult precision the determines interrogation. neuronal excited which optical independently with of be resolution can optical the each photosensitized, are cells mul- tiple Where (2–7). illu- resolution blue-light temporal millisecond using with excitation, mination, single-photon by triggered potentials be action can neurons, CR-expressing in that shown been conductance. has light-gated excitable It this in recapitulate heterologously to expressed encoded neurons been each mammalian have channel, gene, the single of a forms by modified Several (1). nation C microscopy laser-scanning | photostimulation | excitability molecular typically intrinsic not is by photocurrents limited CR of stimulation TPE that excitation indicating cross- two-photon absorption IR two-photon at CR’s using section of by estimate empirical activated An be (TPE). also exci- visible-light can using ion tation, by light-gated activated conventionally a is (CR), that channel channelrhodopsin-2 that demonstrate We Rickgauer Peter John saturation at channelrhodopsin-2 of excitation Two-photon w.nsog/ci/di/1.03/pnas.0907084106 / 10.1073 / doi / cgi / www.pnas.org eue osiuaeato oetasi utrdCR-expressing cultured in potentials action can neurons. stimulate excitation, to visible-light tempo- like used TPE, TPE be that scanning show We low-power summate. using rally pho- the by how influences and stimulated photostimulation, tocurrents state TPE evaluate focused current-conducting of we CR’s resolution depletion, spatial of ground-state pho- saturation of CR model how of a measurements and physiological tocurrents direct using By photon]. o,N 08544; NJ ton, otiue yDvdW ak ue2,20 sn o eiwMy1,2009) 12, May review for (sent 2009 25, June Tank, W. David by Contributed 08544; NJ Princeton, University, eateto oeua ilg,PictnUiest,Pictn J08544; NJ Princeton, University, Princeton Biology, Molecular of Department eew s hl-elrcrig fpoournsi cul- in photocurrents of recordings whole-cell use we Here a using (TPLSM) microscopy laser-scanning Two-photon dif- however, is, cells individual of excitation localized Spatially anlhdpi- C)i irbal eie cation derived microbially illumi- blue-light to response a in opens transiently that is channel (CR) hannelrhodopsin-2 λ = c rneo ersineIsiue alIanLbrtr,PictnUiest,Pictn J054 and 08544; NJ Princeton, University, Princeton Laboratory, Icahn Carl Institute, Neuroscience Princeton 2 mi rsne,wt au (260 value a with presented, is nm 920 z fclpaeo neetpredclrt h axis the to perpendicular interest of plane -focal a,b,c n ai .Tank W. David and [1GM= a,b,c,d,1 10 −50 ± (cm 0GM) 20 4 s)/ b h ei-ilrIsiuefrItgaieGnmc,PictnUiest,Prince- University, Princeton Genomics, Integrative for Institute Lewis-Sigler The hsatcecnan uprigifrainoln twww.pnas.org/cgi/content/full/ at online information supporting 0907084106/DCSupplemental. contains article This h uhr elr ocnito interest. 1 of conflict no declare paper. performed authors the D.W.T. The wrote D.W.T. and and J.P.R. J.P.R. and research; data; designed analyzed J.P.R. D.W.T. research; and J.P.R. contributions: Author (m 0m vs. ms (>10 long is state current-conducting o oeue ntedr ortr oteectbegon state ground excitable the to s return (14). to 60 s) dark (7–8 least the time-constant at in reported molecules the of for than intervals longer dark measurements, allowing between this 13). by initially addressed We is experimentally (1, state. molecules ground issue small excitable of the be population in and the should homogenous that photocycle measured assumes to model second Our contributing a molecules through of record- current fraction of ms the 25 initial where the ings, to analysis our the restrict in pho- we S1 Accordingly, (Fig. transient molecules photocycle CR one the most) illuminated only (at of undergoing describe population a to from intended arising tocurrent is here model illumi- present The 13). we that (1, photocycles assuming multiple undergo molecules illumination, nated stationary under tocurrents absorption CR stimulation. neuronal the methods for TPE-based of optimizing for estimates foundation a and provides cross-section membrane analysis particular induced the for analyze adapted to currents; model, geometries, this scan condi- use and will experimental illumination We typical TPE. to in used appropriate tions is that saturation-limited of activation model CR simplified a present we section, this In Theory imaging. by TPLSM neurons in employed cultured commonly are in than a values potentials squared-intensity As lower action using scan- scan. stimulate that the can demonstrate of TPE we duration ning findings, and these of trajectory illustration scan practical the to sensitive of way this is in much stimulated current recover (whole-cell) integrated excitation, can the blue-light although whole-cell membrane under cell available photocurrent excitable the focus TPE the low-intensity over a when Scanning rapidly focus reduced. the is around intensity region laser Photostimulation the the to focus. confined of better plane is the currents near of than (12) from focus away of focus TPLSM plane amplitudes typical of the photocurrent at plane TPE larger focused excite the that can near find instensities we than here, 8); focus ref. of see (also plane the from fluorescence away more generate of can focus) the inversion (at conditions an saturated to leading state, life- excited ground-state the occupancy. the of state shortens that intensity below light time high which fluorophores, when of satura- saturation occurs conducting-state excited-state to as analogy condition in this tion, ground-state to of refer condition We the to depletion. leads fluorophore), typical the of owo orsodnesol eadesd -al [email protected]. E-mail: addressed. be should correspondence whom To Rmlcl ihan with molecule CR A pho- CR of dynamics the addressed models empirical Several under fluorophores of TPE that observed been has it Previously, = ,2fr1,2poo)adilmntdwt intensity with illuminated and 2-photon) 1-, for 2 1, PNAS etme ,2009 1, September poo bopincross-section absorption m-photon d eateto hsc,Princeton Physics, of Department o.106 vol. o 35 no. 0ne lifetime nsec <10 IAppendix). SI 15025–15030 I (t σ ), m

