the large number of 
 photoswitchable biomolecules discovered and 
 developed in recent years covers a great variety of cellular functions , 
 like catalysis of metabolic processes , cytoskeletal 
 polymerization and motors , nucleic acids dynamics , intracellular signaling , and , perhaps most dazzling , membrane excitability , which has been 
 at the focus of optogenetics and optopharmacology .
the dream of precisely and remotely photocontrolling 
 every aspect of the cell s inner workings in intact tissue 
 appears within reach and offers the promise of interrogating complex 
 cellular processes to discover their molecular mechanisms . in order to take full advantage of light - regulated 
 proteins ,
multiphoton 
 excitation with near - infrared ( nir ) light provides sub - micrometric 
 resolution in three dimensions , deep 
 penetration into tissue , and patterned 
 illumination .
however , to be adapted to two - photon 
 stimulation technology , the light response of natural photoswitchable 
 proteins like channelrhodopsin-2 ( chr2 ) must often be adjusted by 
 mutating the tight binding pocket of the natural chromophore , which 
 has fixed photochemical characteristics . in contrast , synthetic photoswitches developed by optochemical genetics 
 and optopharmacology are based on chromophores that act on the protein 
 surface and thus offer excellent opportunities for rationally tuning 
 their photochemical behavior by chemical substitutions that do not 
 affect the functional properties of the protein .
remarkably , two - photon stimulation 
 of synthetic photoswitchable proteins has not been investigated despite 
 the advances of neurotransmitter uncaging and optogenetics using pulsed nir illumination . to demonstrate the multiphoton activation of synthetic photoswitches , 
 we chose ion channels because they constitute highly sensitive transducers 
 of chromophore isomerization ( potentially up to the single channel 
 level ) .
in particular , we focused on the well - characterized light - gated 
 glutamate receptor ( liglur ) , a gluk2 kainate receptor - channel 
 that is chemically conjugated to a maleimide  azobenzene 
cis photoisomerization of this photoswitchable tethered ligand ( ptl ) 
 allows the efficient activation of the receptor upon one - photon absorption 
 of violet or blue radiation ( open liglur , figure 1b ) , a process that can be reverted back either by absorption 
 of green light or thermal relaxation in the dark ( closed liglur , figure 1b ) .
( a ) 
 structures of the photoswitchable tethered ligands applied 
 to the two - photon control of liglur : mag ( 1 ) , mag2p ( 2 ) , and maga2p ( 3 ) . 

violet ( one - photon ) 
 or nir ( two - photon ) light excitation induces glutamate recognition 
 and channel opening via trans 
this process is reverted 
 by illumination with visible light ( one - photon excitation ) for liglur - mag 
 and by thermal back - isomerization for liglur - mag2p and 
 liglur - maga2p . to control liglur using multiphoton excitation , here
we have 
 investigated 
 the performance of mag and two new mag derivatives ( 2 and 3 , figure 1a ) upon pulsed 
 nir illumination .
compounds 2 and 3 were 
 devised to enhance the two - photon excitation response of the symmetrically 
 substituted azobenzene chromophore in mag , which is expected to be 
 poor .
we introduced an asymmetric 
 aminoazobenzene with sufficiently strong push  pull character 
 as to enhance its two - photon absorption cross - section ( mag2p ) .
in addition , the presence of the electron - donating tertiary amine 
 in the 4-position should dramatically decrease the thermal stability 
 of its cis state in physiological conditions , thus resulting in fast spontaneous cis 
trans back - isomerization and , as such , enabling single - wavelength operation 
 of the switch .
this behavior is also expected for 3 containing 
 the same azobenzene core as mag2p . however
, a novel scheme 
 was exploited in this compound to enhance its nonlinear optical response , 
 which consists in the introduction of a light - harvesting antenna to 
 sensitize the trans  cis isomerization of the 
 system by absorption of nir radiation and subsequent resonant electronic 
 energy transfer ( ret ) to the trans - azobenzene group . because of its maleimide  azobenzene  glutamate  antenna 
 structure , we named compound 3 as maga2p . 

