Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.
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We thank X. Li, D. Li, X. Chen, Z. Zhang, W. Gan, Y. Sun, S. Wang, and X. Chen for comments on the optics and biological experiments, and I.C. Bruce for manuscript editing. We also thank Mirrorcle Technologies for assistance with MEMS design and fabrication. The work was supported by grants from the National Natural Science Foundation of China (31327901, 31521062, 3142800018, and 31570839), the Major State Basic Research Program of China (2013CB531200 and 2012CB518200), and the National Science and Technology Major Project Program (2016YFA0500400).
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Integrated supplementary information
Supplementary Figure 1 Comparison of two-photon excitation efficiency of GCaMP-6f at 800-nm, 920-nm, and 1,030-nm excitation.
(a) Imaging the same sets of neuronal somata in an AAV-transfected mouse expressing GCaMP-6f in V-1 cortex at different excitation wavelengths. (b)-(d) Frequencies (b), amplitudes (c), and averaged time courses (d) of Ca2+ transients from ten somata shown in (a). (e) As in (a), except for imaging neuronal dendrites at a different focal plane. (f)-(h) Frequencies (f), amplitudes (g), and average time courses (h) of Ca2+ transients from ten dendrites shown in (e). Imaging parameters are shown in Supplementary Table S6.
(a) Schematic for compensation of the HC-920 dispersion with an H-ZF62 glass tube. (b) Dispersion parameter of the glass H-ZF62. (c) Pulse-width of the 920-nm femtosecond laser output from a 1-m HC-920 without dispersion compensation. (d) Pulse-width of the 920-nm femtosecond laser before and after a 1-m HC-920 with and without dispersion compensation. See Supplementary Note 1 for details.
(a) Illumination arm of the FHIRM-TPM. Upper: on-axis excitation light path from the output of the HC-920 to the specimen. Middle: off-axis scanning light path from the surface of the MEMS to the specimen. Lower: schematic of the high NA achromatic miniature compound objective assembly. (b) Detection arm of the FHIRM-TPM. Left: without the collection lens, some of the emission light at the edge of a large FOV (130 μm) is lost. Right: The collection lens relays all emission light within the FOV onto the SFB. See details in Supplementary Note 2.
(a) Snapshots from videos captured by the camera showing a representative mouse without (left) or with the FHIRM-mTPM mounted on its head (right). Trajectories extracted from these videos are presented in the below. The speed of movement is coded in color. (b) Average moving speed (left) and total travel distance (right) of mice with and without the FHIRM-mTPM. Data are presented as box-and-whisker plot from 8 mice with or without FHIRM-TPM. Two-tailed paired t-test. p for speed = 0.103. p for distance = 0.324.
Supplementary Figure 5 An integrated platform for comparing the performance of benchtop TPM, miniature wide-field microscope, and FHIRM-TPM.
(a) 3D schematic of excitation ligh path for benchtop TPM and FHIRM-TPM. HWP: half wave plate; AOM: acoustic-optics modulator; PBS: polarization beam splitter. (b) Optical diagram of the light path shown in (a). (c)-(e) Schematic of the light path of the real setup switchable among a benchtop TPM (c, d), a miniature wide-field fluorescence microscope (e, f), and an FHIRM-TPM (g, h). See more details in Supplementary Note 3.
Supplementary Figure 6 Comparison of dendrite activities in V1 cortex under head-fixed and freely moving conditions in a mouse in a dark arena.
(a): 50-s-averaged FHIRM-TPM image of V-1 cortical L2/3 neurons. (b): Two-dimensional trajectories of the mouse within the arena (duration: 100 s). Traces were calculated from video records captured by camera-1, and movement speeds are color-coded. (c) and (d): Time courses (duration: 100 s) of Ca2+ transients from three dendrites (circled in a) under head-fixed (c) and freely-moving conditions (d). Time-dependent change in the speed of the mouse is also shown at the bottom. (e): Average frequencies of all dendritic Ca2+ transients under head-fixed and free-moving conditions. Data are presented as box-and-whisker plot from15 dendrites. Two-tailed paired t-test. *** p <0.001.
Supplementary Figure 7 Preliminary results of the mTPM equipped with another customized miniature optical objective.
Left: Cortical neurons of a mouse were loaded with Cal-520, and imaged with the FHIRM-TPM equipped with another customized miniature objective (NA 0.7, Domilight, China) under head-fixed conditions. A FOV of 170 ×170 mm2 was achieved. Right: Representative Ca2+ transients from selected 4 somata shown in left image. The image speed was 20 Hz and the image depth was 150 mm with a 170-μm-thickness coverslip.
(a) Left, the scheme of the home-made chamber. Right, the photo of a mouse mounted with the chamber and cranial window. (b) The step-by-step schematics of how to mount the microscope onto the head of the mouse. Step 1, the cranial window (coverglass) was removed from the skull and a drop of ACSF was placed on the surface of the exposed brain tissue. Step 2, we moved the microscope with a 3-axial motorized stage, and continuously imaged until the interested ROI identified. The holder was pulled up during this stage. Step 3, we moved the holder down to attach to the chamber. Step 4, we injected glue to the edge of the holder to stabilize the holder and the chamber. Next, we fastened two screws to tightly connect the holder to the microscope. Step 5, we put a drop of 1.5% agarose around the end of the objective and the surface of the brain. (c) Positions of the objective and the agarose in different conditions. Left: Upon imaging the superficial tissue (0~150 μm) without the coverglass, the gap between the objective and the surface of the brain was filled with the agarose. Middle: Upon imaging the deep tissue (more than 150 μm) without the coverglass, the objective was directly attached the surface of the brain, with the agarose dropped around the objective and the top of the brain. Right: Under the condition of imaging through a coverglass, the agarose was filled in between, and surrounding the objective and the coverglass. Due to the limited working distance of the miniature objective (~200 μm) and the thickness of the coverglass (>100 μm), imaging depth is limited (~0-100 μm for the GRINTECH objective). (d) The shift speed of FOV in head-fixed, free-moving conditions with and without the agarose. Data are presented as box-and-whisker plot from 3 dendrites. ***p <0.001.
Supplementary Figures 1–8, Supplementary Notes 1–3 and Supplementary Tables 1–6.
The technical drawings of the FHIRM-TPM.
A protocol for the assembly of miniature two-photon microscope headpiece.
3D morphology of neuronal dendrites and spines imaged with the benchtop TPM, the miniature wide-field fluorescence microscope, and the FHIRM-TPM.
Ca2+ signals from somata imaged with the benchtop TPM, the miniature wide-field fluorescence microscope, and the FHIRM-TPM.
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Zong, W., Wu, R., Li, M. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat Methods 14, 713–719 (2017). https://doi.org/10.1038/nmeth.4305
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