We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 μm3 at a lateral resolution of ~1 μm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.
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The technical drawings of the FHIRM 2.0 are available in Supplementary Dataset 1. All other details required for setting up the complete system are provided in the Methods and Supplementary Notes. Supporting data for Figs. 1c–f and 2b–g and other Extended data figures are available from the corresponding authors upon request. Owing to the size of the datasets, they are not available on a public server.
The control software codes are available online at https://github.com/clarkewayne/FHIRM-TPM-2.0-GINKGO-1.0.2.
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We thank D. Zhang, F. Zeng, Z. Feng, W. Gao, W. Huang, J. Wang and Z. Zhou from Peking University; T. Gao from Southern Medical University; and Y. Guo, Q. Fu, X. Li, W. Jiang, Y. Li and Y. Zhang from Beijing Transcend Vivoscope Biotech Co. for valuable comments on the optics, biological experiments and data processing; I.C. Bruce for manuscript editing; and Domilight Optics for assistance with objective fabrication. The work was supported by grants from the National Natural Science Foundation of China (grant nos. 31327901, 92054301, 81925022, 61975002, 31830036, 31821091 and 8182780030), the Major State Basic Research Program of China (grant nos. 2016YFA0500400 and 2016YFA0500403), the Beijing Natural Science Foundation (grant no. Z20J00059) and the National Postdoctoral Program for Innovative Talents (grant no. BX20190011).
Y.H., D.W. and Y.X. are employees of the company Transcend Vivoscope, which develops and sells microscopes. The other authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Design of the FHIRM-TPM 2.0 system, headpiece and performance test of the low-magnification, large FOV objective.
a, Design of the microscope with inset showing a profile of the FHIRM-TPM 2.0 headpiece. ETL: electrically tunable lens; HC-920: hollow-core photonic crystal fiber to deliver 920-nm femtosecond laser pulses; HWP: half wave plate; MEMS: microelectromechanical system; PMT: photomultiplier; SFB: supple fiber bundle. b, ZEMAX simulation of the optical components in the headpiece. c, FOV. The scanning angle of the MEMS is ± 4.5° and the scanning FOV in the focal plane is ~420 µm × 420 µm (xy). d, Focal plane change by altering the diopters of the ETL. The scanning range of the ETL is ± 30 diopters and the corresponding focusing range is ~± 90 µm (z). e, Photograph of the objective (3×, NA 0.5). f, FOV and distortion test using a 100-µm grid standard. Dashed square outlines a 420 μm × 420 μm area corresponding to the scanning field of the MEMS. g, Spatial resolution test using a standard USAF1951 resolution test target. The light source was a fiber-coupled LED with a central wavelength of 940 nm. h, Intensity profile along the cross line shown in g.
a, Schematics of the baseplate (top) and the holder (bottom). b, Schematic illustrating headpiece mounting onto an assembly of baseplate plus a holder glued on a glass coverslip of a chronic cranial window. c, The protocol for mounting, dismounting, and remounting the headpiece. Step 1, the baseplate with a coverslip is fixed over a cranial window with dental cement. Step 2, positioning the headpiece. The headpiece is first screw-fastened to its holder and then placed onto the baseplate using a triaxial motorized stage. Once a region of interest is found, the holder is fixed to the baseplate with dental cement. Step 3, dismounting the headpiece by unscrewing and unplugging from the holder. Step 4, to remount, a drop of water is placed on the coverslip of the baseplate; then, the headpiece is plugged in and screw-fastened to the holder.
a, Top: snapshots of a representative mouse in homecage, with the baseplate-holder assembly only (left), the assembly and a 4.2-g dummy headpiece with no fiber and cable (middle), or the assembly and the whole 4.2-g headpiece with fiber and cable (right). Bottom: mouse trajectories extracted from the videos of 30-min test sessions. Colors code the moving speeds. b, Total travel distance (left) and average speed (right) in these three conditions. Experimental results of 8 mice are presented as box-and-whisker plots, and no significant differences among three groups were found according to One-Way ANOVA test (mean ± SEM, p = 0.57 for travel distance and 0.56 for average speed). Center line, median; limits, 75% and 25%; whiskers, maximum and minimum.
