Nature Methods: doi: /nmeth Supplementary Figure 1

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1 . Supplementary Figure 1 Schematics and characterization of our AO two-photon fluorescence microscope. (a) Essential components of our AO two-photon fluorescence microscope: Ti:Sapphire laser; optional dispersion compensation unit (DCU); micromirror device (DMD) or segmented deformable mirror (SDM); field stop (FS) at an intermediate image plane between SDM/DMD and liquid crystal spatial light modulator (SLM); X galvanometer (X galvo); Y galvanometer (Y galvo); a photomultiplier tube (PMT). (b) The DCU unit is used with DMD, and consists of an anamorphic prism pair (APP) and an equilateral dispersive prism (EDP). (c) Images and the signal profiles of a 2 µm diameter fluorescent bead in the XY focal plane without and with DCU; Scale bar: 2 µm. (d) Two pupil-segmentation patterns used in this paper, where the active area of the SLM (colored squares) is inscribed to the back pupil of the objective (dashed circles).

2 Supplementary Figure 2 Schematics describing the multiplexed aberration-measurement method. See Details on the multiplexed aberration measurement method section of Online Methods.

3 Supplementary Figure 3 AO correction of GFP-labeled C. elegans neurons in vivo. (a) Imaging through the cylindrical body of a C. elegans. (b) Lateral and axial images of neurons before and after two iterations of AO, with a display gain of 1.8 for the images without AO. (c) Corrective wavefront in units of waves for (b). (d) Orthogonal views of neurons in another C. elegans. (e) Enlarged view of the area inside the dashed box in (d). (f) Axial images along the orange line in (e) before and after one iteration of AO. (g) Signal profiles along the pink line in (f). The improvement here is largely confined to the neuron on which AO correction was done because the large surface curvature of the worm led to a highly localized aberration. (h) Corrective wavefront in units of waves in (f). Crosses mark the neurons on which AO correction was carried out using intensity modulation with a DMD. Scale bar: 8 µm.

4 Supplementary Figure 4 AO correction of EGFP-labeled zebrafish larva in vivo. (a) Imaging through the zebrafish larva. (b) Aberration leads to signal degradation at depth after the excitation light passes through myotomes (MT) and notochord (NC). (c) Lateral and axial images of myotomes 170 µm inside a zebrafish larva before and after three iterations of AO correction at the red cross shown. Images without AO have display gains of 2.3, 2.3, and 1.6, respectively, for better visibility. (d) Corrective wavefront in units of waves. (e) Signal profiles along the cyan, pink, and blue lines in (e). Crosses mark the position where AO correction was carried out using intensity modulation with a DMD. Scale bar: 40 µm in (b), 10 µm in (c).

5 Supplementary Figure 5 The single-segment illumination AO method fails in densely labeled mouse brain, whereas the multiplexed method succeeds. (a) Images of neurons taken before and after AO correction using the single-segment illumination method. Orange square encircles the area used for correction. The improvement in imaging quality is minimal. (b) Images obtained by illuminating different pupil segments, arranged according to their respective pupil segments. The fluorescent background caused by the neighboring densely labeled structures obscure the image shift. As a result, the corrective wavefront (c) is very flat and does not reflect the aberrations in the sample. (d) Images taken before and after AO correction using multiplexed intensity modulation with a DMD. Note that the improvements in signal and resolution allow many more fine structures to be observed. (e) Signal vs. displacement map from the multiplexed measurement on the neuron marked with an orange cross in (d). The final corrective wavefront (f) now accurately shows the aberrations caused by the cranial window and the brain tissue. Scale bar: 10 µm.

6 Supplementary Figure 6 AO correction using signal from a fluorescent sea. (a) Using signal from a fluorescent sea, multiplexed intensity modulation with a DMD is used to correct for an artificial aberration introduced to the microscope. (b) Signal from the fluorescent sea increases over successive iterations of correction. (c) Axial images of a 2-µm-diameter bead measured without AO, without AO with 7.5 display gain, after five-iteration AO correction in fluorescent sea, and under ideal aberration-free condition, respectively. (d) Corrective wavefront in units of waves after five iterations of AO correction in (b). (e) Axial signal profile along the white line in (c). Scale bar: 2 µm.

