Abstract
Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.
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Data availability
The main data supporting the findings of this study are available within the paper and its Supplementary information files. The source data files for all data presented in this paper can be found at: https://github.com/JiLabUCBerkeley/JiLabAO.
Code availability
A LabVIEW executable program that allows standalone operation of the AO module and detailed documentations of its operation are available for download at https://github.com/JiLabUCBerkeley/JiLabAO.
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Acknowledgements
We thank J. Wu for help with the detection system; the Janelia JET team for designing and assembling the dispersion compensation unit; E. Carroll for help with galvo electronics; S. Chen for surgical assistance and K. Borges and Q. Zhang for helpful discussions. This work was supported by the Howard Hughes Medical Institute (C.R., A.C., Y.L., R.G.N., W.S., D.E.M. and N.J.); the National Institutes of Health U01NS118300 (C.R. and N.J.); the Burroughs Wellcome Fund under the Career Awards at the Scientific Interface (C.R.); Lawrence Berkeley National Laboratory LDRD 20–116 (J.A.R.) and the Firmenich Next Generation Fund, the Terman Fellowship, NIH grant nos. R01DA045664, R01MH116904 and R01HL150566 (X.C.).
Author information
Authors and Affiliations
Contributions
N.J. conceived and supervised the project. D.E.M., A.C. and N.J. developed the AO control program. C.R. and A.C. developed the AO standalone control program. T.G.B. performed the DM calibration and provided support on its operation. Y.L., R.G.N. and W.S. performed the mouse brain surgery. M.A.M. performed the mouse spinal cord surgery. M.A.M. built spinal cord temperature stimulation device and C.R. developed control program. A.C. built 2P AO setup and collected 2P data. C.R. built 3P AO setup and collected 3P data in brain and beads. J.A.R. designed new 3P system with input from C.R. J.A.R. and C.R. built new 3P system (used for spinal cord experiments). C.R. and M.A.M. designed and performed 3P imaging experiments in spinal cord. C.R. analyzed data and prepared all figures and supplementary material. X.C. supervised spinal cord experiments. C.R. and N.J. wrote the manuscript with feedback from M.A.M. and input from all other authors.
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Competing interests
N.J. and Howard Hughes Medical Institute have filed patent applications that relate to the principle of frequency-multiplexed aberration measurement. T.G.B. has a financial interest in Boston Micromachines Corporation, which produced commercially the DB used in this work. The remaining authors declare no competing interests.
Additional information
Peer review information Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Schematics of the aberration measurement method.
(1) We fix the tip, tilt, and piston of one group of 17 segments, and add to the remaining 20 pupil segments a specific tip angle Θi and tilt angle Φj (i, j = 1,2,…n) chosen randomly from an array of n angles spaced between -Ψ/2 and Ψ/2. (2) We modulate the phase or intensity of all 20 beamlets, at a distinct frequency ωs (s = 1,2,…20), and record the fluorescence signal. (3) We Fourier transform (FT) the recorded signal trace and measure the Fourier magnitudes at each ωs. (4) This procedure is repeated n × n times for all tip and tilt angles. (5) For each beamlet, we plot the Fourier magnitudes versus the displacements (Xi, Yj) and construct a 2D map of interference strength of this beamlet with the reference focus at different focal displacements. Here, Xi = f*tan(2Θi/M) and Yj = f*tan(2Φj/M); f: focal length of the objective; M: magnification from the DM to objective back focal plane. (6) We repeat steps (1)-(5), with the group of 20 pupil segments fixed, while modulating the remaining 17 segments. We fit each map with a 2D Gaussian function and find the displacements leading to maximal interference between the light ray and the reference focus, corresponding to the tip and tilt angles for each segment of the corrective wavefront. (7) We modulate the phases of the first 20 rays by piston-displacing each corresponding mirror segment at a distinct frequency ωs while keeping the phases of the remaining rays constant. The resulting fluorescence trace is recorded. (8) We FT the recorded fluorescence trace and read out the phase offsets that would lead to constructive interference with the reference focus at the modulation frequencies ωs. (9) We modulate the phases of the remaining 17 segments while keeping the phases of the first 20 segments unchanged and obtain the corrective wavefront. (10) We repeat the steps above as needed to obtain the final corrective wavefront and apply it to the DM. See detailed description in Methods.
Extended Data Fig. 2 Schematics of AO 2 P and 3 P fluorescence microscopes, and example system correction.
a,b, Components of AO 2P and 3 P fluorescence microscopes, respectively. DM, deformable mirror; SLM, spatial light modulator (used to introduce artificial aberration); L, lenses; X and Y, galvanometers; PMT, photomultiplier tube. c, Lateral and axial 3P images of a 1-µm-diameter red fluorescent bead, under 1300 nm excitation, taken without and with AO. Post-objective power: 0.13 mW. d, Signal profiles along the purple and yellow lines in c. e, Corrective wavefront applied to the DM. Scale bar, 1 µm. Microscope objective: NA 1.05 25×. Data representative of n > 20 bead images taken during n > 20 imaging sessions.