NEUROSCIENCE m should absorb light at a rate (σm/m)I(t) (15). As molecules nonconducting intermediates that return to an excitable are excited in a single-photocycle model, the concentration ground state within the period of our analysis, then our estimate ρg(t) of molecules in the ground state is reduced exponentially of σm should be taken as a lower bound. according to − σm m Results ρ = ρ m I(t) t [1] g(t) g0 e , As a first step toward developing a method for TPE stimulation ρ of CR, we used the depletion model to estimate the CR absorp- where g0 is the initial ground-state concentration. For molecules on a two-dimensional plane (e.g., on an idealized membrane sur- tion cross-section, which sets the scale for molecular excitabil- σ face) illuminated with a radially symmetric profile I(r, t)m, the rate ity. Although our focus here is 2, the cross-section for two- dN(t)/dt of recruitment to a particular excited state with a popu- photon excitation using IR illumination, the single-photon cross- σ lation N(t), occurring with quantum efficiency η, can be calculated section 1 under blue-light illumination was also measured for by integrating over the surface, and is given by completeness. Under spatially uniform illumination, the solution to Eq. 4 for  ∗ dN(t) σm the time-dependent photocurrent I (T), expressed as a fraction = η mρ ∗ I(r, t) g(r, t) dA, [2] of the total available photocurrent I , is given by dt m S max ∗   ∗  τ T T ρ I (T) k − τ − τ where g(r, t) now has an explicit spatial dependence. N (T), the = − k g − k ∗ ( 1) e e , [5] number of current-conducting molecules at time t = T after I τg − τk max k=1,2 light onset, can be calculated by weighting the excitation rate with m −1 the channel’s impulse-response function and integrating in time, where τg = m(σmI ) is the ground-state lifetime for one- giving photon (m = 1) and two-photon (m = 2) illumination, and    τ τ = T T−t T−t 1, 2, for k 1, 2 refer to the latency and current decay time ∗ dN(t) − τ − τ N (T) = e 2 − e 1 dt, [3] constants respectively, as in Eq. 3. Our general strategy was to 0 dt analyze the shape of initial transient whole-cell membrane cur- − where molecules enter the current-conducting population rent under voltage-clamp ( 50 mV) in CR-expressing HEK293T ∗ cells under a set of spatially uniform illumination conditions of (denoted by ) with a latency τ1 after excitation, and these cur- increasing intensity. As intensity increases, τ is shortened and rents decay with a characteristic time constant τ2. If the single- g channel current is i∗, then the instantaneous current at time T is depletion becomes more rapid, which changes the initial transient I∗(T) = i∗N∗(T), and the total available current from a popula- shape in a systematic way that depends upon the specific value ∗ = ∗η of σm (Fig. 1B); fitting the set of traces to the model for known tion of Nt molecules is Imax i Nt. The instantaneous fraction σ of available photocurrent is thus illumination intensities allows estimation of m. Wide-field illumination of CR-expressing cells by using blue ∗ ∗ light from an LED (λ = 470 ± 13 nm, 1.1−18.3 × 1018γ/cm2 s) I (T) = N (T) ∗ . [4] stimulated fast-rising inward currents that reached a transient I ηNt max peak amplitude, and subsequently decayed toward a steady-state In the following, we will use solutions to Eq. 4 under different value with reduced amplitude (Fig. 1A). Transient currents (here, excitation profiles. For clarity, we note that σm represents the t = 0−10 ms) stimulated by four or more different intensities molecular absorption cross-section and not the molecular action were then fit simultaneously (i.e., as families) to Eq. 5; one rep- cross-section (ησm); if ground state absorption yields short-lived, resentative family with fits overlaid is shown in Figure 1C. Fits

Fig. 1. Estimating σ2 with measurements of saturation-limited excitation. (A) CR photocurrents stimulated experimentally by using wide-field blue-light illumination (λ = 470 ± 13 nm; blue traces, above) or IR TPE focused to a large-diameter spot (λ = 920 ± 6 nm; red traces, below); experimental illumination geometries are depicted at right (ωtp = 35 μm). Overline indicates illumination epoch (500 ms); trace shading denotes incident intensities (I) or peak squared intensities (I2; see SI Appendix). Each trace is the average of 5–10 repeat trials. (Inset, Lower) Photocurrents excited by using mode-locked (ML) and non-mode- locked (no ML) pulses at constant average power; scale bars are 40 pA, 32 ms. Traces are five-trial averages. (B) Numerically simulated transient photocurrents, 2 generated by using Eq. 5 for each indicated value of σ1 (σ2, lower traces), I values from A (I , lower), and t = 0−10 ms (0−25 ms, lower). Amplitudes in each frame are scaled to maximum values reached during the interval (maximum values are indicated below). (C) Experimental currents from A with overlaid fits to Eq. 5. Boxed values of σ1 (σ2, lower) were obtained by fitting the family of traces simultaneously to Eq. 5, and then used to generate the overlaid fits.

