Miniaturized head-mounted microscope for whole-cortex mesoscale imaging in freely behaving mice

Abstract

The advent of genetically encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive-index-matched skulls, has enabled mesoscale cortical activity imaging in head-fixed mice. However, neural activity during unrestrained behavior substantially differs from neural activity in head-fixed animals. For whole-cortex imaging in freely behaving mice, we present the 'mini-mScope', a widefield, miniaturized, head-mounted fluorescence microscope that is compatible with transparent polymer skull preparations. With a field of view of 8 × 10 mm² and weighing less than 4 g, the mini-mScope can image most of the mouse dorsal cortex with resolutions ranging from 39 to 56 µm. We used the mini-mScope to record mesoscale calcium activity across the dorsal cortex during sensory-evoked stimuli, open field behaviors, social interactions and transitions from wakefulness to sleep.

Figure 1:. mini-mScope: A miniaturized head-mounted device for whole-cortex mesoscale activity mapping in freely behaving mice:

(a) Photograph of the fully assembled mini-mScope and the corresponding See-Shell implant. Scale bar, 5 mm (b) Left: Computer-aided design (CAD) rendering of the mini-mScope showing the internal components. The device is attached to the See-Shell implant via magnets. Three blue LEDs are attached to a filter holder that has a 480 nm excitation filter to excite GCaMP6f in the cortex. The green LED is used to obtain reflectance measurements for hemodynamic correction. Resulting emission signals (~520 nm) are focused through a collector lens and passed through an emission filter onto a CMOS sensor that can be manually focused. Scale bar indicates 5 mm. Right: A still image of a mouse bearing the mini-mScope and engaging in natural behaviors. (c) Fluorescence image of the whole dorsal cortex of a Thy1-GCaMP6f mouse captured by the mini-mScope. Scale bar indicates 2 mm. Image representative of n = 29 mice. (d) Comparative image of a head-fixed Thy1-GCcaMP6f mouse imaged using a standard epifluorescence macroscope through an intact-skull preparation. Scale bar indicates 2 mm. Image representative of n = 2 mice. (e) Left: Resolution test target overlaid onto the mini-mScope FOV. Right: Resolutions obtained within each specified grid. (f) Image of fluorescein dye infused agar phantom captured by the mini-mScope. Colored lines indicate mediolateral sections along which illumination profiles were obtained FOV. (g) Plot of normalized illumination profiles from sections denoted in f.

Figure 2:. Comparison of calcium dynamics imaged with the mini-mScope to conventional widefield epifluorescence macroscope:

(a) Top: Image of the dorsal cortex taken with mini-mScope of a Thy1-GCaMP6f mouse. Image representative of n = 29 mice. Bottom: image taken with a conventional widefield epifluorescence macroscope during the same experimental session. Image representative of n = 8 mice. Black squares indicate ROIs selected for ΔF/F traces analyzed in b-e. Scale bar indicates 2 mm. (b) ΔF/F traces of ROI 1 shown in a recorded for 120 seconds. In (b-e), black and red traces indicate filtered traces (Savitzky-Golay filter, 3^rd order polynomial, 5 point moving average) of ΔF/F recorded from the mini-mScope and the macroscope respectively. Blue traces with markers indicate raw ΔF/F values under 1% isoflurane anesthesia. (c) Plot of the ΔF/F versus Frequency obtained by computing the discrete Fourier series of 120s of spontaneous calcium activity recorded under isoflurane anesthesia (from traces shown in b.) (d) Histogram of ΔF/F values over 2-minute videos captured at the same 1% isoflurane anesthesia conditions with the mini-mScope and the conventional macroscope in the three ROIs indicated in a. (e) Histogram of z-scores of fluorescent intensity values over 2-minute videos captured at the same 1% isoflurane anesthesia conditions with the mini-mScope and the conventional macroscope in the three ROIs indicated in a. (f) Image of the dorsal cortex taken with mini-mScope of a Thy1-GCaMP6f mouse. Black squares indicate ROIs selected for hemodynamic corrections illustrated in g. Scale bar indicates 2 mm. (g) Uncorrected ΔF/F, reflectance ΔF/F and the hemodynamic corrected ΔF/F traces for the two ROIs shown in i.

Figure 3:. Sensory stimulus evoked responses imaged by the mini-mScope:

(a) Schematic of an anesthetized Thy1-GCaMP6f mouse with a vibrational stimulus applied to the right hind limb. (b) Composite of raw grayscale image of the brain and the pseudo-color frame where the largest average ΔF/F occurred within the 1-second stimulus period. White circle indicates ROI analyzed in d and e. Red dot indicates Bregma. Scale bar 3 mm. (c) Montage of average cortical calcium response to the vibration stimulus. Scale bar 2 mm. (d) ΔF/F traces of the contralateral ROI drawn in b. Red line denotes the average traces, grey lines denotes individual trials. Black bar shows the time and duration of the vibrational stimulus. (e) ΔF/F traces of ipsilateral ROI drawn in b. Blue line denotes the average response, grey lines denote each individual trial. (f) Peak ΔF/F within 1-second of vibration stimulus presentation (n=20 trials in one mouse, data is representative of 3 mice). ** indicates p < 0.01, p = 9.1x10⁻³, two-sample t-test. (g) Schematic of a mouse with a visual stimulus applied to the left eye. (h) Composite of raw grayscale image of the brain and the pseudo-color frame where the largest average ΔF/F occurred within the 1-second stimulus period. White circle indicates ROI analyzed in j and k. Scale bar 3 mm. Data were acquired in the same mouse as the data in a to f. (i) Montage of average cortical calcium response to the visual stimulus. Scale bar 2 mm. (j) ΔF/F traces of the contralateral ROI drawn in h. Red line denotes the average trace, grey lines denote each individual trial. Black bar shows the time and duration of the visual stimulus. (k) ΔF/F traces of ipsilateral ROI drawn in h. Blue line denotes average ipsilateral response, grey lines denote individual trials (n=20 trials in one mouse, data is representative of 3 mice). (l) Peak ΔF/F within 1-second of visual stimulus presentation. The bolded line corresponds to the average peak response whereas the grey lines indicate the peak response for each trial. ** indicates p < 0.01, p = 1.31x10⁻⁵, two-sample t-test.

Figure 4:. Mesoscale imaging of cortex during free and social behavior:

(a) Photograph of a mouse with a mini-mScope (red dot annotation) interacting with a companion mouse (blue dot annotation). Scale bar, 5 cm. (b) Traces of instantaneous locations of the two mice during trial. The segments of darker blue and red indicate positions of the mice when they were within 5 cm of each other. Scale bar, 10 cm. (c) Percentage of time spent still, moving, grooming, and rearing during open field behavior (n = 6 trials). (d) Percentage of time spent in contact and not in contact with the companion mouse behavior during trials (n = 3 trials). (e) Representative image of the cortex with locations of the seed pixels used for analysis in f-k are indicated. Scale bar 2 mm. (f) Seed pixel correlation maps of motor cortex (M1), barrel cortex (BC), retrospenial cortex (RSC) and visual cortex (VC) during movement and no movement. Top row: Average of correlations during 1 s epochs of movement; bottom row: Average of correlations during 1 s epochs of no movement. Scale bar 2 mm. (g) Change in percentage of hemisphere with r > 0.5 with respect to seed pixels analyzed. * indicates p < 0.05 (p = 4.6x10⁻³ M1, p = 6.3x10⁻⁴ FL, p = 3.8x10⁻³ HL, p = 4.6x10⁻³ RSC, p = 0.10 BC, p = 1.1x10⁻³ VC, two-sample Mann-Whitney U-test, n = 11 trials, n = 3 mice). Error bars denote mean ± SD. (h) Average inter-seed correlations associated with seeds (n = 11 trials, n = 3 mice). Error bars denote mean ± SD. (i) Seed pixel correlation maps of M1, BC, RSC and VC during non-social and social behavior epochs. Scale bar 2 mm. (j) Percentage of hemisphere with r > 0.5 with respect to seed pixels analyzed. * indicates p < 0.05 (p = 0.048 M1, p = 0.17 FL, p = 0.058 HL, p = 0.091 RSC, p = 0.082 BC, p = 0.37 VC, two-sample Mann-Whitney U-test, n = 8 trials, n = 8 mice). Error bars denote mean ± SD. (k) Average inter-seed correlations associated with the seeds (n= 6 trials, n = 6 mice). Error bars denote mean ± SD.

Figure 5:. Combined electrophysiological recording and mesoscale imaging of brain activity during wakefulness and sleep:

(a) Montages of change in glutamate activity over 1s period during awake, REM and NREM sleep states. Scale bar 2 mm. (b) Representative traces of raw glutamate signals from V1, hippocampal LFP, and video-based movement signals during states of waking, NREM sleep and REM sleep in a freely moving mouse. F₀ is the baseline glutamate signal calculated by averaging the fluorescence over the entire recording time. (c) Magnification of glutamate, LFP and movement signals selected in b. (d) Grouped mean raw glutamate signal from the entire cortex (normalized to baseline) during wakefulness, NREM sleep and REM sleep (n = 3 mice, Friedman paired nonparametric test; post-hoc multiple comparison with Dunn’s correction * indicates p < 0.05, p = 0.02 wakefulness vs REM). (e) Spectral analysis of glutamate signal in three states. (f) Correlation maps of cortical activity between quiet wakefulness, NREM sleep and REM sleep at two different days. Cortex was divided into 21 ROIs. (g) Mean correlation of cortical activity during waking, REM and NREM sleep (n = 3 mice). (h) Glutamate activity, hippocampal LFP frequency spectrogram and animal movement tracking during transition from REM to wakefulness (i) Montage of cortical glutamate changes during the transition shown in h. Scale bar 2 mm.