Extended Data Fig. 3 Correcting artificial aberrations for 2 P microscopy using phase versus intensity modulation and signal from fluorescent features of different sizes.
a,b, Artificial aberration introduced with the SLM and corrective wavefront on the DM, respectively. c,d, Axial images of 0.5-µm- and 10-µm-diameter fluorescent beads, respectively, without AO, with AO (phase modulation), and under ideal aberration-free conditions (without artificial aberrations applied to the SLM). Digital gains were applied to No AO images to increase visibility. Post-objective power: 6 and 2.3 mW, respectively. e,f, 2 P signal versus iteration number for 0.5-µm- and 10-µm-diameter beads, respectively, using phase and intensity modulation. Scale bars: 1 µm in c and 5 µm in d. Microscope objective used: NA 0.8 16×. n = 1 bead in c and d, respectively.
Extended Data Fig. 4 AO recovers spatial frequency components in 2 P images of neuronal structures in the living mouse brain.
a-d, Imaging of dendrites in the cerebral cortex of Thy1-YFP-H mice. a,c, Maximum intensity projections of dendrites at 365–375 µm and 490–513 µm below dura, respectively, under 920 nm excitation, without and with AO (same images as in Fig. 1). b,d, Spatial frequency space representations of the 2 P images in a and c, respectively (left), and their radially averaged profiles (right). Scale bars, 5 µm. Microscope objective: NA 1.05 25×. Representative results from 13 fields of view and 3 mice.
Extended Data Fig. 5 AO improves 3 P imaging of beads in a capillary tube.
a, Schematics of sample geometry of 1-µm-diameter fluorescent beads in an air-filled capillary tube. b, Lateral and axial (along red dotted line) images of beads without and with AO (phase modulation). Post-objective power: 0.13 mW. Digital gains were applied to No AO images to increase visibility. c,d, 3 P signal improvement (AO/No AO) and axial full width at half maximum (FWHM) of a representative bead (white arrowhead in b) as a function of the iteration #, respectively. e, Corrective wavefront, unwrapped (modulo-2π) for visualization purposes. Scale bar, 5 µm. Microscope objective: NA 1.05 25×. Representative results from 2 imaging sessions.
Extended Data Fig. 6 AO improves in vivo 3 P imaging of cortical neurons in the mouse brain.
a, Lateral and axial images of a neuronal cell body (Thy1-YFP-H), at 757 µm below dura, under 1300 nm excitation, without and with AO (same cell body as in Fig. 2a). Post-objective power: 17 mW. b, Signal profiles along the purple and yellow lines in a. c, Maximum intensity projection (MIP) of same neuron as in a, 747–767 µm below dura, under 1300 nm excitation, without and with AO. Post-objective power: 17 mW. d, MIP of the yellow square in c, at 747–757 µm below dura, without and with AO. Insets in d, zoomed-in views of dendrite in white box. 10× digital gain was applied to the inset without AO to improve visibility. Post-objective power: 20 mW. e, Lateral and axial images of a neuron in the mouse cortex (Thy1-GFP-M), at 687 µm below dura, under 1300 nm excitation, taken without and with AO. Post-objective power: 35 mW. f, Signal profiles along the purple and yellow lines in e. g, Corrective wavefront in e. h, MIP of a neuron in the mouse cortex (Thy1-GFP-M, different animal than in e), at 624–644 µm below dura, under 1300 nm excitation, without and with AO. Post-objective power: 13 mW. i, Signal profiles along the purple and blue lines in h. j, Corrective wavefront in h. Insets in a and e: spatial frequency space representation of the corresponding fluorescence images. Scale bars, 10 µm. Microscope objective: NA 1.05 25×. Representative results from 32 fields of view and 8 mice.
Extended Data Fig. 7 Effect of iterations on 3 P fluorescence signal improvement for phase and intensity modulation-based aberration correction in the mouse brain in vivo.
a-f, 3 P images of a neuron in the mouse cortex (Thy1-YFP-H), 623 µm below dura, under 1300 nm excitation, without AO correction and after running aberration measurement a total of N = 1–5 iterations. a,d, Lateral and axial images of the neuron using phase and intensity modulation, respectively. Post-objective power: 20.8 (a) and 23.6 mW (d). b, e, 3 P signal improvement (AO/No AO) with iterations, for phase and intensity modulation, respectively. The plotted signal is the average pixel intensity within a 16×16-pixel area around the image maximum. c,f, Corrective wavefronts measured with phase and amplitude modulation, respectively. Scale bars, 10 µm. Microscope objective: NA 1.05 25×. Representative results from 3 fields of view and 2 mice.