15026 www.pnas.org / cgi / doi / 10.1073 / pnas.0907084106 Rickgauer and Tank Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 ltdb hl-elilmnto thg neste N4stimu- 9 (N=4 cells; three intensities from locations high lated at illumination whole-cell by stim- in ulated amplitude stimulated the of currents 10% area, exceeded surface sometimes for configuration cell this accounted total the focus However, of of 0.1% membrane. plane than the less illuminated in the illuminated (approx- area of the scale although area should the focus the with at imately) excited photocurrents ampli- currents the of molecules, CR tude CR- CR membrane-bound transmembrane a of of excitation Because photostimulated require surface and recorded. ms), upper were (32 the exposures currents on brief stationary for held cell expressing center was geometric focus The the LUMPlanFl/IR). of Olympus N.A., 40×/0.8 1 Rectto (λ excitation IR rfiears h uldaee ftree el Fg 1A, (Fig. cells targeted of diameter ω full the across squared-intensity profile uniform approximately an generating plane, ple xiain(m excitation erisseta w-htnasrto ek(9 M (17). GM) most than (290 higher peak also absorption is value two-photon This spectral CR, its to homologous near pump proton microbial a , 10 tmlto fC htcret yuigahg-..objective (≈1 focus high-N.A. TPE a small a using generate to by lens photocurrents CR of stimulation occur can as (8), focus significantly degraded. the be can near resolution this rate (12), conditions saturated the away under excitation exceeds of focus rate the integrated from the the when near However, generating volume (11). diffraction-limited objective, focus a aperture within numerical mostly high fluorescence a through excitation pulses focusing by achieved repre- is Typically, imaging volume. 1) TPLSM high-resolution excitation Fig. smaller in a precise requires shown spatially but photostimulation (as photostimulation, TPE waist to approach beam one sents large a with stimulation xie wyfo h ln ffcs(ee rmteopposing cell the the from away from focus (here, of plane focus the of stepped we plane membrane), cell the from away excited arising membrane. in-focus not the were of photocurrents excitation from these exclusively that suggested excess This Volvox ekapiue htwr 04%o h ekamplitude Eq. to simultaneously peak the same the of of illumination 10–40% cells. blue-light were high-intensity by that stimulated amplitudes peak rne2.5−3 (range o eectto-iie titniiscmail ihTPLSM with compatible intensities imaging. at should excitation-limited photocurrents be CR of not GM stimulation 40–100 TPE DsRed2; that EGFP, implying [e.g., (18)], TPLSM to common rophores ec htwsntosre nsnl-htnectto trials excitation single-photon in observed the in not S2 (Fig. was that dence depen- power-squared nearly a showed photocurrents 1A, of rise-rates (Fig. non-mode-locked 1A). photocurrents using stimulate not (Fig. but did power pulses illumination average comparable sustained reduced at a Trials during toward decay amplitude by followed stationary amplitude, peak transient a reached that photocurrents inward stimulated cells CR-expressing htsiuainwt ifato-iie P Focus. TPE Diffraction-Limited a with Photostimulation an yielded cells) 8 from recordings (175 for families estimate these of 35 to ikae n Tank and Rickgauer ist aiiso uvs ahicuig6 of including across estimate 42, each to an curves, averaged yielded of total, cells) families recordings 5 5 (286 to intensities Fits more or 1C. Fig. in shown (0.3–1.8 − tp oestimate To etse o hscniinwudafc ptal rcs TPE precise spatially affect would condition this how tested We odtriewehrteecret nlddacomponent a included currents these whether determine To aiiso rnin htcret (here, photocurrents transient of Families −50 7 = × (cm 10 35 hneroosn[1.7 channelrhodopsin × −17 4 ) nti pia ofiuain P tmlto of stimulation TPE configuration, optical this In μm). 10 s)/photon cm σ 49 .Oe hsrneo squared-intensities of range this Over Appendix). SI σ = × 1 2 γ 2 = ,wihi oprbeto comparable is which ), eue ogfcllnt est ou pulsed focus to lens focal-length long a used we , 2 f5 of 10 ) ersnaiefml ihfisoeli is overlaid fits with family representative a 2); /cm 920 −48 hsvlei iia to similar is value This ]. 4 ± computing 5, ± s cm 2 m oalredaee pti h sam- the in spot large-diameter a to nm) 6 1 ,cret tmltdb P reached TPE by stimulated currents ), × 4 s/γ 10 × ,o 260 or ), −17 σ 10 × 2 10 −16 cm = τ 55 σ g 2 2 (16)]. o h aeo two-photon of case the for 2.6 ≤ μm ausrpre o fluo- for reported values (median σ I ± 1 ± 2 2 esrdfrpurified for measured ≤ t ntepaeo focus; of plane the in 0.2 0GM 20 –5m)wr fit were ms) 0–25 = 1 × .Maximum Inset). × σ ± 10 2 10 57 eotdfor reported E;range SEM; −48 [1GM γ Whole-cell 2 /cm cm Inset; 4 4 s s/γ 2 = ). itn h oa urn eeae eradaa rmteplane the from away and near generated current total the dicting 15–25 by equator cell the to ceai lutaino hsgoer ssona ih) smaue exper- measured as right), at (Left shown imentally is geometry of this of plane illustration the distance schematic with a epoch), by (N.A. stimulated separated focus indicates focus TPE overline stationary red power; a average using by stimulated tocurrents tda ufc n ufc ,rsetvl,adtebaktaerepresents trace black the and (1 respectively, current 2, summed