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A miniaturized mesoscope for the large-scale single-neuron-resolved imaging of neuronal activity in freely behaving mice

Abstract

Exploring the relationship between neuronal dynamics and ethologically relevant behaviour involves recording neuronal-population activity using technologies that are compatible with unrestricted animal behaviour. However, head-mounted microscopes that accommodate weight limits to allow for free animal behaviour typically compromise field of view, resolution or depth range, and are susceptible to movement-induced artefacts. Here we report a miniaturized head-mounted fluorescent mesoscope that we systematically optimized for calcium imaging at single-neuron resolution, for increased fields of view and depth of field, and for robustness against motion-generated artefacts. Weighing less than 2.5 g, the mesoscope enabled recordings of neuronal-population activity at up to 16 Hz, with 4 μm resolution over 300 μm depth-of-field across a field of view of 3.6 × 3.6 mm2 in the cortex of freely moving mice. We used the mesoscope to record large-scale neuronal-population activity in socially interacting mice during free exploration and during fear-conditioning experiments, and to investigate neurovascular coupling across multiple cortical regions.

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Fig. 1: Principle of the systematically optimized miniaturized mesoscope (SOMM).
Fig. 2: SOMM performance validation.
Fig. 3: Verification of SOMM against tabletop a one-photon widefield system and two-photon microscope.
Fig. 4: SOMM detects cortex-wide neuronal activities in freely behaving mice during social interaction.
Fig. 5: SOMM reveals electrical-stimulus-evoked activities in freely behaving mice across tens of cortical regions.
Fig. 6: Dual-colour and simultaneous hippocampus–cortex SOMM imaging.

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Data availability

Example data as well as all CAD files for manufacturing the SOMM are available via GitHub at https://github.com/yuanlong-o/SOMM (ref. 57). The Allen CCF atlas is available at http://atlas.brain-map.org. The raw and analysed datasets generated during the study are too large to be publicly shared, but they are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The custom code comprising the SOMM-optimization and calcium-analysis pipeline is available as Supplementary Information. Future updates to the code will be published at https://github.com/yuanlong-o/SOMM, together with optical and optomechanical designs, optimization code used for designing SOMM, and SOMM video-processing code.

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Acknowledgements

We thank P. Strogies and J. Petrillo at The Rockefeller University’s Precision Instrumentation Technology (PIT) for fabrication of mechanical components. This work was supported by the National Natural Science Foundation of China under number 62088102 and 62301293 (Y.Z.), the Ministry of Science and Technology of the People’s Republic of China under number 2020AA0105500, China Postdoctoral Science Foundation under number 2023T160366 (Y.Z.), the Shuimu Tsinghua Scholar Program (Y.Z.), the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award numbers 1RF1NS110501 (A.V.) and 1RF1NS113251 (A.V.), the Kavli Foundation through the Kavli Neural System Institute (A.V.) and through a Kavli Neural Systems Institute postdoctoral fellowship (T.N.)

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Authors and Affiliations

Authors

Contributions

Y.Z. designed and implemented the SOMM-optimization pipeline, performed simulations, designed the optics and mechanics in SOMM, contributed to SOMM assembly and wrote the manuscript. L.Y. contributed to SOMM assembly and calibration experiments, performed freely behaving mouse experiments, analysed data and wrote the manuscript. Q.Z. contributed to behaviour experiments, analysed data and wrote the manuscript. J.W. provided critical support on system setup and signal processing and wrote the manuscript. T.N. provided important suggestions, critical support on system setup and contributed to manuscript editing. R.Z. and G.X. performed cranial window surgeries, viral injections and contributed to freely behaving mouse experiments. M.W., H.X. and Z.G. provided critical support on system setup. A.V. conceived and led the project, conceptualized and guided Y.Z. on the implementation of the principled optimization approach, designed experiments, guided data collection and analysis and wrote the manuscript. Q.D. led and co-funded the project, co-supervised Y.Z., supervised L.Y., J.W., R.Z., G.X., M.W. and H.X., and wrote the manuscript.

Corresponding authors

Correspondence to Qionghai Dai or Alipasha Vaziri.

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The authors declare no competing interests.