a, Schematic of the testing system. ND, neutral density; ETL, electrically-tunable lens; PD, photodetector. b, The principle for ETL diopter measurement using a PD-coupled fiber placed at some distance (h) beneath the ETL. With a collimated incident beam, blue lines show the convergent exit beam at plus diopters of the ETL, and green lines show the divergent exit beam at minus diopters. c, PD readout signal measured as a function of voltage applied to the ETL (from –2.0 to 2.0 V in 0.1-V steps). d, Relationship between diopters and the driving voltage. PD readout is converted into diopter values based on Eq. 1 in Supplementary Note 2. e–i, Dynamic responses of the ETL driven by voltage commands of different waveforms, amplitudes, and frequencies. Panels f and g show expanded views of the transition edges in panel e. Each curve in e–i is the average of at least 10 cycles.
Agarose gel containing 400-nm fluorescent beads served as the test sample. a, xy image of the beads, averaged from 20 frames. b, xy (left) and yz imaging (right) of four fluorescent beads. Data are from a 55 × 55 × 50 μm3 z-stack data set. c, d, Normalized and averaged lateral (c) and axial (d) profiles of the four beads, yielding a full width at half-maximum of 1.13 ± 0.03 μm (c) and 12.18 ± 0.2 μm (d). The plot shows the mean ± SEM from four beads. e, Long working distance illustrated by imaging a volume of 77 × 77 × 1,100 μm3. For panels d and e, the test sample was placed on a precision Z-axis motorized stage. f, g, Volumetric reconstruction from z-stack imaging with the ETL (g) compared to that with the motorized stage (f).
Images showing dendrites and spines in the frontal association cortex (FrA) of an awake, head-fixed mouse expressing GCaMP6s and bottom showing the time courses of Ca2+ changes (duration, 100 s) in four selected spines (marked with numerals in the image). Data were obtained +2.8 mm anteroposterior, +0.75 mm mediolateral at 40 μm below the pial surface. Scale bars, 30 μm (left) and 5 μm (right). Data were derived from one animal and are representative of three similar experiments.
In a freely moving mouse expressing GCaMP6s, three planes at 40, 100, and 200 µm below the pial surface imaged at 3.3 Hz per cycle with an inter-plane transit time of ~1.5 ms. Left, maximum projections from 31 frames of each layer; right, segmentation maps of manually-identified dendrites (106, plane 1) or neurons (229, plane 2; 153, plane 3). Time courses of Ca2+ activity in the segmented areas are displayed beneath the images and maps. Scale bars, 80 μm. Data were derived from three imaging depths in one animal and are representative of three similar experiments.
Extended Data Fig. 8 In vivo imaging of neurons in the FrA of a mouse experiencing electric footshock and recurring imaging of the same cohort of neurons over a month in a freely-moving mouse.
a, Top left, Snapshot of neurons in the FrA expressing GCaMP6f. Right, snapshot of the mouse on an electrical shocking grid typically used for fear conditioning. Bottom, Mouse trajectories with moving speed being color-coded. In each of the three episodes (S1, S2, S3), a pair of triangles delimit the beginning and the end of a 2-s footshock. b, Raster plots of Ca2+ activity in 276 neurons during electric footshock. The FHIRM-TMP 2.0 headpiece was mounted immediately before and detached immediately after image acquisition. c, Imaging the same FOV of mPFC on different days over a month. Expanded views are shown below. Scale bars, 60 µm for top panels and 15 µm for bottom panels. d, Maximum intensity projection of 2,000 time-lapse frames obtained on day 30. Traces show Ca2+ activity in five selected somata (marked with numerals in the image) on different days. Data in a,b were representative of 9 experiments in three animals, and those in c,d were derived from one animal and are representative of two similar experiments in two animals.