surface and the 1 surface at ated au) re iei iuainfaeidctstelcto fsrae1 as 1, surface of location the indicates common frame single in represented a simulation to in indicated scaled line the are Green frame at value). each (Right) in simulated values and all (Left) powers; sample-plane measured as above, defined as mltd hr h ln ffcswsaoetecl (+z cell the above was focus maximum of a plane an the and where equator, membrane, amplitude cell the the near near amplitude amplitude intermediate minimum a generating equator, (measured currents photostimulated at of amplitude the equator), microns of tens several cell. was pho- focus the of above that plane powers the illustrate plane when sample even 2B, current; mW) moderate (40 Fig. at measured stimulated in be shown could the tocurrents cell, one to from significantly recordings contributed excitation (+z z 2. Fig. mmrn urn nertdfrom integrated current (membrane oe n cldfrvisibility. for scaled and power N n urn ieconstants time current and nFg ) ntil sn 04 Wo oe,cret ihthe with currents power, of mW 20–40 (−z charge, using equator total cell trials largest the In below 2). stepped Fig. was in focus of plane the where au,smltdcret hw ngenadbu ersn Eq. represent blue and green in shown currents simulated value, hs rnscudb erdcdi ueia iuain pre- simulations numerical in reproduced be could trends These vrarneof range a Over = t t 1 s curdwe h ln ffcswssprtdfrom separated was focus of plane the when occurred ms) =15 1 s ntal nrae ihicesn itnefo the from distance increasing with increased initially ms) =15 nFg )adrpae hs esrmns Out-of-focus measurements. these repeated and 2) Fig. in / el,2–0m) hstedwsas bevdi / cells 3/3 in observed also was trend This mW). 20–40 cells, 4/4 Pho- (A) saturation. at photocurrents CR of excitation Out-of-focus A.( oun n iuae ueial (Right numerically simulated and column) aea te iuain,btuig01m average mW 0.1 using but simulations, other as Same Inset) PNAS z Q + fclpaepstos(eaiet h cell the to (relative positions plane -focal T ) iuain used Simulations 2). etme ,2009 1, September τ mmrn urn nertdfrom integrated current (membrane 1 μm. z = rmtepaetruhtecl qao (a equator cell the through plane the from 1msandτ t = 0tot 2 = h 0m.(B ms. 20 = o.106 vol. = 5m)potdagainst plotted ms) 15 10 μm, oa charge, Total ) oun.A each At column). o 35 no. σ = 2 .,4 mW 40 0.8, = nFg 2; Fig. in 5 GM, 250 6 evalu- 15027 t =0 Q T z

NEUROSCIENCE of focus. Cell-membrane geometry was approximated by two par- whole-cell current (black trace), thus had a larger amplitude than allel planes (top and bottom membrane surfaces) separated by predicted by the area excited at the focus. a distance h =10μm along the optical axis (Fig. 2A), with an At moderate power (40 mW), the maximum integrated current equatorial plane at a distance z from the plane of focus. Under illu- amplitude occurred in both simulations and experiments when mination with a Gaussian squared-intensity profile, the solution the whole cell was below the plane of focus (z = 30–35 μmin to Eq. 4 for a specified value of z is given by simulations, z = 20–25 μm in experiments), at a distance that was ∗    reduced as the total sample-plane power was reduced (Fig. 2B). In  T t T−t I (z, T) 1 1 − τ − τ = = (−1)k 1 − e g e k dt, [6] simulations using very low power (P 0.1 mW), where the effects I∗ (z) 2 t of saturation should be reduced, integrated current amplitudes max k=1,2 0 were highest when the plane of focus coincided with the mem- τ = σ 2 −1 brane (Fig. 2B, Inset), as expected of TPE under nonsaturated where g 2( 2I0 (z)) is the ground-state lifetime under two-photon excitation. In this formulation, increasing the sep- conditions. aration between the plane of focus and an excitable surface not only decreases the incident peak squared intensity, I2, but Stimulation with a Moving Focal Spot. For some types of measure- ∗ 0 also increases Imax(z) because the area illuminated is larger (see ments, out-of-focus excitation manipulated to maximize whole- SI Appendix). cell current activation (as shown in Fig. 2) could represent a In simulations using 40 mW of power, positioning the plane of second approach to TPE photostimulation. A third approach, focus at one surface (e.g., green trace at z =+5 μm, Fig. 2A, Inset) which could make better use of the nonlinear properties of TPE, concentrated incident TPE on that surface, generating a current would be to use low-power TPE to stimulate currents primarily at that rapidly attained a peak, with an amplitude limited by the small the focus, and to scan the focus, in time, across the photosensitized illuminated area near the focus. Simultaneously, diffuse excitation membrane. on the opposing surface (blue trace, Fig. 2A, Inset) stimulated a We tested this approach by scanning a low-power, high- current that rose less rapidly, but which reached a larger ampli- N.A. focus across a strip-shaped region spanning the visualized tude at peak. The summed two-surface current, approximating the diameters of patched cells (Fig. 3A;1mW,N.A.= 0.8). Small

