Here we present a fluorescence microscope light path that enables imaging, during free behavior, of thousands of neurons in mice and hundreds of neurons in juvenile songbirds. The light path eliminates traditional illumination optics, allowing for head-mounted microscopes that have both a lower weight and a larger field of view (FOV) than previously possible. Using this light path, we designed two microscopes: one optimized for FOV (~4 mm FOV; 1.4 g), and the other optimized for weight (1.0 mm FOV; 1.0 g).
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The imaging data used to generate the figures in this paper are freely available at the following repository: https://academictorrents.com/details/aa2a3d2f5ff0b3974871db47db57bc3dabf3c192.
The code used to perform the analysis and generate the figures in this paper is freely available under the MIT License at the following repository: https://github.com/FeeLab/Feescope-Paper-Code. The firmware source code along with build instructions is available under the CC BY-NC-SA 4.0 license at the following repository: https://github.com/FeeLab/Fee-Scopes.
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We thank D. Aharoni and J. Newman for many helpful discussions and for their technical expertise. In addition, we thank M. Wilson, J. Newman, J. Voigts, N. Nikbakht, A. Bahle, M. Happ, A. Tanner and J. Kornfeld for comments on the manuscript. We thank W. Guo (MIT) for providing transgenic mice and Q. Xu (MIT) for baseplate designs and for running mice. M.S.F. acknowledges funding through the McKnight Foundation. J.R.S. acknowledges funding through the Harold and Ruth Newman Family Hertz Graduate Fellowship. G.F.L. acknowledges funding through the Simons Foundation (grant 542977ASPI). J.J.Z. acknowledges funding through the NIH (R21 EY028381-01).
J.R.S., G.F.L. and M.S.F. have an anticipated financial interest as part of a partnership with Open Ephys, a company working to sell open-source tools based on the technology in this paper. This project continues to be open source.
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Extended Data Fig. 1 Simulation and measurement of optical performance.
a, Ray trace simulation of Featherscope lens assembly (above) and point spread function calculated at the image plane (below). The grid spacing is equal to the pixel width. The cross-shaped behavior of the PSF at the edges of the field of view is characteristic of astigmatic aberrations. b, Image taken with the Featherscope of a USAF 1951 resolution target. The line widths of the largest and smallest elements visible in the inset are 3.91 μm and 2.19 μm, respectively. c, measurements of Featherscope focal surface compared to simulations. Bead height at best focus is plotted in blue for the set of beads used in the PSF measurement in Fig. 1. Simulation results for a spherical surface of best focus is plotted in orange. d, Same as (a) but for the Kiloscope lens assembly. e, Same as in (b) but with the Kiloscope. The line widths of the largest and smallest elements visible in the inset are 6.96 μm and 4.38 μm, respectively. f, Same as in (c) but for the Kiloscope.
Extended Data Fig. 2 Illumination coupling optics.
a, optical assembly for adapting NA and mode scrambling the excitation light before coupling it into the microscope. The beam profile is illustrated below at various points in the assembly. Spacing between the axicons was adjusted for optimal microscope performance, and it was noted that the optimal configuration resulted in a central dark spot in the output beam. This spot is not present at the object plane due to imperfections in the fiber. b, Radial profile of the signal collected with the Featherscope while imaging a thin layer of fluorescent dye using different optical configurations. Optical vignetting (orange) illustrates the effects of light losses within the microscope optics and was measured by illuminating the dye with a diffuse light source opposite the objective. Total vignetting (yellow) illustrates the decrease in fluorescent signal caused by the combination of optical vignetting and a Gaussian-shaped excitation light profile. The curve with correction optics (blue) illustrates the flat signal intensity profile that can be achieved with an axicon pair that increases the excitation intensity at the edges of the field of view. c, same as in (b) but for the Kiloscope.
Extended Data Fig. 3 Analog-to-digital converter functionality.
a, Time series recording of two test signals at 100 Hz (orange) and 1000 Hz (blue) using the onboard analog-to-digital converter, sampling at 25.5 kS/s for each signal. b, Power spectral density of the test signals. The spectrum was calculated using Thomson’s multitaper estimate with Slepian tapers. The time series deviates from a pure tone with a root-mean-square residual of 0.2% of the full scale, calculated using the full bandwidth of the recorded signal.