a naphthalene derivative was selected as antenna because of ( i ) its 
 high two - photon absorption cross - section , ( ii ) the large spectral overlap between its emission and the absorption 
 of the trans isomer of the aminoazobenzene group 
 in 3 , and ( iii ) its reduced size , to minimize potential 
 steric hindrance effects on the glutamate - binding site of the receptor .
the preparation of compounds mag2p and 
 maga2p was achieved via a multistep modular synthetic sequence 
 allowing 
 structural diversity in the final compounds as well as the additional 
 incorporation of a photo - harvesting antenna in 3 ( scheme 1 ) . in both cases
, we took the n , n - orthogonally diprotected l - lysine 4 as scaffold , to which the different functional fragments 
 of the target compounds were sequentially introduced : o - protected aminoazobenzene 5 , fully protected glutamate 
 derivative 6 , naphthalene derivative 7 , 
 and furan - protected maleimide 8 .
these fragments were 
 obtained from commercial products as described in the supporting information . with respect to the synthesis 
 of mag , several changes
first , a branching point was inserted between the 
 glutamate and azobenzene moieties to facilitate the incorporation 
 of additional functional units to the ptl structure .
second , we introduce 
 herein the use of 6 and 8 as more robust , 
 versatile , and convenient precursors of glutamate and maleimide moieties 
 during the multistep synthesis of novel mag derivatives .
reagents and conditions : ( a ) 5 , hatu , dipea , thf ( 89% ) ; ( b ) 37% hcl , meoh ( 93% ) ; ( c ) 6 , edci , hobt , dipea , thf ( 88% ) ; ( d ) 20% piperidine / dmf ( 87% ) ; 
 ( e ) 7 , edci , dipea , thf ( 81% ) ; ( f ) 20% piperidine / dmf 
 ( 64% ) ; ( g ) clcoch3 , pyridine , thf ( 69% ) ; ( h ) 37% hcl , meoh 
 ( 93% ) ; ( i ) 6 , edci , hobt , dipea , thf ( 71% ) ; ( j ) rhcl(pph3)3 , etoh / h2o , reflux ; ( k ) hgo , hgcl2 , acetone / h2o , reflux ; ( l ) 8 , ph3p , diad , thf ( 81% , over the three steps , for 13a , 27% for 13b ) ; ( m ) toluene , reflux ; ( n ) tfa , ch2cl2 ( 81% over the two steps for 2 , 
 86% for 3 ) .
abbreviations : hatu , o-(7-azabenzotriazol-1-yl)-n , n , n,n-tetramethyluronium hexafluorophosphate ; dipea , 
 diisopropylethylamine ; edci , n - ethyl - n-(3-dimethyldiaminopropyl)-carbodiimide hcl ; hobt , 1-hydroxybenzotriazole 
 hydrate ; diad , diisopropyl azodicarboxylate .
the synthesis of both mag2p and maga2p began 
 by the coupling reaction of 4 with aminoazobenzene 5 to afford the common intermediate 9 , from which 
 the synthetic pathways diverged .
for the synthesis of mag2p , acid removal of the tert - butyl carbamate protection 
 of 9 was followed by the coupling reaction of the resulting 
 amine with glutamate derivative 6 , basic deprotection 
 of the terminal amine , and its acetylation to furnish intermediate 12a . in the case of maga2p ,
the best results were 
 obtained by deprotecting first the amino terminus and proceeding through 
 its reaction with the antenna fragment 7 to deliver 11 .
removal of the carbamate protection and coupling with 6 then furnished compound 12b . from intermediates 12a and 12b ,
the next synthetic steps were analogous 
 for both ligands : removal of the allyl protecting group , introduction 
 of the furan - protected maleimide 8 under mitsunobu conditions , 
 release of the maleimide moiety via a retro - diels  alder reaction , 
 and cleavage of the tert - butyl carbamate and ester 
 protections , thus finally affording the target compounds mag2p and maga2p .