Fig. 3. Photocurrents stimulated by focused TPE. (A) Strip scan. (i) TPLSM fluorescence image of a CR-expressing cell (gray = volume-filling Alexa 594, pro- jected in z; yellow = CR-EYFP, single z-focal plane). Highlighted region indicates the boundary of a strip-scan stimulation trajectory (schematically represented in red), in which the TPE focus was oscillated at 1 kHz (y) while scanning across the cell (x). (ii) TPE-stimulated photocurrent recorded during a strip-scan (gray = 5 repeats overlaid; black = average), and trial-averaged TPE fluorescence recording during strip scans (yellow = CR-EYFP; dye-filled region indicates thresholded intracellular Alexa 594 fluorescence). Horizontal axis denotes scan time (0–1024 ms; scan distance is 57 μm). (B) Temporal summation of pho- tocurrents stimulated by a moving TPE focus: maximum photocurrents (scaled) predicted for CR molecules excited in a temporal sequence, occurring over a total time of Ts, approximating progressive excitation by a moving focal spot (Eq. 7). (Inset) Schematic representation of fast and slow scans over a fixed = 2 = × 54γ2 4 2 × 54γ2 4 2 number of molecules. (C) Photocurrents stimulated by focused TPE scans [N.A. 0.2; I0 3.8 10 /cm s ,(Left); 2.5 10 /cm s ,(Right)], illustrating the effect of scan consolidation (i.e., reducing total scan time, Ts). Peak currents are indicated and connected by dotted lines. Raster scan times (grayscale; shorter = faster = left) were varied by changing the number of lines in a fixed-area raster. Currents stimulated using spiral-scan trajectories, scanning inward from the cell boundary to the center, are shown in red [Ts = 27 ms, (Left); Ts = 16 ms, (Right)]. Traces are registered in time by the measured onset of fluorescence during each scan, and represent average response from three or more trials.

15028 www.pnas.org / cgi / doi / 10.1073 / pnas.0907084106 Rickgauer and Tank Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 τ ekapiueb atro -.Apiue nrae more increased Amplitudes the 3-4. increased of low ms factor at 15–30 a rapidly to by time-to-peak amplitude the peak reduced that rents ihso-cnigcret ( T currents slow-scanning with odto nwhich in condition 0%o h hl-elilmntdcret(6 current illuminated representing whole-cell currents the peak of stimulated % typically <10 which ms), 100 ftesm cells. illumination same blue-light the the whole-cell of of by (52–84%) currents stimulated half ms, amplitudes over were <16 were peak in that geometries amplitudes membrane membrane peak the attain of where could scan cells full a with four with each In compatible recordings, cells). averaged 7 (20 5.8 across of factor increase to a photocurrent peak by the allowed ms <100 in stimulation htsann cosauiomcnetaino oeue (N molecules of concentration reasoned uniform we a photocurrents, across scanning scan-stimulated that of amplitude the n atsas n hl-elcretapiue nrae as reducing increased by amplitudes consolidated current were whole-cell scans deflections slow and these both scans, in trajectory fast observable scan were and the membrane cell of photocur- excitable intersections general, and In from raster. arising a in deflections lines rent of number the geometries changing by cell after patterned (see trajectories spiral-like followed et ob eeae ihlwpwrectto,w measured we duration, excitation, varying of low-power scans with by stimulated generated currents be to rents “consolidation.” time, as less to in refer area here same we the which over scanning by significantly increase nti omlto,asa eunelasting sequence scan a formulation, this In plcto fC sbu-ih tmlto fato potentials findings, action our of of demonstration practical stimulation a As blue-light neurons. excitable is in CR of application atrsa ie(T whole-cell TPLSM time typical scan a For raster 3B). (Fig. current available the of o,adsd facl)fo ige(x single a from cell) a bot- of (top, side areas and membrane tom, all from currents stimulate To surface. n h fetv ..o h betv iha rs(N.A. iris an with objective the of N.A. (in effective axis the optical ing the along volume TPE the extended we yEq. by prxmtdby approximated .T siaehwtettlsa time, scan Fig. total (e.g., the scan the how in estimate later To activated 3A). currents with summate not oeue oa)a osatvlct hudect molecules (i.e., excite rate should constant velocity constant a a at at total) molecules was focus the 1 when primarily within excited were photocurrents inward ikae n Tank and Rickgauer Neurons. Cultured in Stimulation Spike Two-Photon el) osldtn h cn(.. reducing (i.e., scan the consolidating cells), httesa-tmltdcretapiuewudbe would amplitude current scan-stimulated the that odrneof range fold the in S1 Fig. (see ieNA =.)adTLMiaeaqiiinsa ie (T times scan acquisition image TPLSM and (=0.8) N.A. tive cnigpootmlto ae uhlne hnC’ current CR’s than if population (τ longer decay-constant However, large much currents. a takes large-amplitude across photostimulation scanning generate scanned to be molecules must of focus the focus, the (78% mW). excited 1–7 cells, also 3 was across boundary cell deflections the of with associated cence h ekcretapiue hc clsivreywith inversely scales that which predicts amplitude, current also peak It the amplitude. current stimulated whole-cell the 2 ots hte osldto ol lo agrapiuecur- larger-amplitude allow would consolidation whether test To i.3C Fig. oprdwt atrsann cosclsuigtefl objec- full the using cells across scanning raster with Compared ne odtosweecret r eeae rmrl at primarily generated are