Abstract
In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.
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Data availability
Numerical source data are provided with this paper, and image source data are available at https://osf.io/ug68m/?view_only=09643eaee3604796bbbb89b988a9241f. Source data are provided with this paper.
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Acknowledgements
We thank J. An, S. Wang, M. Sun, M. Zhai, R. Chen, R. Wei and J. Wang at Peking University, Y. Chen, C. Fu, Q. Fu, Y. Gao, D. Wu, W. Jiang, and Y. Zhang at Beijing Transcend Vivoscope Biotech, and K. Zheng at the Advanced Innovation Center for Capital Medical University for their valuable comments on the optics, biological experiments, and data processing, and the Nanjing Brain Observatory for data-processing services. The work was supported by grants from STI2030-Major Projects (2021ZD0202205 for H.C., 2022ZD0212100 for R.W.), the National Natural Science Foundation of China (32293211 for H.C., 61975002 for A.W., 81925022 for L.C., 31830036 for A.W., 92054301 for L.C., and 8200907151 for R.W.), the Beijing Natural Science Foundation (Z20J00059) for L.C., CAMS Innovation Fund for Medical Sciences (2019-I2M-5-054) for H.C., and the National Postdoctoral Program for Innovative Talents (BX20190011) for R.W.
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Contributions
H.C., A.W., and C.Z. conceived the project and supervised the research; C.Z. and S.C. designed and built the optical system; C.Z. designed the miniature optical configuration; L.Z. designed and supervised the biological experiments; S.C. and D.Z. performed the experiments and led data analysis and figure-making; A.W. oversaw the fiber optics and assisted with mechanical assembly; R.W. provided support on the system builds and light-shielding design; Y.H. designed the electronics of gating-mode imaging under the supervision of Y.Z.; F.Z. performed data analysis under the supervision of J.Z.; Y.L. made the collimator connecting optical fiber; D.W. manufactured the optical fibers under the supervision of F.Y.; H.C., C.Z., L.Z., A.W., L.C., S.C., and D.Z. wrote the paper; all authors participated in discussion and data interpretation.
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Y.H. and Y.L. are employees of Transcend Vivoscope, which develops and sells microscopes.
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Extended data
Extended Data Fig. 1 Optical design of the miniature microscope maximizing scattered fluorescence collection.
(a) The optical design of the fluorescence collection arm. (b) The optical design of the illumination arm. (c-h) Trajectory analysis of ballistic and scattered photons. A point source was implanted at the depth of 1 mm in the inhomogeneous scattering brain tissue, and a large-area detector (18 ×18 mm2) was placed at the position of the supple fiber bundle to receive all the scattered photons. Without considering scattering, the optical path structure changed from ballistic photons passing through SIMO alone (c), to passing through SIMO plus the front lenses of the Abbe condenser (e), and then to passing through SIMO plus the front lenses and rear lens of the Abbe condenser (g). SIMO, simplified infinity miniature objective; WM, white matter. Corresponding irradiation distribution and cross-section profile of scattered photons on the detector were shown in (d), (f) and (h) respectively. The position of the cross section was indicated by a black dashed line in the irradiation distribution diagram. The full widths at half-maximum of the cross-section profile in (d), (f) and (h) were 5.2, 3 and 1.1 mm respectively.
Extended Data Fig. 2 Tests of resolution and full FOV imaging.
(a-d) Left, x-y and y-z images, averaged from 50 frames; right, normalized and averaged lateral and axial profile of beads (n = 5 in a-b, n = 12 in c-d), yielding a full width at half-maximum in silicon oil immersion at the center of FOV (a), in water immersion at the center of the FOV (b), at the 4 corner positions of a 220×220 μm2 FOV (c), and at the 4 corner positions of a 400×400 μm2 FOV (d). Data are shown as mean ± s.e.m. (e) 50-frame averaged x-y image of neurons labeled with GCaMP6s in posterior parietal cortex (PPC) (L6, depth of 590 μm) with a 400 × 400 μm2 FOV at a frame rate of 8.35 Hz and 200 × 200 pixels per frame. The experiment was independently repeated n = 4 times. (f) Time courses of calcium activities of the indexed neurons expressing GCaMP6s in (e). Traces were 3-frame moving averaged. The optical power after the objective is 4.2 mW.
Extended Data Fig. 3 The design of the m3PM system.