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Nature Biomedical Engineering thanks Mark Rossi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Comparison of agility of animals wearing no device, SOMM, dual-color SOMM, and SOMM with unoptimized cable.

Quantification of animal agility from recordings of behavior in an arena of 40-cm side length. Naïve mice were fitted with the devices on the day 1 and their agility was tested for the agility for three consecutive days. One trial under each condition per animal and day, resulting in a total n = 36 trials. Trial duration: 10 minutes. Inter-trial break: 1 hour. Box center line, median; Box shoulders: 25th and 75th and percentile; whiskers, maximum and minimum. Data points indicate trials. a. Extracted motion trajectories for mice carrying different devices. First column: representative mouse in homecage with the baseplate only. Second column: representative mouse with baseplate and SOMM connected by the optimized cable. Third column: representative mouse with baseplate and dual-color SOMM connected by the optimized cable. Fourth column: representative mouse with baseplate and dual-color SOMM connected by the unoptimized cable (SOMM, unoptimized wire). b. Boxplot of distance traveled across the 4 modalities. N.S., not significant by one-way ANOVA. ***P = 2.21 ×10-11. c. Assessment of moving agility across 3 days through measuring distance traveled inside the arena, across 4 modalities. d. Boxplot of mean speed across the 4 modalities. N.S., not significant by one-way ANOVA. ***P = 2.21 ×10-11. e. Assessment of moving agility across 3 days through measuring mean speed inside the arena. Scale bar: 10 cm in a.

Source data

Extended Data Fig. 2 Evaluation of long-term and cross-day imaging capabilities of SOMM.

a. Position trajectory of an animal wearing SOMM and behaving freely in an arena. b. Segmented neurons overlaid onto Allen CCF atlas, with different colors representing different cortical regions. c. Temporal activity heat map of detected neurons inferred by the processing pipeline in a 27.7-minute recording. Two zoom-in panels show example traces (each with 100 neurons), where one zoom-in is in the middle of the recording duration and the other one is at the end. 993 neurons were detected in total. Neurons are sorted randomly within each cortical region d. Left, segmented neuronal masks overlaid on the deconvolved SOMM recording. A 5-minute recording was conducted each day for three days. 730 neurons were detected in total. The white circles indicate neurons detected exclusively on one of the three days (170, 178, and 174 neurons, respectively). The green circles indicate neurons detected on both Day 1 and Day 2 (124 neurons). The turquoise circles represent neurons detected on both Day1 and Day 3 (112 neurons). The yellow circles represent neurons detected on both Day 2 and Day 3 (123 neurons). The blue circles represent neurons detected all three days (75 neurons). Right, an enlarged view of the red dashed box on the left. Segmentation masks from each day are depicted as grey shadows. Green masks indicate neurons identified on both Day 1 and Day 2, turquoise masks are for neurons identified on both Day 1 and Day 3, yellow masks are for neurons identified on both Day 2 and Day 3, and blue masks are for neurons identified across all three days. MOp, Primary motor area; MOs, Secondary motor area; SSp-ll, Primary somatosensory area lower limb; SSp-ul, Primary somatosensory area upper limb; SSp-tr, Primary somatosensory area trunk; VISam, Anteromedial visual area; VISp, Primary visual area; VISpm, posteromedial visual area; RSPagl, Retrosplenial area lateral agranular part; RSPd, Retrosplenial area dorsal part; VISa, Anterior visual area. Scale bars: 5 cm in a.

Extended Data Fig. 3 System setup for temporally interleaved 2p–SOMM functional ground truth recordings.

a. Schematic of hybrid 2p-SOMM microscope setup used for functional ground truth recordings. LED, light-emitting diode light source; Ti:Sa, titanium: sapphire laser; EOM, electro-optical modulator; M, mirror; DM, dichroic mirror; BS, beam splitter; Fm, emission filter; Fx, excitation filter; CL, collection lens; TL, tube lens; S, triggerable shutter. SOMM was mounted after a de-magnification module that consisted of a tube lens and an objective identical to the magnification module. Under such a configuration, the 2p focal plane (P1) was optically conjugated with the SOMM focal plane (P2). b. Various defocus configurations for SOMM, for verification across the DOF. We changed the position of SOMM focal plane (P2) relative to the static 2p scanning plane (P1) from -150 µm to +150 µm, which is the entire DOF of SOMM. c. Representative examples of verification data captured using 2p (blue) and SOMM (red) for a range of defocus distances. We first conducted imaging of a layer of 4-µm fluorescent beads using 2p. Then, we captured the fluorescent beads for a range of defocus distances using SOMM as shown in b, and matched the FOV with that of 2p. Scale bar: 50 µm.

