Adaptive optics improves multiphoton super-resolution imaging.

Two photon excitation instant SIM /adaptive optics microscope Our microscope is based upon a previously reported design 15 that uses a 2D galvanometric scanner for excitation and a matched 2D emission side galvanometric scanner for rescanning, thereby directly providing super-resolution images that are captured on a widefield detector (an EM-CCD, Andor, DU-855K-CS0-#VP, back-thinned, 1004 pixels x 1002 pixels, 8 μm x 8 μm). The optical components, layout, and microscope base in this study are identical to the previous setup, with two important additions: a Shack Hartmann sensor (SHS, Imagine Optic, HA-7364, HASO3-First, 40 X 32 microlenses) for direct wavefront sensing and a deformable mirror (DM, Imagine Optic, 52 actuators, +/− 50 μm stroke, 15 mm pupil) for aberration correction ( Supplementary Fig. 1 ). To measure the wavefront, we diverted the descanned emission fluorescence to the SHS using a flip mirror (Thorlabs, GVS211), reimaging the emission scanner (and thus the back focal plane of our 1.2 NA water objective) to the SHS with a pair of achromatic lenses (L8 and L9, f = 250 mm and f = 100 mm, Thorlabs, AC508-250-A-ML and AC254-100-A-ML) placed in a 4f imaging configuration. Emission filters (F2, Semrock, Supplementary Table 2 ) placed after L9 rejected excitation and isolated fluorescence before collection on the SHS. To control aberrations, we imaged the back focal plane of our objective lens onto a DM with lens pair L5 and L6 (Thorlabs, both AC508-200-A-ML, both f = 200 mm), placed in a 4f configuration. Lens pair L3 and L4 (Thorlabs, both AC508-250-A-ML, both f = 250 mm, also in a 4f imaging configuration) ensured that the DM was optically conjugate to the excitation scanner. We matched the diameter of the excitation beam to the diameter of the emission beam at the DM with a 5-fold beam expander (L1 and L2, Thorlabs, AC254-040-B-ML and AC254-200-B-ML, f = 40 mm and f = 200 mm, with pinhole, Edmund Optics, 36-392, 100 μm diameter placed at the co-focal point between the lenses to spatially filter the beam) and iris (placed after L2 for fine adjustment of beam diameter), thereby allowing aberration correction in both excitation and emission paths with the same DM setting. Once the appropriate correction was applied to the DM, emission fluorescence was diverted away from the wavefront sensor by the same flip mirror mentioned above, and imaged onto the EM-CCD via lens L7 (Thorlabs, AC508-250-A-ML, f = 250 mm), filtering out excitation light with filter FI (Semrock, Supplementary Table 2 ). In some experiments an additional filter was used (Semrock, Supplementary Table 2 ) in order to further isolate fluorescence in a particular spectral band. Data acquisition, wavefront correction, and deconvolution For all imaging, the acquisition time was set to 0.5 s / frame. In most experiments, 5-10 frames were averaged to improve SNR. The imaging field of view spanned 1004 * 48 nm x 1002 * 48 nm = 48.2 μm x 48.1 μm. The axial step size used for acquiring stacks and further acquisition parameters (excitation wavelength, fluorescence filter, total axial extent of stack) are provided in Supplementary Table 2 . The electron multiplication gain of the EM-CCD was set to 200 for all measurements. For wavefront correction, the image exposure on the SHS was set to 0.5 s to 1.5 s depending on the sample brightness. The excitation laser power measured post objective varied: ~5 mW for fluorescent beads, ~30 mW for cells and live samples (nematodes, zebrafish embryos and algae), and ~90 mW for fixed, thick tissue ( Drosophila brain lobe and mouse leg muscle). For datasets of fluorescent beads and cells in gels, we used Richardson-Lucy deconvolution, as previously described 14 . We assumed Gaussian PSFs with lateral and axial FWHMs derived from 100 nm fluorescent beads ( Supplementary Table 1 ), and ran each deconvolution for 20 iterations. For time-lapse volumetric AO 2P-ISIM embryo data, we used Richardson-Lucy deconvolution, but implemented the algorithm in the Fourier domain with a graphics processing unit (GPU) card 27 to speed processing. We modeled the system PSF as the product of the two-photon excitation PSF and wide field emission PSF, i.e., PSF SYSTEM = PSF EXC X PSF EXC X PSF EMISSION , where PSF EXC and PSF EMISSION were simulated with PSF Generator (ImageJ plugin, http://bigwww.epfl.ch/algorithms/psfgenerator/ ) using the ‘Born and Wolf’ model with appropriate numerical aperture (1.2 NA), refractive index (1.33) and wavelength (930 nm for PSF EXC , and 550 nm for PSF EMISSION ). The resulting system PSF has FWHM of 220 nm, corresponding well to the measured case ( Supplementary Table 1 ). The number of iterations was set to 60. For other biological samples, datasets were deconvolved with Huygens deconvolution software (Scientific Volume Imaging), which provided better de-noising capability, perhaps due to