The FHIRM-TMP 2.0 headpiece was mounted immediately before and detached immediately after image acquisition. Statistical data are shown as mean ± STD. a, Left, short-term stability analysis of x- and y- displacements of FOV. Data were from 6 sessions over a period of 4 days (180-s per session, > 4-hour intervals) of an open-field free exploration experiment. Right, FOV displacement among 6 sessions. b, Within-session (left) or day-to-day FOV displacement (right) during the social behavior experiments in Fig. 2. c, Within-session (left) and long-term FOV displacement (right) during the experiment shown in Extended Data Fig. 8c. Center line, median; limits, 75% and 25%; whiskers, maximum and minimum. Data a–c are representative of 3 separate experiments in three animals.
a, Averaged Ca2+ activities of ON (red) and OFF (blue) neuronal ensembles at the onset and the ending of all social interaction epochs. b, Raster plots of representative neuronal Ca2+ activities (top) and averaged Ca2+ traces (bottom) of ON and OFF neurons (Direct-ON and Direct-OFF) of Day 3. Red traces, Direct-ON; blue traces, Direct-OFF; gray shadows, epochs of social interaction. Statistical data are shown as mean ± STD. Data were derived from one animal and are representative of three similar experiments in three animals.
Supplementary Notes 1–6 and Table 1.
In vivo imaging of dendrite and spine activity in FrA expressing GCaMP6s at an 8-Hz frame acquisition rate. Data were obtained at 40 μm below the pial surface. The data were smoothed with a Gaussian kernel (σ = 1 pixel) and corrected for motion artifacts before being projected with three-frame running average with the Running Z Projector plugin (ImageJ).
In a freely behaving mouse, mPFC neurons expressing GCaMP6s at depths of −100 μm and −160 μm were imagined at frame rates of 10 Hz (5-Hz cycle rate, 512 × 512 pixels, top) and 20 Hz (10-Hz cycle rate, 256 × 256 pixels, bottom). Left, time-lapse images of Plane 1; middle, time-lapse images of Plane 2; right, Ca2+ activity of the marked neurons. Note that FOV was kept unchanged when imaging at different frame rates. The ImageJ plugin Running Z Projector was applied to perform three-frame running average.
In vivo volumetric imaging of mPFC neurons expressing GCaMP6s at an 8-Hz frame acquisition rate. The corresponding volume was 420 × 420 × 180 µm3. The data were smoothed with a Gaussian kernel (σ = 0.8 pixel) and corrected for motion artifacts before being projected after ten-frame running average with the Running Z Projector plugin (ImageJ).
Time-lapse imaging of FrA neuron activity expressing GCaMP6f (upper left) and behavior video (right) of a mouse experiencing electric footshock (2 s an epoch). Lower left, mouse trajectories extracted from the videos with colors coding for moving speeds. The Ca2+ imaging data were processed with NoRMCorre for piecewise rigid motion correction.
In a free-moving mouse expressing GCaMP6s, two planes at −80 and −160 µm from the pial surface of the mPFC were imaged at 3.3-Hz cycle rate with interplane transit time ~1.5 ms. Ca2+ imaging data were corrected for piecewise rigid motion shifts with NoRMCorre. The corresponding video of mouse behavior is shown to the bottom.
The imaging of dual-plane Ca2+ activity expressing GCaMP6s (top) and mouse behavior on day 1, day 3 and day 5 (bottom). Two planes at −150 μm and −250 μm were imaged at 5-Hz cycle rate. Ca2+ imaging data were corrected for piecewise rigid motion shifts with NoRMCorre.
Technical source files and complete assembly flow chart of FHIRM_TPM 2.0.
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Zong, W., Wu, R., Chen, S. et al. Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging. Nat Methods 18, 46–49 (2021). https://doi.org/10.1038/s41592-020-01024-z