1 ) is comprised of subsystems for two - photon near - infrared ( nir ) excitation , visible fluorescence detection and wavefront measurement , and continuous wave ( cw ) visible excitation . in the two - photon subsystem ,
pulsed light from a ti : sapphire laser ( coherent , chameleon ultra ii ) , intensity controlled by a pockels cell ( conoptics , 350 - 80-la-02 ) , is expanded to a 1/e diameter of 8 mm before being reflecting at 8 from the normal off of a nir - responsive spatial light modulator ( slm nir , boulder nonlinear systems , hsp256 - 1064 ) .
the slm is used to apply the corrective pattern needed to retain a diffraction - limited two - photon excitation ( tpe ) focus in the specimen . a pair of nir achromatic relay lenses ( focal lengths f1 = 150 mm and f2 = 125 mm ) operating in a 2f1 + 2f2 configuration are then used to image the slm onto the 5 mm mirror of a galvanometer ( y galvo , cambridge technology , 6215h ) .
another pair of f1 = f2 = 85 mm relay lenses then image the slm onto a second 5 mm galvo mirror ( x galvo , cambridge technology , 6215h ) .
a final pair of f1 = 89 mm and f2 = 350 mm relay lenses creates a magnified image of the slm at the rear pupil plane of the imaging objective ( nikon , cfi apo lwd 25xw , 1.1 na and 2 mm wd ) .
mutual conjugation of the slm , both galvos , and the objective rear pupil insures that the corrective phase pattern from the slm is stationary at the objective rear pupil , even as the galvos scan the focused nir light laterally across the specimen .
the visible excitation subsystem begins when four cw lasers (  = 440 nm , 50 mw , crystalaser ;  = 488 nm , 200 mw , coherent sapphire 488 lp ;  = 514 nm , 300 mw , mpb communications , model 2ru - vfl - p-300 - 514-r ; and  = 561 nm , 200 mw , coherent sapphire 561 lp ) are expanded to a common a 1/e diameter of 2 mm and combined into a single co - linear beam using dichroic beamsplitters ( semrock , lasermux family ) .
an acousto - optic tunable filter ( aotf , aa opto - electronic , aotfnc-400.650-tn ) selects one or more wavelengths and controls the power of each .
the linearly polarized output of the aotf is expanded to a 1/e diameter of 10 mm , inserted into the microscope beam path using a dichroic beamsplitter ( d2 in supplementary fig .
1 , semrock di01-r442/510 - 25x36 or di01-r488/561 - 25x36 ) , and reflected from a spatial light modulator responsive to visible light ( slm vis , boulder nonlinear systems , hsp256 - 0532 ) .
the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode .
this pinhole is manually removed when imaging in the two - photon mode . after passing through a polarizing beam splitter ( pbs , thorlabs , pbs251 ) , a pair of f1 = 150 mm and f2 = 125 mm relay lenses image slm vis onto y galvo .
consequently , slm vis , both galvos , and the objective rear pupil are also mutually conjugate , and corrective phase pattern from slm vis is stationary at the objective rear pupil , even as the galvos scan the focused visible light laterally across the specimen . in the fluorescence detection subsystem , the focused fluorescence emission generated by either tpe or visible light
is collected by the objective and initially follows the reverse path of the nir and visible excitation beams .
after y galvo , however , another dichroic beamsplitter ( d1 , semrock ff705-di01 - 25x36 ) diverts the emission through the relay lens pair that conjugates y galvo to slm vis .
half of this unpolarized light passes through pbs , reflects off slm vis , and is focused by an f = 300 mm lens before a photomultiplier tube ( pmt , hamamatsu , h7422 - 40 or r10467u-40 ) . the signal from this detector forms the image in either the confocal or the tpe imaging mode ,
the other half of the fluorescence is reflected by pbs and is sent to the shack - hartmann ( sh ) wavefront sensor , positioned such that the lenslet array ( 10  10 lenses , 0.5 mm pitch , f = 46.7 mm , edmund optics , 64 - 483 ) is conjugate to the objective rear pupil and the two galvos . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen .
displacements of the foci on the sh camera ( andor ixon3 897 emccd ) then solely represent the local wavefront gradients , as desired .