regularization. We used the following settings: (1) modeling a theoretical PSF based on the microscopy parameters: ‘confocal’ PSF type, 48 nm pixel dimensions, 1.2 NA objective, water immersion, and 1.33 index of refraction; (2) using classic maximum likelihood estimation (CMLE) as the restoration method; (3) setting the quality stop criterion to 0.1 and the targeted signal-to-noise ratio to 20; and (4) setting 20-30 iterations for convergence. Setting up, characterizing, and using the AO Loop The DM, SHS, and relay optics between them form the heart of the AO system ( Supplementary Fig. 1 ). Both DM and SHS are controlled through the software CASAO, provided by the vendor. For measuring and correcting aberrations in biological samples, we also wrote a master control program in LabVIEW (National Instruments) for communications between the piezo stage and CASAO software development kit (SDK), enabling plane-by-plane AO correction during image volume collection. AO correction includes three steps. First, we establish an interaction matrix relating the amplitude of the Zernike wavefront modes and the voltages applied to the DM. Second, the wavefront is measured with the SHS and the necessary corrective voltages to the DM are computed. Third, we apply the corrective voltages to the DM, canceling or reducing aberrations. We provide more detail on these steps below: Determining the interaction matrix. A 1 μm diameter fluorescent bead (Molecular Probes, F13081 ) deposited on a glass coverslip was excited by a 488 nm laser (Spectra-Physics, Excelsior-488-200c-CDRH) and the transmitted fluorescence signal isolated with a 500 nm longpass filter (Semrock, FF01-500/LP-25) was detected by the SHS. An iris was placed in an imaging plane to selectively image only the wavefront of a single bead. Second, +/− 0.2 V was applied to each actuator successively to push or pull on the surface of the deformable mirror, and the consequent change in wavefront was recorded by the SHS. We obtained a total of 104 wavefront shapes corresponding to the push/pull of all 52 actuators of the DM. Third, each measured wavefront shape was decomposed into the amplitudes of 36 Zernike modes (up to 7 th order). Using the function ‘IM-CM’ provided in CASAO, the above decomposition and voltages were used to compute the interaction matrix. Each wavefront was collected in ~500 ms. The entire process took ~1 minute, but only needed to be performed once during instrument setup. Measuring the system wavefront and correcting system aberrations. A single layer of 100 nm fluorescent beads was deposited on a coverslip, and this sample immersed in water and used to measure system aberrations (i.e. those aberrations inherent to the optical setup). After exciting the beads with 2P illumination, the descanned fluorescence signal from the beads (i.e., the nonlinear guide-star) was detected by the SHS (exposure time set to 0.5 s) and decomposed into Zernike modes. Using the interaction matrix, the DM was then set to cancel the system aberrations. This DM setting was recorded and subsequently used as a basis for future AO correction on biological samples. Measuring the distorted wavefront and correcting aberrations in biological specimens. We used two types of AO correction: plane-by-plane (every plane in the sample is corrected) or fiducial-based (a fiducial at a particular location in the sample is used for correction and the resulting correction applied throughout the image volume). Differences between these modes of correction are the area over which the measurement and correction are derived (the entire imaging field vs. a selected subregion covering the fiducial) and the frequency of measurement/correction (a unique measurement/correction for each plane vs. a single measurement in one plane and the same correction applied throughout the entire volume). The general protocol is similar in either case: i) set the deformable mirror to correct system aberrations and set the flip mirror to divert the fluorescence emission to the SHS ( Supplementary Fig.1 ). ii) Scan the focal point across a defined area, descan the fluorescence with the emission-side scanner, and record the descanned fluorescence signal with the SHS. The typical exposure time on the SHS for biological specimens varied between 0.5 s – 1.5 s depending on the sample brightness. iii) Decompose the wavefront into Zernike modes, and use the interaction matrix to compute the corrective voltages that, when applied to the DM, will set the 4 th to 36 th Zernike modes to zero. Steps ii to iii were often repeated (and sometimes 3 iterations were used) to achieve better correction. iv) Add these voltages to the base state of the mirror and shift the flip mirror so that the fluorescence will now be collected by the EM-CCD. v) Finally, form the 2P-ISIM image by scanning the excitation focus across the imaging area again, now rescanning the emission for super-resolution.

