We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein–labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.
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We thank our colleagues at Janelia Farm Research Campus, Howard Hughes Medical Institute: E. Betzig for helpful discussions; H. Dana, B. MacLennan, G. Ranganathan, K. Smith for help with mice samples; and P. Keller and M. Ahrens for providing zebrafish samples. We thank C. Fang-Yen for advice on C. elegans samples. This work was supported by Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Integrated supplementary information
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).
See “Details on the multiplexed aberration measurement method” section of Online Methods.
(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.
(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).
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.
(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.
Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 2269 kb)
Average of five trials of calcium imaging without and with AO correction at 150 μm depth in mouse primary visual cortex (same dataset as in Fig. 2d,e). (AVI 32007 kb)
Z stacks of basal dendrites of layer 5 pyramidal neurons at 458-500 μm depth imaged without and with AO correction in mouse primary somatosensory cortex (same dataset as in Fig. 3c; scale bar: 5 μm) (AVI 7813 kb)
Z stacks of basal dendrites of layer 5 pyramidal neurons at 463-483 μm depth imaged without and with AO correction in mouse primary somatosensory cortex (same dataset as in Fig. 3d,e; scale bar: 5 μm) (AVI 6004 kb)
Average of five trials of calcium imaging without and with AO correction at 490 μm depth in mouse primary visual cortex (same dataset as in Fig. 3f,g,h). (AVI 16467 kb)
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Wang, C., Liu, R., Milkie, D. et al. Multiplexed aberration measurement for deep tissue imaging in vivo. Nat Methods 11, 1037–1040 (2014). https://doi.org/10.1038/nmeth.3068
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