the choice of pbs to split the fluorescence signal equally between the sh sensor and the imaging pmt is an obvious one , given that slm vis requires linearly polarized light to modulate the phase properly .
however , while this configuration is perhaps the simplest for an slm - based system , the 50% signal loss at the pmt is a substantial price to pay . on the other hand ,
the question of the optimum split ratio is a complex one , as a number of factors influence how much signal the sh sensor requires to accurately measure the displacement of each lenslet - defined focal spot . increasing the number of lenslets increases the complexity of the aberration that can be measured , but divides the sh signal among more elements .
decreasing the size of each ao corrective subvolume provides more local measurement of the aberration , but decreases the total integrated signal collected for each such measurement .
finally , increased imaging depth generally leads to greater aberration and thus more dramatic improvement after ao correction , but also results in more scattered background and less ballistic ( focused ) light at the sh sensor , requiring more signal to accurately measure the focal spot displacements . in short , while the 50/50 ratio of the pbs configuration represents a simple compromise that works well for the specimens studied here , other configurations , including a system with a variable split ratio , can be envisioned for other biological systems . before measuring and correcting sample - induced aberrations ,
we measure these system aberrations by the phase retrieval method , since it provides an independent means to determine the correction necessary to recover an ideal diffraction - limited focus for an ideal , non - aberrating point object . to correct the aberrations in the visible light path
, the pinhole near the pmt is removed , and a 3d image of an isolated , 200 nm diameter fluorescent bead on a glass slide is obtained by scanning the visible focus in a series of xy planes , and stepping the sample in z to different planes with a piezoelectric flexure stage ( physik instrumente , p-622.2cd ) . the sampling interval must be smaller than the nyquist limit : 
 nx , y=/(4na),nz=/[2n(1 - 1-(na / n)2 ) ] in each direction , and the field of view must be large enough that aberrated images of the bead ( see below ) are not cropped at the edges .
the 3d image is then inspected , particularly for axial asymmetry indicative of spherical aberration , and the correction collar on the objective is adjusted .
this process of 3d imaging and collar correction is repeated until the spherical aberration is minimized .
next , the bead is moved to the z plane of best focus , and a series of seven 2d images are taken while applying seven different zernike polynomial phase patterns of 2 p - p amplitude on slm vis : flat phase ; positive defocus ; negative defocus ; positive x astigmatism ; negative x astigmatism ; positive y astigmatism ; and negative y astigmatism . from these images , the wavefront correction for system aberration in the visible excitation path is retrieved using the gerchberg - saxton algorithm . thereafter
, this pattern is applied to slm vis , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to correct for aberrations in the nir light path ,
a ccd camera ( avt , guppy f-146 ) is placed at the intermediate image plane located at the focus of the first relay lens after x galvo .
seven 2d images of this focus are taken while applying the seven zernike polynomial phase patterns listed above to slm nir , and the wavefront correction for system aberration in this portion of the nir excitation path is retrieved using the gerchberg - saxton algorithm .
thereafter , this pattern is applied to slm nir , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to calibrate the sh sensor , the visible and nir wavefront corrections for system aberration
are applied to slm vis and slm nir , respectively . a 2d image of a field of fluorescent beads
is then taken in the two - photon imaging mode while integrating the signal at the sh camera .
the resulting sh image consists of an array of foci , matching the elements of the lenslet array .
the centroids of these foci are determined to sub - pixel precision , and serve as the calibration reference .
thereafter , the displacements of these centroids from their reference positions indicate the local gradient of the sample - induced wavefront error , from which the wavefront itself can be calculated using a generalized matrix inversion method .
note that all of the wavefronts presented in this work represent the corrections for sample - induced aberrations , i.e. , after compensation for system aberrations . because aberrations can vary rapidly as a function of position within biological samples
, we image large volumes by dividing them into smaller sub - volumes , and determine an averaged ao correction unique to each sub - volume .
stacked , closed - loop ultrasonic piezomotor stages ( physik instrumente , m-663.465 ) are used initially for x - y positioning of the sample to the focal point of the objective , as well as for lateral translation between sub - volumes . a closed loop ball - screw driven stage ( physik instrumente , m-110.2dg ) provides similar functions in z. within each sub - volume , x galvo and y galvo scan the focus laterally , while a piezo flexure stage ( physik instrumente , p-622.2cd ) steps between scan planes to build a 3d image of the sub - volume . at each voxel ,
the fluorescence photons reaching pmt generate current spikes which are first amplified ( femto messtechnik gmbh , dlpca-200 ) and then integrated over the pixel dwell time in a custom , fast - resetting analog integrator .