figure 2a plots the electronic 
 absorption spectra of the initial trans state of 
 compounds 13 and of the photo - harvesting 
 antenna tethered to maga2p ( see also figures s1 and s2 
 in the supporting information ) . owing to 
 the 4-amino substituent introduced in the azobenzene core of trans - mag2p and trans - maga2p , the absorption maximum of the 
 azoaromatic  * electronic transition of these 
 compounds clearly bathochromically shifts with respect to trans - mag ( 50 nm in dmso ) .
this allows the trans  cis photoisomerization of mag2p and 
 maga2p to occur upon illumination with blue light instead 
 of violet radiation . as shown in figure 2b
, 
 excitation of both ligands at 473 nm led to a noticeable decrease 
 of their  * absorption band , a typical signature 
 of photoinduced cis isomer formation .
this was further confirmed by h 
 nmr measurements in dmso - d6 , which revealed 
 that the relative concentration of cis - mag2p and cis - maga2p in the resulting photostationary 
 mixtures was 58% in both cases .
such photoproducts can be transformed 
 into their corresponding trans isomers by irradiation 
 with green light , as previously reported for mag ( figure s3 in the supporting information ) . in the case of mag2p and maga2p , however , 
 spontaneous thermal cis 
trans isomerization 
 plays a significant role in the recovery of the initial state of the 
 ligands , and it strongly competes with cis 
this effect is ascribed to the introduction of a 4-amino 
 substituent in the azobenzene moiety of mag2p and maga2p , and it is expected to be dramatically 
 enhanced in aqueous media .
thus , while 
 the lifetimes of cis - mag2p and cis - maga2p in the dark at room temperature are 
 75 min in dmso ( see figure s4 in the supporting 
 information ) , they further drop off down to the millisecond 
 time scale in aqueous buffer (  = 118 and 96 ms in 80% pbs : 
 20% dmso , respectively ; figure 2c ) .
cis isomerization of mag2p and maga2p at high frequencies in aqueous media 
 with a single irradiation source , which we have exploited to demonstrate 
 the high photostability of these light - responsive ligands ( figure 
 s5 in the supporting information ) .
( a ) absorption 
 spectra in dmso of trans - mag , trans - mag2p , trans - maga2p , and 
 the free naphthalene photo - harvesting antenna .
( b ) absorption spectra of trans - mag2p and trans - maga2p ( solid lines ) , and the photostationary states obtained upon 
 photoexcitation of these compounds in dmso at exc = 473 nm ( dashed lines ) .
( c ) variation of the absorption at  
 = 450 nm of trans 
cis mixtures of mag2p and maga2p in the dark at 25 c in 80% pbs:20% 
 dmso . at these conditions , thermal cis 
trans back - isomerization takes place , thus restoring the initial concentration 
 of the trans state of the ligands , which presents 
 a larger extinction coefficient at abs = 450 nm . 

solid lines correspond to the experimental data , while dashed lines 
 were obtained from monoexponential fits .
although the incorporation of a photo - harvesting antenna 
 negligibly 
 affects the intrinsic photochemical behavior of the azobenzene group 
 of maga2p with respect to mag2p , it provides 
 ligand 3 with some additional optical properties .
thus , trans - maga2p displays an extra band in the absorption 
 spectrum ( max = 385 and 380 nm in dmso and 80% pbs:20% 
 dmso , respectively ) , which arises from the naphthalene sensitizer 
 ( figure 2a and figure s1 in the supporting information ) .
the fluorescence emission 
 of this group is however strongly quenched upon covalent attachment 
 to the ligand , with a 20-fold decrease in fluorescence quantum 
 yield measured in aqueous buffer ( antenna = 0.43 
 and trans - maga2p = 0.02 ; 
 figure 3a ) .
this indicates the occurrence of 
 efficient ret processes from the photoexcited naphthalene antenna 
 to the azo moiety of trans - maga2p , in 
 agreement with the large frster radius calculated for this 
 donor  acceptor pair ( see figure s6 in the supporting information ) .
cis isomerization should take place in this 
 ligand , as demonstrated in figure 3b : 60% 
 increase in trans 
cis photoconversion was 
 determined for maga2p with respect to mag2p upon 
 selective irradiation of the naphthalene antenna at exc= 355 nm .