Preparation of bead samples

We employed three bead samples: a monolayer of fluorescent beads on a coverslip surface for assessing system resolution, a monolayer of fluorescent beads on a curved tube surface for verifying our ability to correct significant coma/astigmatism, and fluorescent beads embedded in a 3D polyacrylamide gel that allowed us to produce and correct substantial spherical aberration. To prepare a bead monolayer on a conventional coverslip, 24 × 50 mm #1.5 coverslips (VWR, #48393241) were coated with 100 mg/ml poly-L lysine (Sigma, P8920) and ~50 μL of 100 nm diameter yellow–green fluorescent beads (Invitrogen, F8803, 1:1000 dilution in water) were deposited on the coated coverslips. After several minutes, the coverslips were gently washed with water to remove excess, unbound beads. During imaging, the fluorescent beads were immersed in water by dripping ~500 μL deionized water on the beads. To prepare bead monolayers on a curved surface, a monolayer with 100 nm diameter beads (Invitrogen, F8803) or 200 nm diameter beads (Invitrogen, F8811) was deposited on a 24 × 50 mm #1.5 coverslip as described above. Next, a 0.9 mm diameter glass tube (Microcells, CAT. # 8100-100) was sandwiched between a glass slide and the coverslip, and pressure applied until the coverslip buckled, thereby forming a curved surface with radius ~270 mm. Beads in polyacrylamide were prepared by suspending 100 μL of 100 nm diameter yellow–green fluorescent beads (Invitrogen, F8803, 1:100 dilution) and 25 μL of 1 μm diameter yellow-green fluorescent beads (Molecular Probes, F13081 ,1:1000 dilution) together into a solution containing 125 μL Bis : acrylamide (30 : 0.8), 3 μL 10% ammonium persulfate (APS) and 0.5 μL TEMED. The solution was mixed, vortexed vigorously, and then deposited on a #1.5 glass-bottomed dish (Matek, P35G-1.5-14-C). The gel was allowed to solidify for an hour and imaged immediately thereafter. The assumed refractive index was 1.452. Preparation of Algae Zygnema (Carolina Biological Supply, 152695) were sandwiched between a coverslip and a glass slide. To avoid overcrowding of algae, two pieces of plastic plate with thickness ~500 μm were inserted between the coverslip and glass slide, along the two borders of coverslip. Algae were kept moist during imaging by immersing them in their original buffer (alga-Gro freshwater).

Preparation of larval and embryonic nematodes

C. elegans were grown at 20°C on NGM media plates containing E. coli OP50, picked at the larval stage, immobilized in 50 mM levamisole and mounted on an agarose pad sandwiched between a slide and coverslip (VWR, #48393241, #1.5 thickness). Neuronal imaging was performed on strain DCR4315 [olaEx2537 [pncs-1::GFP; punc-122::mCH]] and gut granule imaging on strain BV24 [Itls44 [ple-1p-mCherry::PH(PLC1delta1) + unc-119(+)]; zuls178 [(his-72 1 kb::HIS-72::GFP); unc-119(+)] V] . The latter strain was incubated overnight on NGM plates containing a 1:1 solution of CellTracker Orange and E. coli OP50. Nematode embryos (strain BV24) were prepared for imaging as previously described 15 , except that #1.5 coverslips were used instead of well chambers.

Preparation of endothelial cells in gels

Primary mouse endothelial cells were cultured and stained as previously described 28 , with minor modifications. C57bl/6 mouse aorta sections were embedded in 2.5 mg/mL bovine collagen I (Corning, Bedford, MA) in DMEM (ThermoFisher), which was supplemented with 1 μm fluorescent microspheres ( F13081 , Molecular Probes, Life Technologies, OR), in MatTek dishes (MatTek, Ashland, MA). Gels were covered with EGM-2 endothelial growth medium (Lonza, Houston TX) and maintained at 37°C for 4 days. Gels were fixed in 4% paraformaldehyde in cytoskeleton buffer (CB; 20 mM PIPES pH 6.9, 145 mM KCl, 3 mM MgCl 2 , 2 mM EGTA) at 25°C for 40 minutes. Cells in gels were permeabilized in the presence of 100 nM Alexa-488-phalloidin (ThermoFisher) in CB supplemented with 0.5% Triton X-100 for 2 hours at 25°C. Gels were rinsed 3 times in CB with 0.1% Tween and one time in CB buffer alone. For microtubule staining, 0.5% glutaraldehyde in CB buffer was used as fixative. Autofluorescence was quenched with 1 mg/mL sodium borohydride in CB, and following CB+Tween washes, gels were blocked with blocking buffer (CB supplemented with 3% bovine serum albumin and 0.1% Tween) for 1 hour at 25°C or 16 hours at 4°C. Gels were incubated with anti-tubulin antibody (1:300, DM1A, AbCam) in blocking buffer for 2-4 hours, followed by three washes with CB buffer, and then Alexa 488-anti-mouse antibody (1:300, ThermoFisher) in blocking buffer for 2 hours at 4°C, followed by three washes in CB. Animals were maintained and euthanized according to guidelines approved by National Heart, Lung and Blood Institute Institutional Animal Care and Use Committee Preparation of Drosophila brain lobe Drosophila melanogaster (strain Canton-S) brain lobes were dissected from third instar larvae and fixed in 4% paraformaldehyde/PBS for 20 mins at room temperature. After several washes with 0.1% Triton X-100/PBS the brain lobes were incubated in Alexa Fluor 488 Phalloidin (Cat# A12379, ThermoFisher Scientific) for 2 hours at room temperature. The samples were washed in 0.1% Triton X-100/PBS and mounted in ProLong (Cat# P36934 , ProLong Gold Antifade Mountant, ThermoFisher Scientific).