Extended Data Fig. 4 Temporally interleaved 2p–SOMM functional ground truth recordings with an electrical tunable lens (ETL).

a. Schematic of the hybrid 2p-SOMM microscope setup used for functional ground truth recordings. LED, light-emitting diode light source; Ti:Sa, titanium: sapphire la-ser; EOM, electro-optical modulator; M, mirror; ETL, electrical tunable lens; DM, dichroic mirror; BS, beam splitter; Fm, emission filter; Fx, excitation filter; CL, collection lens; TL, tube lens; S, triggerable shutter. SOMM was mounted after a de-magnification module that consisted of a tube lens and an objective identical to the magnification module, but in reverse orientation. In this configuration, the 2p focal plane (P1) was optically conjugated with the SOMM focal plane (P2), and P1 could be shifted while leaving P2 stationary. Top right and bottom right: imaging session configuration. For each imaging session (that is, different imaging sites), we conducted seven trials; within each trial, we maintained the SOMM focal plane at 150 µm below the dura, while shifting the 2p focal plane using the newly added ETL to positions below the dura at depths 0 µm, 50 µm, 100 µm, 150 µm, 200 µm, 250 µm, and 300 µm, and rapidly alternating between one- and two-photon excitation (every 30 ms) to acquire 2p and SOMM signals nearly simultaneously on the time-scale of calcium indicator dynamics. 2p imaging power: 120 mW post the objective. 2p imaging field of view: 600 × 450 μm. b. Examples of two imaging sessions. SOMM-detected neurons were classified into different depths, based on paired 2p ground truth depths. c. Distributions of temporal correlation coefficients between SOMM traces and corresponding 2p traces as a function of depth. White circle: median. Vertical grey bar: Interquartile range. Transparent disks: data points. Transparent violin-shaped areas: Kernel density estimate of data distribution. n = 173, 441, 423, 355, 87 neurons at depths of 50 µm, 100 µm, 150 µm, 200 µm, 250 µm, across 7 imaging sessions and 2 mice. d. Neuron detection precision scores achieved by SOMM. Mean ± SD of precision scores is 0.78 ± 0.06, n = 7 imaging sessions across 2 mice. Central red mark: Median. Bottom and top edges: 25th and 75th percentiles. Whiskers extend to extreme points excluding outliers (1.5 times above or be-low the interquartile range). Data points are overlaid. Scale bar: 100 µm in b.

Extended Data Fig. 5 Assessing neuronal detection ability of SOMM across different cortical depths.

a. Each depth’s neuronal detection ability of SOMM versus the 2-photon (2p) ground truth is characterized. The 2p detection plane was shifted using an electrically tunable lens (ETL), while the SOMM detection plane remained stationary. For each depth, the 2p and SOMM frames are temporally interleaved, allowing the 2p signal to serve as the functional ground truth. Boundaries of neurons detected by 2p are delineated in blue, whereas the corresponding SOMM neurons are marked in red. These boundaries are superimposed on the standard deviation projection of the 2p recordings. 2p imaging power: 120 mW post the objective. 2p imaging field of view: 600 × 450 μm. b is the same as a but from the other animal. c. Neuron detection sensitivity scores achieved by SOMM as a function of depths. Mean ± SD of sensitivity scores are 0.75 ± 0.18, 0.68 ± 0.09, 0.60 ± 0.11, 0.53 ± 0.10, 0.43 ± 0.18 from 50 to 250 µm imaging depth at a step of 50 µm. Error bar: SD. n = 7 imaging sessions across 2 mice. d. Pie chart of the depth distribution of neurons detected by SOMM. n = 1479 neurons across imaging sessions from 2 mice. e. Histogram of the normalized standard deviation of neuronal activity for neurons that were detected by the SOMM (indicated in red), in contrast to those that were not detected by the SOMM (shown in blue). f. Histogram of the normalized peak intensity of neuronal activity for neurons that were detected by the SOMM (indicated in red), in contrast to those that were not detected by the SOMM (shown in blue). Scale bar: 100 µm in a, b.