the integrator output is digitized by an fpga - based reconfigurable i / o board ( national instruments , pcie-7852r ) just prior to integrator reset from the same board at the end of the dwell period . in the two - photon imaging mode , ao correction occurs simultaneously with image acquisition . the sh camera exposure time is chosen to be just long enough to yield an snr sufficient to accurately measure the gradient of the wavefront .
calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir .
currently , the fastest closed - loop update time for new ao corrections is 14 ms , limited in bright samples by the read - out speed of the emccd - based sh camera . in the future
in the confocal mode , ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera .
after the wavefront correction is calculated and added to the system corrections at slm vis and slm nir , the nir light is blocked , and the visible light is passed in order to image the entire sub - volume . in either imaging mode , the aberration - corrected 3d point spread function ( psf ) of the microscope
is first determined by imaging an isolated 200 nm diameter fluorescent bead on a glass slide with system corrections applied to both slms . for regions of the sample where ao correction recovers near diffraction - limited resolution
, these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab .
this provides a sharper 3d representation of the imaging volume that depicts the sample and the relative amplitudes of its spatial frequencies more accurately .
volume renderings of the data are created in amira ( fei visualization sciences group ) . for data sets with intensities covering a large dynamic range , a gamma function
the imaging conditions and visualization parameters for all figures and videos are listed ( supplementary table 1 , 2 ) .
the emccd which serves as the sh camera is set to internal triggering mode and serves as the master timing source -- timing pulses from the fire out ttl output of the emccd are read by the fpga card in a control computer ( pc ) to synchronize all operations ( supplementary figs .
analog outputs from the fpga card provide user - defined waveforms that control x galvo , y galvo , and the z sample piezo during imaging .
these are conditioned by individual scaling amplifiers ( srs , sim983 , and sim900 mainframe ) to match their 16-bit resolution to the control range of each device .
additional analog outputs to the pockels cell and the aotf control the intensity and blanking of the nir and two selected wavelengths of visible light .
a digital output synchronizes the integrator to the scanning , while an analog input digitizes the integrated signal at each voxel .
all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc .
the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro .
wild - type and transgenic lines were maintained according to institutional animal care and use protocols .
the following lines were used : roy ; gmc604et ; gmc930tg , which expresses yfp in the membranes of a sparse set of neurons ( fig . 1 , supplementary fig . 7 and supplementary video 1 ) ; tg(-actin : mgfp ) , which expresses gfp in the membranes of all cells ( fig .
2a and supplementary video 3 ) ; tg(4.9sox10:egfp ) , which expresses egfp cytosolically in a subset of oligodendrocytes in the brain ( fig .
tg(4.9sox10:egfp ) crossed with tg(-8.4neurog1:nrfp ) , which expresses rfp in neuronal nuclei ( fig .
3 and supplementary video 4 ) ; s1101t - gal4 x uas - memcerulean , uas - centrin2-yfp , which expresses yfp - tagged centrin2 in a broad subset of neurons , including in the retina ( supplementary fig . 5a 
e ) ; and huc - gal4 x uas - mitocfp / memyfp , which expresses cfp in the mitochondria and yfp in the plasma membrane of a broad subset of neurons ( supplementary figs .
embryos were transferred at 12h or 24 h post - fertilization to e3 solution ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) containing n - phenylthiourea ( sigma ) to inhibit pigmentation . for imaging embryos
were anesthetized using tricaine ( sigma ) in e3 solution and mounted in 0.7% low - melting agarose ( sigma - aldrich a4018 ) as described by godinho .