Extended Data Fig. 4 Additional performance characterization of Kiloscope optics.
a, Image taken with the Kiloscope of a fixed coronal brain slice from one Tg(Camk2a-cre)T29-1Stl x Ai95(RCL-GCaMP6f)-D mouse. The image is corrected for vignetting. b, Image of USAF resolution target using Kiloscope optics and a Sony IMX260 image sensor with 1.4 μm pixel width. The smallest element in group 7 is resolvable and has a linewidth of 2.2 μm.
Extended Data Fig. 5 Extraction results from the three birds presented in the paper.
Footprints for each extraction are plotted as colored outlines against a grayscale background of maximum intensity of median filtered data over the course of a recording session. Footprints were extracted using EXTRACT and manually curated based on videos of each footprint’s activity. The high variability in the number of extracted footprints is likely due to variations in virus expression levels between individuals. All scale bars are 100 μm. a, 46 footprints after curation. b, 177 footprints after curation. c, 225 footprints after curation.
Extended Data Fig. 6 Extraction results from the four mice presented in the paper.
Footprints for each extraction are plotted as colored outlines against a grayscale background of the maximum intensity of median filtered data over the course of a recording session. Footprints were extracted using EXTRACT and manually curated for panel (a) based on videos of each footprint’s activity. For panels (b–d), we inspected each footprint by eye and removed visually anomalous footprints and footprints overlapping with blood vessels. The high variability in the number of extracted footprints is likely due to variations in virus expression levels between individuals. All scale bars are 1 mm. a, 4 mm cranial window, 2382 footprints after curation. b, 3 mm cranial window, 1029 footprints. c, 4 mm cranial window, 401 footprints. d, 3 mm cranial window, 112 footprints.
Extended Data Fig. 7 Movement observed using the Kiloscope.
Time series of horizontal and vertical displacement of the FOV, calculated using NoRMCorre, during the recording shown in Fig. 2.
Extended Data Fig. 8 Spatial distribution of coactive neural populations in the Kiloscope dataset shown in Figure 3.
a, Ridgeline plot from Fig. 2d; highlighted in green is a single large correlated event for each NMF component. b, Locations of footprints that participate in each highlighted event. Pixels containing one active footprint are colored blue, two or more overlapping footprints colored red, and inactive footprints colored gray. Footprints are thresholded at the 10% level. Listed in each panel are the number of footprints active in the associated event and the total number of footprints in the NMF component. All scale bars are 750 μm.
Extended Data Fig. 9 Decoding position within the maze with octant resolution.
a, Circular maze with octants labeled; in this case, direction is not coded. b, Decoding matrix for a set of binary SVM models trained to predict the labels in (a) based on the activity in Fig. 2h. White is set at chance level. c, Map of decoder accuracies using the 32 closest footprints less than 270 μm to a given point. Pixels that do not meet a significance threshold (Benjamini-Hockberg false discovery rate = 5%) are plotted in black.
Extended Data Fig. 10 Receptive fields of selected footprints from the Kiloscope.
a, the circular maze that the mouse explored during the recordings in Fig. 2e–h. b, receptive fields plotted for a collection of footprints from Fig. 2e–h. The plotted intensity is the fraction of the time a given footprint was active at a given location. Footprints were selected by eye for large-scale structure in their receptive fields. c, locations of the selected footprints within the FOV. Footprints were selected blindly without knowledge of their location within the FOV. Regions of overlap between footprints are plotted in red. Scale bar is 750 μm.
Median-subtracted video and audio corresponding to the bird data in Fig. 2a–d.
Median-subtracted video corresponding to the mouse data in Fig. 2e–h. Video is temporally downsampled by a factor of 4.
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Scherrer, J.R., Lynch, G.F., Zhang, J.J. et al. An optical design enabling lightweight and large field-of-view head-mounted microscopes. Nat Methods 20, 546–549 (2023). https://doi.org/10.1038/s41592-023-01806-1