7 Supplementary Table 1 Experimental parameters used for aberration measurement. Data AO method (modulation device) Modulation type Sample (labeling) Excitation power (mw) a Signal integration time (sec) Figure 1a DC Beads Figure 1a PM (DMD) Intensity Beads Figs. 2a,d PM (DMD) Intensity Mouse (GCaMP6s) Figs. 3a,c PM (SDM) Phase Mouse (YFP) Figs. 3f,g PM (SDM) Intensity Mouse (GCaMP6s) Supp. Fig. PM (DMD) Intensity C. elegans (GFP) b Supp. Fig. PM (DMD) Intensity C. elegans (GFP) f Supp. Fig. 4 PM (DMD) Intensity Zebrafish (GFP) Supp. Figs. PM (DMD) Intensity Mouse (GCaMP6s) a Excitation power was measured post microscope objective.

8 Supplementary Note: Multiplexed aberration measurement in C. elegans and zebrafish in vivo. The ultimate test of any AO method is in live biological samples, in which microscopic and sometimes macroscopic motions abound and photodamage as well as photobleaching limit the amount of light exposure. In addition to the mouse brain, we also tested our method in C. elegans and zebrafish, two of the most popular model organisms for bioimaging. Two examples of AO correction on GFP-labeled neurons in live C. elegans are shown (Supplementary Fig. 3). In both cases, we imaged neurons through the aberrating cylindrical bodies of the worms. In one example (Supplementary Fig. 3b,c), AO correction after two iterations improved both the signal (1.8 ) and resolution of two neurons in the deeper body wall. In the other (Supplementary Fig. 3d h), we corrected the aberration through the more densely labeled worm head by parking the focus at a bottom neuron. After one iteration of AO correction, the improvement in signal (2 ) and resolution was most prominent for the neuron on which the AO correction is done because the large surface curvature of the worm leads to a highly localized aberration (Supplementary Fig. 3f h). As an example of a very densely labeled sample, we applied the multiplexed aberration measurement method to in vivo imaging of 4-d-old zebrafish larvae, in which all cell membranes were labeled with EGFP. The axial views through the body of one larva showed notable signal and resolution deterioration for myotomes below the notochord (Supplementary Fig. 4a,b). After three rounds of AO correction at a myotome 170 µm inside the larva, the signal increased up to 2.3 in both lateral and axial imaging planes. More importantly than mere signal increase, the resolution improvement left various structural features much better resolved (Supplementary Fig. 4c,e). For example, the membrane folds could be easily observed after AO correction, the aberration-induced ghosts were eradicated, the individual membranes were much better separated, and the periodic structures of the myotome were much better visualized. Comparison with the single-segment illumination method. The signal-versus-displacement maps presented in Figure 1 reflect only the focal intensity variation relative to the ray displacement in focal plane and do not reflect or rely on specific fluorescent structures of the sample. Therefore, we can achieve AO correction from fluorescent samples of arbitrary structural complexity, including densely labeled fluorescent samples, for which the singlesegment illumination method 3 fails, because the reduction of excitation NA during single-

9 segment illumination allows the originally out-of-focus fluorescent structures to appear in the image and makes it difficult to determine wavefront gradients 2. For the same densely labeled mouse brain in vivo, the single-segment illumination method failed to improve signal quality, whereas the multiplexed method, which operates at the full NA of the objective, was able to improve image quality notably (Supplementary Fig. 5). An extreme example of a densely labeled fluorescent sample is a fluorescent solution ( fluorescent sea ). Without any structural feature, a fluorescent-sea sample cannot provide the image shift information that the single-segment illumination method needs to work. In contrast, the multiplexed aberration measurement method can be used to recover diffraction-limited resolution using signal in a fluorescein solution (Supplementary Fig. 6). Here an aberration was introduced to the system by overlaying a phase pattern on the SLM, which reduced the signal from a fluorescent sea by 1.7. Parking the laser at a fixed spot inside the fluorescent sea, and after five iterations with parallel intensity modulation, we fully recovered the signal from the fluorescent sea (Supplementary Fig. 6b). Because signal from a smaller object is much more sensitive to aberration than is a fluorescent sea 7, we tested how well this AO correction works by imaging a 2-µm-diameter fluorescent bead without and with AO correction obtained from the fluorescent sea and compared the resulting images with that from the bead under ideal, aberration-free imaging condition (Supplementary Fig. 6c,e). The multiplexed method improved signal from this bead by 7.5, almost fully recovering the imaging performance under ideal condition. Finally, deep inside scattering samples where excitation power becomes limited, the multiplexed method would perform better even for sparsely labeled samples because it does not require more power than what is necessary to visualize fluorescent structures, whereas the singlesegment illumination method needs higher excitation power during aberration measurement to compensate for the reduction of NA.