( a ) fluorescence emission spectra in 80% pbs:20% dmso of trans - maga2p and the photo - harvesting antenna 
 tethered to this ligand
. the spectra are normalized relative to the 
 excitation intensity and the absorption at the excitation wavelength 
 ( exc = 355 nm ) .
( b ) trans  cis photoconversion efficiency of trans - mag2p and trans - maga2p upon irradiation at 
  = 355 nm in dmso , which allows nearly selective excitation 
 of trans - maga2p sensitizer ( see figure 2a ) .
we 
 next tested mag , mag2p , and maga2p to photoswitch 
 liglur in living cells using one- and two - photon stimulation .
we expressed 
 gluk2-l439c - egfp in hek293 cells and incubated them in mag , mag2p , or maga2p in order to allow the selective conjugation 
 of the ptls to the cysteine introduced at position l439c of the receptor . 

for each compound , we recorded the corresponding photocurrents generated 
 upon light - induced opening of liglur channels using whole - cell patch 
 clamp ( see the supporting 
 information ) .
one - photon liglur currents were obtained 
 when the receptor was conjugated with the new compounds ( figures 4 and 5a ) .
the magnitude of 
 the photocurrent response was not reduced after repeated stimulations , 
 demonstrating the photostability of these compounds after protein 
 conjugation ( figure 4b , c and figure s8 in the supporting information ) .
figure 4 shows that for one - photon excitation , the wavelength dependence 
 of current amplitude measured is different for each ptl .
photocurrent 
 amplitudes at different wavelengths were quantified from electrophysiological 
 recordings obtained for the three compounds ( figure 4a  c ) , and the corresponding one - photon action spectra 
 were calculated ( figure 4d ) .
introduction of 
 the 4-amino substituent in the azo core allows the one - photon action 
 spectra of mag2p and maga2p to red - shift 60 
 nm with respect to that of mag , as recently reported for a similar 
 compound ( figure 4d and table s1 in the supporting information ) .
an additional peak is observed for maga2p at  
 = 360 nm , which lies very close to the absorbance band of the naphthalene 
 moiety ( see figure 2a ) .
thus , sensitization 
 of the azobenzene photoisomerization by the antenna also occurs when 
 the photoswitch is conjugated to liglur .
in addition , the time course 
 of the mag2p and maga2p one - photon currents 
 ( blue and red traces in figure 5a ) confirms 
 that fast spontaneous cis 
trans back - isomerization 
 and channel closure takes place after the illumination is switched 
 off , while it requires irradiation with green light for mag ( black 
 trace in figure 5a ) . by fitting
the one - photon 
 current decays in the dark with monoexponential functions , the lifetimes 
 of cis - mag2p and cis - maga2p tethered to liglur were determined to be 150 and 265 ms , 
 respectively ( table s1 in the supporting information ) .
these values are larger than those measured in solution ( see above ) , 
 which suggests that the ligand - binding site interaction slows down 
 the thermal cis 
this effect is enhanced for maga2p probably due to additional hydrophobic interactions and/or steric 
 hindrance effects arising from the tethered naphthalene antenna .
whole - cell 
 voltage - clamp current recordings in hek293 cells expressing 
 gluk2-l439c after conjugation to ( a ) mag , ( b ) mag2p , and 
 ( c ) maga2p .
current responses to one - photon light pulses 
 of wavelengths ranging from 325 to 575 nm were quantified .