Extended Data Fig. 6 Statistical validation of SOMM’s capability to detect calcium events across a 50-250 µm imaging depth range.

a. Example traces from 5 cortical depths were recorded by hybrid “temporally interleaved” 2p and SOMM microscope, where 2p signals are blue and SOMM signals are red. For each modality, calcium transient events were identified by OASIS (vertical lines). Seven traces are randomly selected for each cortical depth. Each recording was 100 s long. Scale bar: 10 seconds. Neurons are randomly ordered. b. Calcium event detection scores (precision, sensitivity, and F-score) achieved by SOMM on experimental functional verification dataset as a function of depth. Mean ± SD of precision scores are 0.78 ± 0.27, 0.73 ± 0.31, 0.69 ± 0.33, 0.65 ± 0.35, 0.54 ± 0.31 from 50 to 250 μm imaging depth at a step of 50 μm. Mean ± SD of sensitivity scores are 0.74 ± 0.27, 0.68 ± 0.27, 0.67 ± 0.28, 0.54 ± 0.29, 0.51 ± 0.31 from 50 to 300 μm im-aging depth at a step of 50 μm. Mean ± SD of F scores are 0.71 ± 0.24, 0.65 ± 0.26, 0.62 ± 0.27, 0.52 ± 0.26, 0.50 ± 0.24 from 50 to 250 μm imaging depth at a step of 50 μm. n = 1479 neurons across 7 imaging sessions from 2 mice. c. Linear fits to the normalized peak intensity of each neuron (blue cross) as a function of calcium transient detection F score. The solid red line represents the fitted model and the dashed red line represents the 95% confidence bounds. n = 1479 neurons across 7 imaging sessions from 2 mice. d is the same as c but shows linear fits to the standard deviation of normalized temporal activity of each neuron as a function of the calcium transient detection F score. n = 1479 neurons across 7 imaging sessions from 2 mice.

Extended Data Fig. 7 SOMM detects cortex-wide neuronal activities in freely-behaving mice during social interaction.

a. Segmented neurons overlaid with Allen CCF atlas with different colors representing different cortical regions. b. Temporal activity rendering of detected neurons inferred by the SOMM processing pipeline in a 2.7-minute recording. Two zoom-in panels showed example traces (each with 100 neurons). 1057 neurons are presented. Neurons are randomly sorted in each cortical region. c. Tuning analysis at a single-cell level. Left, temporal activities of neurons that are upregulated (red) and downregulated (blue) by social interactions. Deep blue shadows represented the periods when two animals touched each other. Right, spatial distributions of those neurons aligned in the CCF atlas. d-f and g-i are the same as a-c but for different animals. MOp, Primary motor area; MOs, Secondary motor area; SSp-bfd, Primary somatosensory area barrel field; SSp-ll, Primary somatosensory area lower limb; SSp-ul, Primary somatosensory area upper limb; SSp-tr, Primary somatosensory area trunk; SSp-un, Primary somatosensory area unassigned; VISam, Anteromedial visual area; VISp, Primary visual area; VISpm, posteromedial visual area; RSPagl, Retrosplenial area lateral agranular part; VISa, Anterior visual area; VISrl, Rostrolateral visual area. Scale bar, 30 seconds in b, 20 seconds in c, 1 minute in e, f, h, and i.

Extended Data Fig. 8 SOMM reveals electrical-stimulus-evoked activities in freely behaving mice across tens of cortical regions.