Extended Data Fig. 8 AO enables in vivo 3 P imaging of dendritic spines and axonal boutons in deep layers of the mouse cortex.
a, Maximum intensity projection (MIP) of a neuron in the mouse cortex (Thy1-YFP-H), at 601–616 µm below dura, under 1300 nm excitation, without and with AO. Post-objective power: 17 mW. b, Signal profiles along the purple and blue lines in a. c, Corrective wavefront in a. d, MIP of the orange box in a, at 609–619 µm below dura, without and with AO. Post-objective power: 25.6 mW. e, Spatial frequency space representations of the images in d (top) and their radially averaged profiles (bottom). f,g, Zoomed-in views of the red and gray boxes in d, respectively. 4× digital gain was applied to images without AO to improve visibility. White arrowheads: dendritic spines; orange arrowheads: axonal boutons. h, MIP of a neuron in the mouse cortex (Thy1-YFP-H), at 863–875 µm below dura, under 1300 nm excitation, taken without and with AO. Post-objective power: 42 mW. i, Zoomed-in views of the red box in h. 3× digital gain was applied to the image taken without AO to improve visibility. j, Signal profiles along the purple and blue lines in h. k, Corrective wavefront in h. l, Spatial frequency space representation of the images in h (left) and their radially averaged profiles (right). Scale bars: 10 µm in a, d, h, and i; 2 µm in f and g. Microscope objective: NA 1.05 25×. Representative results from 20 fields of view and 5 mice.
Extended Data Fig. 9 AO improves in vivo 3 P imaging of hippocampal structures at different depths in the mouse brain, with 1700 nm excitation.
a-l, 3 P images of neurons in the mouse hippocampus at different depths. a,d,g,j, Lateral and axial images of neurons without and with AO, at 917, 960, 1010, and 1020 µm below dura, respectively. Post-objective powers: 26.5 (a), 10 (d), 27 (g), and 24 mW (j). b,e,h,k, Signal profiles along the green and yellow lines in a, d, g, and j, respectively. c,f,i,l, Corrective wavefront in a, d, g, and j, respectively. For a, a Gad2-IRES-Cre × Ai14 (Rosa26-CAG-LSL-tdTomato) mouse was used; for d, g, and j, neurons in wildtype mice were infected by a mix of AAV-Syn-Cre and AAV-CAG-FLEX-tdTomato. Scale bar, 10 µm. Microscope objective: NA 1.05 25×. Representative results from 9 fields of view and 2 mice.
Supplementary information
Supplementary Information
Supplementary Figs. 1–10, Tables 1–3 and Notes 1–4.
Supplementary Video 1
In vivo image stacks of a cortical neuron in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Fig. 2a. Scale bar, 10 µm.
Supplementary Video 2
Zoomed-in views of in vivo image stacks of cortical neuron dendrites in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Fig. 2b. Scale bar, 10 µm.
Supplementary Video 3
In vivo image stacks of a cortical neuron in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Extended Data Fig. 8a. Scale bar, 10 µm.
Supplementary Video 4
Zoomed-in views of in vivo image stacks of cortical neuronal processes with dendritic spines and axonal boutons in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Extended Data Fig. 8d. Scale bar, 10 µm.
Supplementary Video 5
Stack of 3P fluorescence and third-harmonic images going from cortex to the hippocampus of a Thy1-YFP-H mouse brain in vivo. This image sequence of combined 3P fluorescence and third-harmonic signals was obtained from 250 to 754 µm below the dura of a mouse whose hippocampal neurons are shown in Fig. 2f–j. Note the transition from cortex to hippocampus, through the white matter. Scale bar, 20 µm.
Supplementary Video 6
In vivo image stacks of a hippocampal neuron in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Fig. 2f. Scale bar, 10 µm.
Supplementary Video 7
Zoomed-in views of in vivo image stacks of hippocampal neuronal processes in a Thy1-YFP-H mouse measured without and with AO. Same data as shown in Fig. 2g. Scale bar, 10 µm.
Supplementary Video 8
In vivo image stacks of hippocampal neurons in a Gad2-IRES-Cre × Ai14 (Rosa26-CAG-LSL-tdTomato) mouse measured without and with AO. Same data as shown in Fig. 2k. Scale bar, 10 µm.
Supplementary Video 9
AO improves calcium activity recordings in the mouse spinal cord in vivo, 310 µm below dura. Image sequence corresponding to data shown in Fig. 3i–k (four-trial average). Scale bar, 10 µm.
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Rodríguez, C., Chen, A., Rivera, J.A. et al. An adaptive optics module for deep tissue multiphoton imaging in vivo. Nat Methods 18, 1259–1264 (2021). https://doi.org/10.1038/s41592-021-01279-0
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DOI: https://doi.org/10.1038/s41592-021-01279-0
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