( note that 
 in ( a ) resting  = 500 nm induces liglur deactivation , and in 
 ( b ) and ( c ) resting  = 690 nm is not absorbed and allows thermal 
 relaxation of these photoswitches . )
( d ) normalized one - photon action 
 spectra corresponding to mag ( black ) , mag2p ( blue ) , and 
 maga2p ( red ) ( n = 2 cells , n = 38 cells , and n = 410 cells , 
 respectively ) . before averaging over different cells ,
wavelength - dependent 
 current amplitudes were normalized to the maximum photocurrent along 
 the spectral range measured for each cell .
errors are sem . ( a ) one- and ( b ) two - photon whole - cell voltage - clamp recordings 
 on hek293 cells expressing liglur conjugated with mag ( black ) , mag2p ( blue ) , and maga2p ( red ) .
bars indicate stimulation 
 pulses applied to open ( one - photon pulses in violet and blue , two - photon 
 pulses in gray ) and close liglur ( one - photon pulses in green ) .
two - photon excitation conditions : 
 mag (  = 820 nm , 10 scans of 0.4 s , 38 mw on sample ) , mag2p (  = 900 nm , 0.4 s scan , 30 mw on sample ) , and maga2p (  = 880 nm , 0.4 s scan , 42 mw on sample ) . using a custom - built multiphoton setup where a 
 tightly focused 
 fs laser is raster scanned over the cells of interest , all three ptls 
 display robust and liglur - specific photocurrents in living cells that 
 first demonstrate two - photon stimulation with nir light of a synthetic 
 photoswitchable protein ( figure 5b and figures 
 s9 and s10 in the supporting information ) .
the amplitude of the responses follows the characteristic power 
 dependence of two - photon absorption processes ( figure s11 in the supporting information ) and corresponds to 1020% 
 of the photocurrent under one - photon excitation ( table s2 in the supporting information ) . in order to optimize 
 the multiphoton stimulation conditions we characterized the two - photon 
 action spectrum of each ptl ( figure 6a ) .
the 
 wavelength that yields maximal two - photon responses of mag is around 
 820 nm .
repeated cell raster scans are required to get a saturating 
 photocurrent from all available receptors ( black trace in figure 5b ) .
then , the current remains stable without laser 
 illumination until liglur is closed with 500 nm light via one - photon cis 
the mag2p two - photon action spectrum is red - shifted and yields maximum current 
 amplitude around 900 nm .
the reduced currents obtained from maga2p hindered the acquisition of a detailed action spectrum , 
 but are sufficient to identify two spectral ranges allowing two - photon 
 activation of liglur : the first can be found at 880 nm ( corresponding 
 to the direct absorbance of azobenzene , as in mag2p ) , and 
 the second is located around 740 nm and is consistent with the naphthalene - sensitized 
 photoisomerization .
( a ) two - photon action 
 spectra of liglur - mag ( black ) and liglur - mag2p ( blue ) and 
 two - photon activation of liglur - maga2p ( red ) at selected 
 wavelengths .
photocurrent amplitudes were corrected 
 for the different power densities used ( pd ) , averaged over all cells 
 measured , and normalized to the spectral maximum .
( b ) absolute two - photon 
 ( 2p ) responses at the optimal wavelength . for maga2p values 
 are given for sensitized (  = 740 nm ) and direct (  = 
 880 nm ) azobenzene excitation . ( c ) ratio between the two- and one - photon 
 responses ( 2p/1p ) . to compare between different liglur - tethers , 

two - photon responses were corrected for the distinct power densities 
 and excitation times used and averaged over all cells measured . in 
 all spectra here ,
remarkably , multiphoton currents 
 mediated by mag2p and 
 maga2p completely saturate after a few laser scans of the 
 recorded cell ( blue and red traces in figure 5b ) .
in addition , their rapid relaxation allows liglur to close immediately 
 after the end of each stimulus , with time constants similar to those 
 obtained with one - photon illumination ( table s2 in the supporting information ) , which enable fast , repeated 
 activation of the receptor without requiring a second irradiation 
 source for deactivation .
thus , the novel compounds mag2p and maga2p enable single - wavelength , multiphoton gating 
 of liglur .