Preparation and staining of live zebrafish embryos Tg

(XIEef1a1:dclk2–GFP io008 ) Danio rerio embryos were generated by natural spawning and raised at 28.5°C according to standard procedures 29 in egg water (0.06g/L Instant Ocean, Doctors Foster and Smith Cat #CD-116528) supplemented with 0.003% N-phenylthiourea to prevent pigment formation (Sigma, Cat. # P7629). Dechorionated 32 or 40hpf old embryos were soaked in a solution of 1.1 μg/mL CellTracker Orange CMRA (ThermoFisher Scientific, Cat. # C34551 ) in egg water for one hour at room temperature in the dark. Following staining, embryos were washed in egg water twice for 5 minutes and then anesthetized in 600 μM MS-222 (Sigma, Cat. # E10521 ) diluted in egg water. Embryos were then mounted for imaging in 1% low melting point agarose (NuSieve GTG, Cambrex BioScience, Cat. # 50080). Zebrafish experiments were approved by the NIH intramural animal care and use committee (protocol number 15-039).

Preparation of mouse leg muscle Four-week-old

A/J mice (Jackson Laboratory) were sacrificed according to the procedures in NIH protocol ASP 14-018. The hind limbs were then separated at the femur/hip joint with the membrane and skin removed. Limbs were placed in a glass-bottomed #1.5 dish (MatTek Corportation) submerged in DMEM media (Gibco). Legs were fixed for 30 minutes at 37°C with 4% paraformaldehyde (Electron Microscopy Sciences). Legs were washed twice with phosphate buffered saline (PBS), washed with 10 mM CellTracker Green CMFDA (ThermoFisher Scientific, Cat. # C7025) in DMEM and 12 units of rhodamine phalloidin (ThermoFisher Scientific, Cat. # R415) and incubated at room temperature for two hours prior to imaging. Estimating spatial resolution 100 nm diameter yellow–green fluorescent beads were used to characterize system resolution at the coverslip surface. The excitation wavelength was set to 900 nm, the axial interval between successive imaging planes set to 100 nm, and the pixel size in each imaging plane 48 nm for 2P-ISIM operation and 96 nm for conventional (diffraction-limited) imaging. The full width at half maximum (FWHM) values of beads were computed using a custom-written MATLAB script that fits a Gaussian function to vertical and horizontal lines centered on the brightest point in each single bead image. To suppress background, we measured the average background on a coverslip area devoid of beads and subtracted this average from each bead measurement. In biological samples, we used the modulation transfer function (MTF) to estimate spatial resolution. In any single imaging plane or axial reslice, the MTF was noisy or poorly defined due to sparsity of the sample, so we pooled multiple planes for MTF measurement: first, we created an isotropic 3D image stack by axially interpolating the measured image data using the Scale function (Interpolation: Bicubic) in ImageJ. Second, we computed the 2D Fourier transform at different axial depths over an interval of 2 μm or at different lateral distances spanning an interval of ~4 μm. The FFT function in ImageJ was used for calculation. Finally, the Maximum Intensity Projection function in ImageJ was applied to produce the lateral and axial MTFs used in analysis and in figures. Photobleaching/Phototoxicity Assessment At the power levels we used, we observed minor or no photobleaching in any of our live samples (for the nematode dataset presented in Supplementary Video 2 , fluorescence actually increased during the hour long time course). In live zebrafish samples ( Fig. 3 ), fish showed no sign of morphological damage after imaging, and we confirmed that the embryonic heart continued to beat 24 hours after imaging. In live nematode embryos ( Supplementary Video 2 ), we counted 108 nuclei at the onset of imaging and 194 nuclei after 60 minutes. The change in cell number over time is consistent with the known, highly stereotyped timing of divisions in C. elegans and further supports our assertion that photodamage was minimal during imaging.

Supplementary Material 1 SI Video 1 SI Video 2 SI Video 3 SI Video 4 Supplementary Software