currents where conditions Under (T ,adteohrsas(rycl)wr produced were (grayscale) scans other the and Appendix), SI s = 7. nτ fteifre ebaeeg,weefluores- where edge, membrane inferred the of μm eosrtsteefc fcnoiainoe ten- a over consolidation of effect the demonstrates 2 ol ii h ekpoourn o(1 to photocurrent peak the limit would ) > T T s s nareetwt h eednepredicted dependence the with agreement in , 2 2 ntocls h ats cn soni red) in (shown scans fastest the cells; two in IAppendix). SI ,cret tmltderyi h cnshould scan the in early stimulated currents ), σ s T I bv h en sn sbn;3 scans 30 bins; ms 1 using mean, the above = ∗ I s max (T ∗ 5 s and ms) 250 > s dN ) = τ /dt 1 h ouint Eq. to solution the , T τ s 2 s > (1 = 0 s,fse-ciae cur- faster-activated ms), 100 − K τ e , 2 − nEq. in oaini hs scans, these in location y) = τ T 2 s T ). 0m,Eq. ms, 20 s n oalwwhole-cell allow to ) T s .I h typical the In 2). ie ogrthan longer times ol influence could , T ± T s vrtecell the over , s Compared . % N 3%; common A z 4 sweeps, ≥3 yadjust- by ) = < 7 sclosely is − T 0.2–0.5) predicts s 0 of 10% could , e −n = )/n s [7] > 5 t I ,soni ieve n rmtetp ( blue-light top. wide-field using (I stimulation the by illumination stimulated from TPE changes, and voltage scanning membrane view of ings and side in (Upper) to shown excitation used (Lower), boundary blue-light (B outer whole-cell trajectory. the of indicates spiral-scan outline volume- a Red yellow; (CR-EYFP, designate gray). culture 594, in Alexa neuron SCG filling patch-clamped a of image 4. Fig. fsm-agtdsia cn;the scans; trajectories spiral define soma-targeted to imaged of and filled, recordings, whole-cell (see for CR express to duced used neurons. be cultured can in scans potentials spiral action TPE stimulate consolidated to that showed we then, n Wo oe,>9 fteC oeue hudb excited be should molecules CR the 10 of within 40 >99% standard power, of a mW of 1 illu- and focus continuously the is using membrane by containing minated CR When nm). 920 at stimulation. single-cell duration. experimental for of and design perspective the geometry from results stimulation these of review we effects Here, cross-section the absorption explore CR to the and estimate to This used one- depletion. was cur- a framework ground-state in CR incorporates captured that transient quantitatively model be of photocycle can 5/10, kinetics TPE by the min stimulated that rents neuron; demonstrate per results Our trials 10 trials, Discussion spikes 60 generating 4). (Fig. trials, of 10/10) depo- most out max cells in same spikes threshold the (50 of above stimulation cells spike TPE larized above trials, 5/5 voltages in membrane threshold depolarized the illumination match light (N.A. approximately cells to of adjusted was volume pnfo htptho ebaeadwihdcy wyo the on away (τ decays scale which time and channels tens-of-msec membrane most of represents patch that that from produced open is rapidly current but small of a pulse scale, time rising msec the on viewed saturated When a response. yielding excitation further is state with ground depleted, the substantially as current more produce not does the position at same intensity higher or illumination continuing state, conducting 0 2 = iscae ueircria agin(C)nuostrans- neurons (SCG) ganglion cervical superior Dissociated 6 GM (≈260 cross-section absorption two-photon high a has CR 7.9 × P fluorescence TPE (A) TPE. using potentials action of Stimulation 10 seEq. (see μs 54 γ = 2 /cm 10 = PNAS 19 .–.) nsxnuosweewd-edblue- wide-field where neurons six In 0.2–0.5). 4 γ s /cm 2 ; .Bcueo h oglftm fteexcited the of lifetime long the of Because 6). .Oelnsidct tmlto times. stimulation indicate Overlines Right). 2 etme ,2009 1, September s; r3 ssia cn ihTE(N.A. TPE with scans spiral ms 32 or Left) 2 eetargeted were Methods) and Materials .Ti ai nighstoimportant two has finding basic This ). ceai eito fgeometries of depiction Schematic ) z dmnino h P focal TPE the of -dimension o.106 vol. C × urn-lm record- Current-clamp ) . ..objective N.A. 0.8 o 35 no. z -dimension = 15029 0.3,

NEUROSCIENCE implications for localized TPE photostimulation using CR: (i)at Materials and Methods high light intensities that rapidly saturate the excited photocur- Cell Culture and Gene Expression. CR expression used the pLenti-EF1α- rents from an in-focus patch, excitation away from the focus can hChR2-EYFP-WPRE plasmid a gift from K. Deisseroth (Stanford Univer- produce photocurrents in other membranes from the same cell sity, Stanford, CA). Transient CR expression in low-passage HEK293T cells (or in other cells) that can easily exceed focally excited currents; (Invitrogen; cultured in DMEM +10% FBS) was attained by using and (ii) at lower intensities that still excite most available current phosphate precipitation (0.5–1 μg DNA/35 mm plate), and physiology was from an in-focus patch but which also reduce out-of-focus excita- performed within 24–96 h of transfection. Stable expression of CR in disso- ciated neurons from E17 rat SCG [provided by K. McCarthy and L. Enquist tion, scanning the focused spot rapidly across the entire excitable τ (Princeton University, Princeton, NJ); cultured in supplemented Neurobasal membrane area of a cell on a time scale less than 2 will produce medium] was attained