In vivo wide-field calcium imaging of mouse thalamocortical synapses with an 8 K ultra-high-definition camera

Abstract

In vivo wide-field imaging of neural activity with a high spatio-temporal resolution is a challenge in modern neuroscience. Although two-photon imaging is very powerful, high-speed imaging of the activity of individual synapses is mostly limited to a field of approximately 200 µm on a side. Wide-field one-photon epifluorescence imaging can reveal neuronal activity over a field of ≥1 mm2 at a high speed, but is not able to resolve a single synapse. Here, to achieve a high spatio-temporal resolution, we combine an 8 K ultra-high-definition camera with spinning-disk one-photon confocal microscopy. This combination allowed us to image a 1 mm2 field with a pixel resolution of 0.21 µm at 60 fps. When we imaged motor cortical layer 1 in a behaving head-restrained mouse, calcium transients were detected in presynaptic boutons of thalamocortical axons sparsely labeled with GCaMP6s, although their density was lower than when two-photon imaging was used. The effects of out-of-focus fluorescence changes on calcium transients in individual boutons appeared minimal. Axonal boutons with highly correlated activity were detected over the 1 mm2 field, and were probably distributed on multiple axonal arbors originating from the same thalamic neuron. This new microscopy with an 8 K ultra-high-definition camera should serve to clarify the activity and plasticity of widely distributed cortical synapses.

Mechanism and design of a novel 8K ultra-high-definition video microscope for microsurgery

“…The 8K-UHD camera technology can also be used to measure higher-order brain functions. It has already been reported to have been utilized in measuring the brain activity of mice, and that, when using 8K-UHD technology, the neuroactivity in synaptic structures of a size of 0.5 um is 25 times larger than with conventional methods and can be viewed at twice the speed [ 20 ]. When the human eye sees the near field with both eyes, the binocular parallax stereoscopic view is the most important [ 9 ].…”

High‐speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction

“…We further investigate the correlations between pairs of neurons to study whether the ΔF/F detected from each neuron reflects its own activity or is overwhelmed by the background. 28 If the ΔF/F detected from individual neurons mostly reflects the change in out-of-focus fluorescence, correlations between pairs of neurons would be high. We therefore compute the pair-wise correlation coefficients for the calcium traces between each pair of neurons, for each plane, as shown in Figure 6K.…”

Emerging imaging methods to study whole-brain function in rodent models

“…To achieve a large FOV, a complete 2D or 3D image of a sample is usually acquired by moving the illumination spot across the sample [ 104 , 109 , 121 ]. Confocal fluorescence imaging was used by Yoshida et al [ 122 ] to measure GCaMP6 fluorescence from individual axonal boutons in behaving head-restrained mice. By using a multispot confocal design, they could image for the first time a relatively large FOV of 1 mm 2 .…”

“…Nevertheless, in the confocal design, the photons out of the focal plane still contribute to phototoxicity and photobleaching [ 124 ]. Another drawback is related to tissue scattering, which limits the depth of focus to a maximum of 100–200 µm [ 122 , 123 ].…”