however , mag achieves higher two - photon current amplitudes 
 than mag2p and maga2p in the long term ( figure 6b ) , because the thermal stability of its cis isomer allows building up a larger population of open - state 
 channels upon repeated cell raster scans ( figure s12 in the supporting information ) . to compare the efficacy 
 of liglur activation between ptls , we calculated the ratio between 
 two - photon and one - photon maximal responses ( figure 6c ) .
noticeably , mag2p and maga2p ( both 
 via direct and sensitized azobenzene excitation ) display a higher 
 ratio than mag , thereby demonstrating that the efficiency of multiphoton 
 isomerization was enhanced by the design of the new photoswitches . after characterizing the two - photon stimulation of liglur , we pursued 
 physiological applications that exploited the ability of this receptor 
 to rapidly activate neurons and trigger 
 calcium - regulated processes .
the stimulation of 
 individual neurons in micrometric volumes and millisecond time scales 
 has been demonstrated using two - photon neurotransmitter uncaging and optogenetics . to complement 
 this set of tools for investigating brain connectivity , we applied 
 two - photon activation of liglur in neuronal and non - neuronal cells 
 of the brain using the high photocurrents provided by mag and mag2p .
we expressed gluk2-l439c - egfp in cultured hippocampal neurons , 
 incubated them in mag2p and recorded neuronal activity 
 using whole - cell patch clamp ( figures 7 ) .
excitation 
 of the soma with 900 nm light elicits inward currents in voltage - clamp 
 experiments ( figure 7c ) . in current - clamp mode
, 
 these photocurrents triggered action potentials in two out of three 
 tested neurons ( figure 7d ) .
although several 
 properties of liglur - mag2p must be improved in order to 
 reliably photocontrol whole neurons and individual presynaptic terminals 
 ( lifetime of the cis isomer , receptor expression 
 level , and subcellular localization ) , these results indicate that 
 it is possible to activate neurons using two - photon stimulation of 
 synthetic photoswitchable proteins .
( a ) two - photon image (  = 1000 nm ) 
 of a cultured liglur - mag2p hippocampal neuron filled with 
 alexa fluor 594 .
( b ) voltage - clamp recording during one - photon stimulation 
 ( blue bar ) .
( c , d ) two - photon raster scan ( gray bars ) of the same neuron 
 during ( c ) voltage - clamp recording , which shows two - photon current 
 with a transient current spike ( two - photon mean current amplitude : 
 21  3 pa , 19  2% of one - photon current , n = 3 ) , and ( d ) current - clamp recording ( resting potential = 45 
 mv ) .
two - photon excitation conditions :  = 900 nm , 0.25 s scan , 
 and 24 mw on sample . in the same experimental conditions , no spikes were elicited 
 by 
 two - photon stimulation of liglur - mag , probably due to the slow photoresponses 
 shown in figure 5b .
however , the large , step - function 
 photocurrents provided by mag and the calcium permeability of gluk2 make liglur - mag more attractive to trigger calcium - regulated 
 processes including astrocyte activation ( see also figure s7 in the supporting information ) .
in cultured astrocytes expressing 
 liglur - mag ( figures 8) , two - photon excitation 
 at 820 nm triggered bistable currents ( figure 8c ) .
interestingly , whole - cell photocurrents can also be measured 
 during the stimulation of a subcellular region ( figure 8d ) or a spot ( figures 8e  g ) , 
 and these responses are reversible by illuminating the cell at 500 
 nm ( figures 8c , d , h ) . in order to verify whether 
 such stimuli were enough to activate an intracellular calcium response 
 in the astrocyte , we performed two - photon calcium 
 imaging together with two - photon stimulation of astrocytes expressing 
 liglur - mag .
when we stimulated an expressing astrocyte , liglur activation 
 caused a calcium increase that propagated to neighboring cells , generating 
 a calcium wave that expanded to astrocytes throughout the field of 
 view ( figure 8i  k and movie s1 in the supporting information ) .
this effect , which is 
 not observed when locally stimulating non - expressing astrocytes ( figure 
 s13 and movie s2 in the supporting information ) , demonstrates that two - photon liglur activation can be used to 
 manipulate cytosolic calcium levels in cultured astrocytes .