by lentiviral transduction 4–7 days in vitro; vectors the largest current for stimulation. were grown as described (2). Physiology was performed within 2–4 weeks of Both of these effects are captured quantitatively and can be transduction. studied more generally as a function of light intensity and geome- − try in the framework described by Eqs. 1–4, where photocurrents Electrophysiology. Whole-cell recordings under voltage clamp ( 50 mV) and current-clamp (Fig. 4) were made at room temperature under low represent the rate of excitation as integrated, in the mathematical ambient-light conditions, using an N = 0.1 headstage and a BVC700A sense, over the duration and membrane-incident area of illu- bridge/voltage clamp amplifier (Dagan), and then digitized and recorded at mination in a single-photocycle model. In our experiments and 20–40 kHz using the pClamp 10 recording platform (Digidata 1322a, Clam- simulations, transient photocurrents at high light intensity (much pex 10; Axon/Molecular Devices). See SI Appendix for solutions and other higher than focused spot saturation) are largest when the focal recording parameters. spot is off the membrane (Fig. 2). Photocurrent stimulation could Data Analysis. Data were analyzed by using scripts written in Matlab be better confined to the small region near the focus by using (v7.6.0; Mathworks). Photocurrent amplitudes in voltage-clamp recordings lower-intensity excitation (Figs. 2–3), although such currents were were determined relative to the mean holding current during a 5–10 ms win- (naturally) much than smaller in amplitude because the membrane dow preceding the synchronously recorded light-on trigger signal, and low- area contributing to the current is very small. This finding is con- pass filtered by using least-squares polynomial smoothing (Matlab’s sgolayfilt sistent with studies that use focused single-photon excitation of function; order 1, frame size 5). Averaged traces, where shown, represent the CR (19, 20), wherein the spatial precision attainable with similar mean of the time-registered recordings. Groups of recorded currents were fit parked-beam protocols was also improved by reducing excitation simultaneously (as a family) to aligns given in the text by using nonlinear intensity. least-squares optimization (see SI Appendix). Consistent with the high CR cross-section and Eq. 7, we found Optical Stimulation Sources. Blue light for single-photon photostimula- that low-intensity, focused TPE stimulation at moderate N.A. val- tion trials was supplied by a Luxeon V LED (470 ± 13 nm; Star Hex, lambertian ues could generate currents with amplitudes exceeding 50% of the pattern, #LXHL-LB5C, Philips Lumileds) powered by a variable voltage sup- whole-cell stimulated photocurrent by rapidly scanning the focal ply (#72-6615, Tenma) with the current switched on and off by using a metal volume over the excitable cell membrane. Fast-scanning stimula- oxide semiconductor field effect transistor. Pulsed IR excitation for photostim- tion should be compatible with most TPLSM systems, although ulation and fluorescence image acquisition was supplied by a mode-locked ± photocurrents stimulated in this way reached amplitudes that Ti:Sapphire laser tuned to 920 6 nm (Mira 900, Verdi V10-pumped; Coher- τ ent), incorporated into a home-built apparatus based on an Olympus BX51 decreased sharply as whole-cell scan times exceeded 2. Alter- microscope (see Fig. S1 in the SI Appendix). Laser-scanning excitation for pho- nate approaches to achieving large-amplitude currents with spatial tostimulation and fluorescence image acquisition was controlled by using τ precision might use molecules with longer 2 values [e.g., C128 software written in LabView (National Instruments) to issue command signals mutants (3)] (see Eq. 7), although long τ2 values should also render driving deflections of galvanometer-driven scan mirrors (6210 series, Cam- such molecules more sensitive to collateral excitation away from bridge Technology). Actual galvanometer positions during these scans were the plane of focus (see Eq. 6). These properties could be comple- monitored by digitizing and recording the position feedback signals from the mented or mitigated with spatially targeted expression strategies, galvo amplifiers (Micromax 677, Cambridge Technology) synchronously with e.g., by using molecular trafficking signals (21). the electrophysiology data. Flexible methods for TPE photostimulation of CR or other ACKNOWLEDGMENTS. We thank D. Dombeck for insightful discussions, might take advantage of optical techniques recently devel- K. Deisseroth (Stanford University, Stanford, CA) for constructs, L. Enquist oped for TPLSM imaging, including focal-array scanning (22), (Princeton University) for tissue culture advice and facilities, H. Coller (Prince- ton University) for VSV-G and R8.9 plasmids, and K. McCarthy (Princeton multiple z-focal plane excitation (23), or beam-shaping with a University) for SCG neurons and helpful discussions throughout. This work spatial light modulator (24). Our framework may provide some was supported by the National Institutes of Health Grant 1R01MH083686-01, guidance in the development of these techniques. and a National Science Foundation Graduate Research Fellowship (to J.P.R.).