(a) The microscope system’s design with inset shows a profile of the m3PM headpiece. HWP, half-wave plate; PBS, polarizing beam splitter; HC-1300, hollow-core fiber for 1300 nm transmission; SFB, supple fiber bundle; DM, dichroic mirror; MEMS, micro-electro-mechanical systems; TL, tube lens; SL, scan lens; SIMO, simplified infinite miniature objective. (b) The x-y plot of the laser beam after Fiber-01. Inner, electron micrograph cross-section and an image of 1300 nm laser light delivered by Fiber-01 after collimation by a doublet. (c) The laser pulse temporal profile at the exit of the 1.5 m Fiber-01, after slight adjustment of the built-in dispersion compensation of the laser. (d) The x-y plot of the laser beam after Fiber-02. Inner, electron micrograph cross-section and an image of 1300 nm laser light delivered by Fiber-02 after collimation by an aspheric lens. (e) The laser pulse temporal profile at the exit of the 1.6 m Fiber-02 without dispersion compensation.
Extended Data Fig. 4 The performance of the gating acquisition mode.
(a,b) Single-frame images of pollens in the gating-on acquisition mode (a) and the gating-off acquisition mode (b). The experiment was independently repeated n = 3 times. (c) The signal-to-noise ratio (SNR) of the circular regions in (a) and (b), n = 200 frames respectively. Data are shown as mean ± s.e.m.
Extended Data Fig. 5 Neuronal activities in hippocampus CA1 at 1200 μm depth.
(a) Three-dimension reconstruction of a 1236-μm stack of GCaMP6s-labeled neurons in the PPC and underlying hippocampal CA1. Data were obtained from a head-fixed mouse bearing the m3PM. Green, GCaMP6s fluorescence; magenta, third harmonic generation (THG) signal. (b) Top, 50-frame averaged x-y images of CA1 shown in (a) at a depth of 1200 μm. Bottom, time courses of calcium activities of the indexed neurons expressing GCaMP6s in the x-y image. Traces were 3-frame moving averaged. The optical power after the objective is 51.5 mW. The experiment was independently repeated n = 3 times.
Extended Data Fig. 6 Triple-plane imaging of somatic activity in the dorsal hippocampus CA1.
(a) Schematic of the m3PM headpiece with the 2-gram z-scanning module (ZSM). The ZSM consists of a fast electrically tunable lens (ETL) and a pair of relay lenses. We integrated the ETL, the relay lens (RL) pair and the MEMS to form a 4 F (conjugated) system, in which the equal pupil plane of the ETL was conjugated to the surface of MEMS. The ZSM can be easily removed from the headpiece without affecting the performance of the headpiece itself. HC-1300, hollow-core optical fiber for 1300 nm transmission; MEMS, microelectromechanical systems; SL, scan lens; TL, tube lens; DM, dichroic mirror; SIMO, simplified infinite miniature objective; SFB, supple fiber bundle. (b) a snapshot of the m3PM headpiece. (c) Triple-plane imaging of somatic activity in the hippocampus CA1 at 885, 905 and 927 μm depth with a cycle rate of 4.4 Hz. We acquired a frame on each plane and looped for 3500 times. Top, 50-frame averaged x-y images. Bottom, time courses calcium activities of the indexed neurons expressing GCaMP6s in x-y images. Traces were 3-frame moving averaged. The optical power after the objective is 25.6 mW. The experiment was independently repeated n = 4 times.
Extended Data Fig. 7 Imaging blood vessels in PPC and underlying hippocampus with a maximum depth of 1428 μm.
(a) Three-dimension reconstruction of m3PM images of the brain vasculature in PPC plus underlying hippocampal CA1. Blood vessels were labeled with FITC-Dextran. The dotted box indicated CC position. (b) The 5-frame averaged x-y frames at representative depths except for 50-frame averaged x-y frames at 1428 μm depth. The experiment was independently repeated n = 2 times.
Extended Data Fig. 8 Analysis of motion artifacts.