( a ) two - photon 
 image (  = 1000 nm ) of a cultured liglur - mag 
 astrocyte filled with alexa fluor 594 .
red and blue squares define 
 the whole cell and subcellular raster scan areas , respectively .
( b ) whole - cell voltage - clamp recording of the astrocyte 
 during one - photon stimulation ( liglur opening , purple bar ; liglur 
 closing , green bar ) .
( c  h ) two - photon currents measured on 
 the same astrocyte ( liglur opening , gray bar ; one - photon liglur closing , 
 green bar ) : ( c ) cell scan stimulation at  = 820 nm , 10 scans 
 of 0.7 s and 37 mw on sample ( mean current amplitude : 60  20 
 pa , 30  10% of one - photon current , n = 2 ) ; 
 ( d ) subcellular scan stimulation at  = 820 nm , 10 scans of 
 0.25 s and of 68 mw on sample ( mean current amplitude : 17  9 
 pa , n = 2 ) ; ( e  g ) single - point stimulation 
 ( gray bar ) at  = 820 nm , 50 ms and 68 mw on sample ( points 
 1 and 2 are on the cytoplasm , and point 3 is out of the cell as control ) ; 
 ( h ) liglur closing at the end of the stimulation protocol ( green bar ) . 
 ( i  k ) two - photon calcium imaging of cultured astrocytes loaded 
 with fura-2 ( in purple ,  = 800 nm ) overlapped with an image 
 of gfp fluorescence ( in green ,  = 900 nm ) to identify astrocytes 
 expressing liglur - mag .
( j ) images at 1.55 , 4.65 , and 7.75 s after targeting the astrocyte 
 to which the arrow points in ( i ) with two - photon stimulation ( 20 targets , 
 16-pixel diameter , 10 ms per point at  = 800 nm , 60 mw on sample ) . 

we 
 have demonstrated the two - photon activation of azobenzene - based 
 photoswitches in living cells expressing the light - gated receptor 
 liglur . although a symmetrically substituted 
 azobenzene
was reported to photoisomerize under continuous - wave nir 
 excitation , in general these chromophores 
 present low two - photon absorption cross sections
. however , synthetic ptls like mag offer 
 great flexibility to adjust their photochemical properties without 
 altering protein function .
we have rationally designed mag derivatives 
 with visible absorption , fast thermal relaxation , and high two - photon 
 isomerization efficacy based on push  pull substitutions and sensitization of the azobenzene 
 photoisomerization .
these modifications and the reported multiphoton 
 excitation conditions should be directly applicable to all azobenzene - based 
 bioactive ligands , including intracellular 
 photoswitches known to penetrate into cells directly or through specific ion channels , and hyperpolarizing step - function photoswitchable channels like 
 spark or ligaba .
our findings thus enable the use of synthetic photoswitches to manipulate 
 extra- and intracellular biochemical processes with the spatiotemporal 
 precision provided by two - photon stimulation .
a detailed description of the synthesis of 
 target photoswitchable tethered ligands is given in the supporting information .
cis isomerization of mag2p and maga2p in solution 
 was investigated by ( i ) h nmr for the elucidation of the 
 photostationary - state mixtures ; ( ii ) steady - state uv  vis absorption 
 spectroscopy for trans 
trans thermal back - isomerization 
 processes ; and ( iii ) transient absorption spectroscopy for fast cis 
tsa201 cell line , cultured 
 hippocampal neurons , and astrocytes plated on glass coverslips were 
 transfected with gluk2-l439c - egfp .
prior to each experiment , they 
 were incubated with one of the ptls to allow the chemical conjugation 
 with the receptor channel and light sensitization .
a second incubation 
 with concavalin a was done in order to inhibit desensitization of 
 the glutamate receptor . for two - photon 
 stimulation , voltage - clamp 
 and current - clamp recordings under whole - cell configuration
were done 
 with an axon multiclamp 700b amplifier ( molecular devices ) , and data 
 were acquired at 10 khz .
borosilicate glass pipettes were pulled with 
 a typical resistance of 46 m for hek293 tsa201 cells 
 and neurons or 78 m for astrocytes .
bath solution was 
 composed of 140 mm nacl , 1 mm mgcl , 2.5 mm kcl , 10 mm hepes , 2.5 mm 
 cacl2 , and 1020 mm glucose to fix osmolarity to 
 310 mosmkg , ph 7.42 adjusted with naoh . 