1 ) is comprised of subsystems for two - photon near - infrared ( nir ) excitation , visible fluorescence detection and wavefront measurement , and continuous wave ( cw ) visible excitation . in the two - photon subsystem ,
pulsed light from a ti : sapphire laser ( coherent , chameleon ultra ii ) , intensity controlled by a pockels cell ( conoptics , 350 - 80-la-02 ) , is expanded to a 1/e diameter of 8 mm before being reflecting at 8 from the normal off of a nir - responsive spatial light modulator ( slm nir , boulder nonlinear systems , hsp256 - 1064 ) .
the slm is used to apply the corrective pattern needed to retain a diffraction - limited two - photon excitation ( tpe ) focus in the specimen . a pair of nir achromatic relay lenses ( focal lengths f1 = 150 mm and f2 = 125 mm ) operating in a 2f1 + 2f2 configuration are then used to image the slm onto the 5 mm mirror of a galvanometer ( y galvo , cambridge technology , 6215h ) .
another pair of f1 = f2 = 85 mm relay lenses then image the slm onto a second 5 mm galvo mirror ( x galvo , cambridge technology , 6215h ) .
a final pair of f1 = 89 mm and f2 = 350 mm relay lenses creates a magnified image of the slm at the rear pupil plane of the imaging objective ( nikon , cfi apo lwd 25xw , 1.1 na and 2 mm wd ) .
mutual conjugation of the slm , both galvos , and the objective rear pupil insures that the corrective phase pattern from the slm is stationary at the objective rear pupil , even as the galvos scan the focused nir light laterally across the specimen .
the visible excitation subsystem begins when four cw lasers (  = 440 nm , 50 mw , crystalaser ;  = 488 nm , 200 mw , coherent sapphire 488 lp ;  = 514 nm , 300 mw , mpb communications , model 2ru - vfl - p-300 - 514-r ; and  = 561 nm , 200 mw , coherent sapphire 561 lp ) are expanded to a common a 1/e diameter of 2 mm and combined into a single co - linear beam using dichroic beamsplitters ( semrock , lasermux family ) .
an acousto - optic tunable filter ( aotf , aa opto - electronic , aotfnc-400.650-tn ) selects one or more wavelengths and controls the power of each .
the linearly polarized output of the aotf is expanded to a 1/e diameter of 10 mm , inserted into the microscope beam path using a dichroic beamsplitter ( d2 in supplementary fig .
1 , semrock di01-r442/510 - 25x36 or di01-r488/561 - 25x36 ) , and reflected from a spatial light modulator responsive to visible light ( slm vis , boulder nonlinear systems , hsp256 - 0532 ) .
the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode .
this pinhole is manually removed when imaging in the two - photon mode . after passing through a polarizing beam splitter ( pbs , thorlabs , pbs251 ) , a pair of f1 = 150 mm and f2 = 125 mm relay lenses image slm vis onto y galvo .
consequently , slm vis , both galvos , and the objective rear pupil are also mutually conjugate , and corrective phase pattern from slm vis is stationary at the objective rear pupil , even as the galvos scan the focused visible light laterally across the specimen . in the fluorescence detection subsystem , the focused fluorescence emission generated by either tpe or visible light
is collected by the objective and initially follows the reverse path of the nir and visible excitation beams .
after y galvo , however , another dichroic beamsplitter ( d1 , semrock ff705-di01 - 25x36 ) diverts the emission through the relay lens pair that conjugates y galvo to slm vis .
half of this unpolarized light passes through pbs , reflects off slm vis , and is focused by an f = 300 mm lens before a photomultiplier tube ( pmt , hamamatsu , h7422 - 40 or r10467u-40 ) . the signal from this detector forms the image in either the confocal or the tpe imaging mode ,
the other half of the fluorescence is reflected by pbs and is sent to the shack - hartmann ( sh ) wavefront sensor , positioned such that the lenslet array ( 10  10 lenses , 0.5 mm pitch , f = 46.7 mm , edmund optics , 64 - 483 ) is conjugate to the objective rear pupil and the two galvos . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen .
displacements of the foci on the sh camera ( andor ixon3 897 emccd ) then solely represent the local wavefront gradients , as desired .
the choice of pbs to split the fluorescence signal equally between the sh sensor and the imaging pmt is an obvious one , given that slm vis requires linearly polarized light to modulate the phase properly .