a. Segmented neurons overlaid with Allen CCF atlas with different colors representing different cortical regions. b. Temporal activity rendering of 984 inferred neurons in a 9.33-minute recording. Stimuli of cue (brown) and shock (red) are marked in the top and aligned with the neuronal activities. Two zoom-in panels showed example traces (each with 100 neurons) with the presence of cue (brown) and shock (red) indicated as transparent overlays. Neurons are randomly sorted in each cortical region. c. Animal instantaneous locations during the assay. The positions consisted of x (blue) and y (green) dimensions and are plotted with the presence of cues (brown) and shocks (red) indicated as transparent overlays. d. Distribution of the onset delays of cue-evoked activity peaks during the assay. The onset delay time for each of the neurons was counted as the time between the visual stimulus and the arrival of the first activity peak. Conditions with both cues and shocks (left) and only cues (right) are both plotted across cortical regions. e-h and i-l are the same as a-d but in different animals. MOp, Primary motor area; MOs, Secondary motor area; SSp-ll, Primary somatosensory area lower limb; SSp-ul, Primary somatosensory area upper limb; SSp-tr, Primary somatosensory area trunk; VISam, Anteromedial visual area; RSPagl, Retrosplenial area lateral agranular part; VISa, Anterior visual area. RSPd, Retrosplenial area dorsal part.

Extended Data Fig. 9 Dual-color SOMM uncovers neurovascular coupling at mesoscopic scale.

a. Interleaved dual-color recording of neuronal activity (GCaMP6f) and vasculature dynamics (TRITC) using SOMM (Methods). Two groups of LEDs with different colors were switched on alternatingly and in sync with the sensor to separately capture fluorescence in the two different color channels (top). The large circular image is a combination of raw frames of TRITC labeled vessels (top left segment), raw frames of GCaMP6f labeled neurons (middle), and their respective segmentation (bottom right; blood vessels in green and neurons in magenta). The zoom-in panel shows the dilation of a vessel at two-time points with the vessel diameter labeled (right). b. Left, illustration of dual-color SOMM captured vessels (bottom left), neurons (bottom right), and segmented neurons (top). Middle, segmented neurons in the left column are overlaid with Allen CCF atlas with different colors representing different cortical regions. The segmented vessels are overlaid in shallow green. Right, the correlation between principal components of neuronal population activity and vascular dynamics. Left side, heatmaps of correlation coefficients between PCs and vascular dynamics reported by changes of fluorescence due to modulation of blood flow. Right side, three principal components (PCs) of inferred neuronal activity signals. c-d are the same as b but for distinguished animals. MOp, Primary motor area; MOs, Secondary motor area; SSp-ll, Primary somatosensory area lower limb; SSp-ul, Primary somatosensory area upper limb; SSp-tr, Primary somatosensory area trunk; SSp-un, Primary somatosensory area unassigned; RSPagl, Retrosplenial area lateral agranular part; RSPd, Retrosplenial area dorsal part. Scale bar: 0.5 mm and 2 minutes in b-d.

Extended Data Fig. 10 SOMM effectively detects neuronal activity with implanted GRIN lens.

a. Standard deviation projection of deconvolved SOMM movie (left) and background-removed movie (middle) obtained through the processing pipeline (Methods). The mPFC region (-2.0 mm dorsoventral from the top of the skull) was relayed by a GRIN lens and the relayed image was captured by SOMM. Distinct neuronal segments are colored in different colors. The boundary of the GRIN lens is indicated by the red dashed line. The number of neurons is shown in the middle panel. Z-scored temporal traces of these components are illustrated in the right panel, with colors corresponding to those in the middle panel. Neurons are arranged according to the chronological appearance of each neuron’s initial peak. b-c are the same as a but from different imaging trials. Scale bars: 100 µm and 1 minute.

Supplementary information

Main Supplementary Information

Supplementary figures, tables, notes, references and video captions.

Reporting Summary

Supplementary Video 1

Evaluation of the animal movements when wearing SOMM and dual-color SOMM in an open field arena.

Supplementary Video 2

SOMM enables mesoscopic observation of cortical activities during free exploration of an open field arena by solitary animals.

Supplementary Video 3

SOMM enables mesoscopic observation of cortical activities during animal interactions.

Supplementary Video 4

SOMM enables mesoscopic observation of cortical activities during free behaviour in an open field arena under electric foot shocks.

Supplementary Video 5

SOMM enables simultaneous hippocampal and cortical imaging.

Supplementary code

SOMM-optimization and calcium-analysis pipeline.

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Zhang, Y., Yuan, L., Zhu, Q. et al. A miniaturized mesoscope for the large-scale single-neuron-resolved imaging of neuronal activity in freely behaving mice. Nat. Biomed. Eng 8, 754–774 (2024). https://doi.org/10.1038/s41551-024-01226-2

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