1. Nagel G, et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective 13. Nikolic K, et al. (2009) Photocycles of channelrhodopsin-2. Photochem Photobiol membrane channel. Proc Natl Acad Sci USA 100:13940–13945. 85:400–411. 2. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, 14. Bamann C, Kirsch T, Nagel G, Bamberg E (2008) Spectral characteristics of the pho- genetically targeted optical control of neural activity. Nat Neurosci 8:1263–1268. tocycle of channelrhodopsin-2 and its implication for channel function. J Mol Biol 3. Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K (2009) Bi-stable neural 375:686–694. state switches. Nat Neurosci 12:229–234. 15. Xu C, Webb WW (1996) Measurement of two-photon excitation cross sections of 4. Lin JY, Lin MZ, Steinbach P, Tsien RY (2009) Characterization of engineered channel- molecular fluorophores with data from 690 to 1050 nm. J Opt Soc Am B 13:481–491. variants with improved properties and kinetics. Biophys J 96:1803–1814. 16. Ernst OP, et al. (2008) Photoactivation of channelrhodopsin. J Biol Chem 283:1637– 5. Huber D, et al. (2008) Sparse optical microstimulation in barrel cortex drives learned 1643. behaviour in freely moving mice. Nature 451:61–64. 17. Birge RR, Zhang CF (1990) Two-photon double-resonance spectroscopy of 6. Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L (2008) Neural sub- bacteriorhodopsin—assignment of the electronic and dipolar properties of the 1 1 ∗ π π∗ strates of awakening probed with optogenetic control of hypocretin neurons. Nature low-lying Ag-like and Bu-like , states. J Chem Phys 92:7179–7195. 450:420–424. 18. Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Absolute two- 7. Ayling OGS, Harrison TC, Boyd JD, Goroshkov A, Murphy TH (2009) Automated light- photon absorption spectra and two-photon brightness of orange and red fluorescent based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic . J Phys Chem B 113:855–859. mice. Nat Methods 6:219–224. 19. Schoenenberger P, Grunditz A, Rose T, Oertner TG (2008) Optimizing the spatial 8. Theer P, Denk W (2006) On the fundamental imaging-depth limit in two-photon resolution of channelrhodopsin-2 activation. Brain Cell Biol 36:119–127. microscopy. J Opt Soc Am A 23:3139–3149. 20. Petreanu L, Mao TY, Sternson SM, Svoboda K (2009) The subcellular organization of 9. Holekamp TF, Turaga D, Holy TE (2008) Fast three-dimensional fluorescence imaging neocortical excitatory connections. Nature 457:1142–1145. of activity in neural populations by objective-coupled planar illumination microscopy. 21. Lewis TL, Mao TY, Svoboda K, Arnold DB (2009) Myosin-dependent targeting of Neuron 57:661–672. transmembrane proteins to neuronal dendrites. Nat Neurosci 12:568–576. 10. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence 22. Nielsen T, Frick M, Hellweg D, Andresen P (2001) High efficiency beam splitter for microscopy. Science 248:73–76. multifocal multiphoton microscopy. J Microsc 201:368–376. 11. Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: Multiphoton microscopy 23. Amir W, et al. (2007) Simultaneous imaging of multiple focal planes using a in the biosciences. Nat Biotechnol 21:1368–1376. two-photon scanning microscope. Opt Lett 32:1731–1733. 12. Nagy A, Wu JR, Berland KM (2005) Observation volumes and gamma-factors in 24. Papagiakoumou E, de Sars V, Oron D, Emiliani V (2008) Patterned two-photon illumi- two-photon fluorescence fluctuation spectroscopy. Biophys J 89:2077–2090. nation by spatiotemporal shaping of ultrashort pulses. Opt Express 16:22039–22047.

15030 www.pnas.org / cgi / doi / 10.1073 / pnas.0907084106 Rickgauer and Tank Downloaded by guest on October 1, 2021