however , while this configuration is perhaps the simplest for an slm - based system , the 50% signal loss at the pmt is a substantial price to pay . on the other hand ,
the question of the optimum split ratio is a complex one , as a number of factors influence how much signal the sh sensor requires to accurately measure the displacement of each lenslet - defined focal spot . increasing the number of lenslets increases the complexity of the aberration that can be measured , but divides the sh signal among more elements .
decreasing the size of each ao corrective subvolume provides more local measurement of the aberration , but decreases the total integrated signal collected for each such measurement .
finally , increased imaging depth generally leads to greater aberration and thus more dramatic improvement after ao correction , but also results in more scattered background and less ballistic ( focused ) light at the sh sensor , requiring more signal to accurately measure the focal spot displacements . in short , while the 50/50 ratio of the pbs configuration represents a simple compromise that works well for the specimens studied here , other configurations , including a system with a variable split ratio , can be envisioned for other biological systems .
before measuring and correcting sample - induced aberrations , the microscope must be calibrated to compensate for its own aberrations .
we measure these system aberrations by the phase retrieval method , since it provides an independent means to determine the correction necessary to recover an ideal diffraction - limited focus for an ideal , non - aberrating point object . to correct the aberrations in the visible light path ,
the pinhole near the pmt is removed , and a 3d image of an isolated , 200 nm diameter fluorescent bead on a glass slide is obtained by scanning the visible focus in a series of xy planes , and stepping the sample in z to different planes with a piezoelectric flexure stage ( physik instrumente , p-622.2cd ) . the sampling interval must be smaller than the nyquist limit : 
 nx , y=/(4na),nz=/[2n(1 - 1-(na / n)2 ) ] in each direction , and the field of view must be large enough that aberrated images of the bead ( see below ) are not cropped at the edges .
the 3d image is then inspected , particularly for axial asymmetry indicative of spherical aberration , and the correction collar on the objective is adjusted .
this process of 3d imaging and collar correction is repeated until the spherical aberration is minimized .
next , the bead is moved to the z plane of best focus , and a series of seven 2d images are taken while applying seven different zernike polynomial phase patterns of 2 p - p amplitude on slm vis : flat phase ; positive defocus ; negative defocus ; positive x astigmatism ; negative x astigmatism ; positive y astigmatism ; and negative y astigmatism . from these images , the wavefront correction for system aberration in the visible excitation path is retrieved using the gerchberg - saxton algorithm . thereafter
, this pattern is applied to slm vis , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to correct for aberrations in the nir light path ,
a ccd camera ( avt , guppy f-146 ) is placed at the intermediate image plane located at the focus of the first relay lens after x galvo .
seven 2d images of this focus are taken while applying the seven zernike polynomial phase patterns listed above to slm nir , and the wavefront correction for system aberration in this portion of the nir excitation path is retrieved using the gerchberg - saxton algorithm .
thereafter , this pattern is applied to slm nir , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to calibrate the sh sensor , the visible and nir wavefront corrections for system aberration
are applied to slm vis and slm nir , respectively . a 2d image of a field of fluorescent beads
is then taken in the two - photon imaging mode while integrating the signal at the sh camera .
the resulting sh image consists of an array of foci , matching the elements of the lenslet array .
the centroids of these foci are determined to sub - pixel precision , and serve as the calibration reference .
thereafter , the displacements of these centroids from their reference positions indicate the local gradient of the sample - induced wavefront error , from which the wavefront itself can be calculated using a generalized matrix inversion method .
note that all of the wavefronts presented in this work represent the corrections for sample - induced aberrations , i.e. , after compensation for system aberrations .
because aberrations can vary rapidly as a function of position within biological samples , we image large volumes by dividing them into smaller sub - volumes , and determine an averaged ao correction unique to each sub - volume .
stacked , closed - loop ultrasonic piezomotor stages ( physik instrumente , m-663.465 ) are used initially for x - y positioning of the sample to the focal point of the objective , as well as for lateral translation between sub - volumes . a closed loop ball - screw driven stage ( physik instrumente , m-110.2dg ) provides similar functions in z. within each sub - volume , x galvo and y galvo scan the focus laterally , while a piezo flexure stage ( physik instrumente , p-622.2cd ) steps between scan planes to build a 3d image of the sub - volume . at each voxel ,
the fluorescence photons reaching pmt generate current spikes which are first amplified ( femto messtechnik gmbh , dlpca-200 ) and then integrated over the pixel dwell time in a custom , fast - resetting analog integrator .