for hek293 tsa201 cell line , pipet solution contained 120 mm cesium 
 methanosulfonate , 10 mm tea - cl , 5 mm mgcl2 , 3 mm na2atp , 1 mm na2gtp , 20 mm hepes , and 0.5 mm egta , 
 290 mosmkg , ph 7.2 adjusted with csoh . for 
 neurons and astrocytes it consisted of 130 mm potassium gluconate , 
 5 mm nacl , 10 mm hepes , 0.1 mm egta , 2 mm mgso4 , 4 mm mg - atp , 
 0.4 mm naxgtp , 7 mm na2-phosphocreatine , 2 mm pyruvic acid , 
 and 0.1 mm alexa fluor 594 ( molecular probes ) , ph 7.3 adjusted with 
 koh .
all two - photon experiments were 
 performed in the yuste laboratory with a custom - made two - photon laser 
 scanning microscope based on a modified olympus bx50wi microscope 
 with a ti : sapphire laser as light source ( coherent chameleon ultra 
 ii , 140 fs pulses , 80-mhz repetition rate ) .
laser power was modulated 
 by a pockels cell ( conoptics ) and adjusted for each wavelength to 
 be close to 40 mw on sample for mag2p and maga2p and 50 mw on sample for mag , if not specified otherwise . in experiments 
 with mag
, we used a 20x/0.5-na objective ( olympus ) , 
 and with the red - shifted compounds , we used a 20x/0.95-na objective ( olympus ) in figures 5 and 6 and a 40x/0.8-na objective ( olympus ) 
 in figures 7 and 8 . 
 for two - photon stimulation
we defined a roi and applied a unidirectional 
 raster scan using fluoview software , or we performed point stimulations 
 with custom - written labview software .
first , 50 l of 
 dmso was added to a 50 g aliquot of fura-2-am ( life technologies ) . 

next , 0.2 l of this solution and 0.2 l of pluronic acid 
 ( 20% in dmso ) in 2 ml supplemental media were added to the culture 
 dish and incubated at 37 c for 30 min , before washing and liglur 
 conjugation with mag and concanavalin a treatment .
we raster - scanned 
 fura-2 ( 100 frames , 1.55 s / frame ) at 800 nm and 40 mw on sample with 
 a 20x/0.5-na objective for recording the activity 
 of astrocytes and stimulated single nonexpressing or gfp - positive 
 astrocytes using custom written software , with a protocol of 20 stimulation targets on the cell with a16-pixel 
 diameter , corresponding to approximately 11 m diameter .
amplitudes of liglur currents were analyzed 
 using igorpro ( wavemetrics ) , and closing time constants of liglur 
 were determined with a custom - made software using labview . in the 
 two - photon action spectrum of each compound ,
every set of data from 
 one cell was normalized to the action spectrum integral from a chosen 
 representative before cell average .
calcium imaging of astrocytes was analyzed 
 using custom written software ( caltracer ) and imagej .
 synthetic photochromic compounds 
 can be designed to control a variety 
 of proteins and their biochemical functions in living cells , but the 
 high spatiotemporal precision and tissue penetration of two - photon 
 stimulation have never been investigated in these molecules . here 
 
 we demonstrate two - photon excitation of azobenzene - based protein switches 
 and versatile strategies to enhance their photochemical responses . 
 
 this enables new applications to control the activation of neurons 
 and astrocytes with cellular and subcellular resolution . 