(a) Three-dimension reconstruction of a 1038-μm stack of the YFP-labeled neuronal structure in PPC L5/6 and underlying hippocampal CA1 in Thy1-YFPH transgenic mouse. The data was acquired at 2-μm intervals at a frame rate of 8.35 Hz (200 × 200 pixels), and 50 frames were averaged at each slice. (b) Representative 50-frame averaged x-y images at L5 and hippocampal CA1. The experiment was independently repeated n = 4 times. (c) Lateral motion artifacts in PPC L5 and hippocampus CA1 in anaesthetized, head-fixed and freely behaving mice. The anesthesia and head-fixed data were 2,000 frames and the freely behaving data were 13,900 frames. The experiment was independently repeated n = 2 times. (d) Schematic diagram of the analysis method for axial motion artifacts. We obtained a 100-μm stack at 2-μm intervals near the focal plane prior to time lapse imaging. Then, we performed correlation analysis between a time lapse frame with each layer of the volumetric stack. The axial position with the greatest correlation coefficient was regarded as the current focal plane. (e, f) Axial motion artifacts in PPC L5 (e) and hippocampus CA1 (f) under different imaging paradigms. The data source of (e, f) were the same as in (c), the experiment was independently repeated n = 2 times. (g) Calcium imaging in a mouse under head-fixed and freely behaving conditions. Left, a 50-frame averaged frame showing the hippocampal CA1 region, depth and laser power used. Right, time courses of calcium activities of the indexed neurons expressing GCaMP6s in x-y image under head-fixed and freely behaving conditions. The experiment was independently repeated n = 4 times. (h) Relative change of the averaged amplitudes, transient decay time constants and SNRs of single neurons of the freely behaving to head-fixed condition (n = 15 neurons). The box shows 5th and 95th percentile of data in (c, e, f) and 25th and 75th data in (h). Black center line in (c, h) and blue dash line in (e, f), median; whiskers, maximum and minimum.
Supplementary information
Supplementary Information
Supplementary Notes 1 and 2 and Tables 1–3.
Supplementary Video 1
Three-dimensional reconstruction of image stacks of PPC and dorsal hippocampal CA1 in a head-fixed mouse. A 970-μm stack from the cortical surface, through PPC and CC until the bottom of the SP layer of CA1, with 2-μm intervals. Green, GCaMP6s-labeled neurons; magenta, third-harmonic generation signals. The FOV is 250 × 250 μm2. The imaging depth is displayed on the top left corner.
Supplementary Video 2
m3PM achieved dorsal hippocampal CA1 imaging in a freely behaving mouse. Left: the video of a mouse freely moving in the home cage (29 cm long × 17.5 cm wide × 15 cm high). CA1 neurons expressing GCaMP6s at a depth of 978 μm were imaged at a frame rate of 8.35 Hz (200 × 200 pixels). The data were corrected for lateral motion artifacts and then projected with a three-frame moving average with the Running Z Projector plug-in (ImageJ). Scale bar, 50 μm. Right: time courses of calcium activities of the indexed neurons expressing GCaMP6s in the x–y image. Traces were three-frame moving averaged. The optical power after the objective is 35.9 mW. The moving blue line on the traces was synchronized with the behavior video.
Supplementary Video 3
m3PM imaging of PPC layer 6 in mice performing a single-pellet reaching task. Left: the video of a mouse reaching a 20-mg sucrose pellet through a 0.5-cm slit. Middle: PPC layer 6 neurons expressing GCaMP6s at a depth of 650 μm were imaged at a frame rate of 15.93 Hz (128 × 128 pixels). The images were corrected for lateral motion artifacts and then projected with the five-frame moving average with the Running Z Projector plug-in (ImageJ). Scale bar, 50 μm. Right: the representative traces of five selected neurons, each trace was moving averaged with a span of five frames, and each green line indicates one grasp action. The moving blue line on the traces was synchronized with the behavior video on the left and neuronal activities in the middle. Clips were shown at normal (×1), slow (×0.5), and fast speed (×10), for easily viewing grasp behavior.
Source data
Source Data Fig. 1
Fluorescence-collection efficiency.
Source Data Fig. 2
Trace data, statistical data of calcium transients.
Source Data Fig. 3
Trace data, statistical data of calcium transients.
Source Data Extended Data Fig. 2
PSF data, trace data.
Source Data Extended Data Fig. 3
Fiber data.
Source Data Extended Data Fig. 4
Statistical data of the gating acquisition mode.
Source Data Extended Data Fig. 5
Trace data.
Source Data Extended Data Fig. 6
Trace data.
Source Data Extended Data Fig. 8
Statistical data of motion artifacts, trace data, statistical data of calcium transients.
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Zhao, C., Chen, S., Zhang, L. et al. Miniature three-photon microscopy maximized for scattered fluorescence collection. Nat Methods 20, 617–622 (2023). https://doi.org/10.1038/s41592-023-01777-3
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DOI: https://doi.org/10.1038/s41592-023-01777-3
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