the integrator output is digitized by an fpga - based reconfigurable i / o board ( national instruments , pcie-7852r ) just prior to integrator reset from the same board at the end of the dwell period . in the two - photon imaging mode , ao correction occurs simultaneously with image acquisition .
the sh camera exposure time is chosen to be just long enough to yield an snr sufficient to accurately measure the gradient of the wavefront .
calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir .
currently , the fastest closed - loop update time for new ao corrections is 14 ms , limited in bright samples by the read - out speed of the emccd - based sh camera . in the future , a scmos camera may permit faster correction . in the confocal mode ,
ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera .
after the wavefront correction is calculated and added to the system corrections at slm vis and slm nir , the nir light is blocked , and the visible light is passed in order to image the entire sub - volume . in either imaging mode , the aberration - corrected 3d point spread function ( psf ) of the microscope is first determined by imaging an isolated 200 nm diameter fluorescent bead on a glass slide with system corrections applied to both slms . for regions of the sample where ao correction recovers near diffraction - limited resolution , these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab .
this provides a sharper 3d representation of the imaging volume that depicts the sample and the relative amplitudes of its spatial frequencies more accurately .
volume renderings of the data are created in amira ( fei visualization sciences group ) . for data sets with intensities covering a large dynamic range , a gamma function
the imaging conditions and visualization parameters for all figures and videos are listed ( supplementary table 1 , 2 ) .
the emccd which serves as the sh camera is set to internal triggering mode and serves as the master timing source -- timing pulses from the fire out ttl output of the emccd are read by the fpga card in a control computer ( pc ) to synchronize all operations ( supplementary figs .
analog outputs from the fpga card provide user - defined waveforms that control x galvo , y galvo , and the z sample piezo during imaging .
these are conditioned by individual scaling amplifiers ( srs , sim983 , and sim900 mainframe ) to match their 16-bit resolution to the control range of each device .
additional analog outputs to the pockels cell and the aotf control the intensity and blanking of the nir and two selected wavelengths of visible light .
a digital output synchronizes the integrator to the scanning , while an analog input digitizes the integrated signal at each voxel .
all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc .
the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro .
wild - type and transgenic lines were maintained according to institutional animal care and use protocols .
the following lines were used : roy ; gmc604et ; gmc930tg , which expresses yfp in the membranes of a sparse set of neurons ( fig . 1 , supplementary fig . 7 and supplementary video 1 ) ; tg(-actin : mgfp ) , which expresses gfp in the membranes of all cells ( fig .
2a and supplementary video 3 ) ; tg(4.9sox10:egfp ) , which expresses egfp cytosolically in a subset of oligodendrocytes in the brain ( fig .
tg(4.9sox10:egfp ) crossed with tg(-8.4neurog1:nrfp ) , which expresses rfp in neuronal nuclei ( fig . 3 and supplementary video 4 ) ; s1101t - gal4 x uas - memcerulean , uas - centrin2-yfp , which expresses yfp - tagged centrin2 in a broad subset of neurons , including in the retina ( supplementary fig . 5a 
e ) ; and huc - gal4 x uas - mitocfp / memyfp , which expresses cfp in the mitochondria and yfp in the plasma membrane of a broad subset of neurons ( supplementary figs .
embryos were transferred at 12h or 24 h post - fertilization to e3 solution ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) containing n - phenylthiourea ( sigma ) to inhibit pigmentation . for imaging embryos
were anesthetized using tricaine ( sigma ) in e3 solution and mounted in 0.7% low - melting agarose ( sigma - aldrich a4018 ) as described by godinho .
 using a de - scanned , laser - induced guide star and direct wavefront sensing , we demonstrate adaptive correction of complex optical aberrations at high numerical aperture and a 14 ms update rate . 
 this permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens , and recover diffraction - limited imaging over large ( > 240 m)3 volumes . 
 we applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain . 
ONLINE METHODS
Scanning adaptive optical microscope using a de-scanned nonlinear guide star
System Calibration
Image Acquisition, Wavefront Sensing, and Adaptive Optical Correction
Control electronics and timing
Zebrafish care and preparation
Supplementary Material