Large-scale two-photon calcium imaging in freely moving mice

Abstract

We developed a miniaturized two-photon microscope (MINI2P) for fast, high-resolution, multiplane calcium imaging of over 1,000 neurons at a time in freely moving mice. With a microscope weight below 3g and a highly flexible connection cable, MINI2P allowed imaging to proceed with no impediment of behavior in half-hour free-foraging trials compared to untethered, unimplanted animals. The improved cell yield was achieved through a new optical system design featuring an enlarged field of view (FOV) and a new micro-tunable lens with increased z-scanning range and speed that allowed for fast and stable imaging of multiple, interleaved planes as well as 3D functional imaging. A novel technique for successive imaging across multiple, adjacent FOVs enabled recordings from more than 10,000 neurons in the same animal. Large-scale proof-of-principle data were obtained from cell populations in visual cortex, medial entorhinal cortex, and hippocampus, revealing spatial tuning of cells in all areas, including visual cortex.

Functional network topography of the medial entorhinal cortex

“…The weight decrease of this modified miniscope compared with its predecessors was achieved in both the scope itself, now made from a type of machinable plastic, as well as in the connection cable, which now contains a much thinner, tapered fiber bundle with decreased overall diameter (46). While lowering the weight, these design changes also drastically increased the degree to which the head-mounted assembly could be twisted and thereby permitted animals to exhibit more naturalistic foraging behaviors (46). Some of the newest hardware features of MINI2P that are introduced in ref.…”

“…Some of the newest hardware features of MINI2P that are introduced in ref. 46, such as the even further enlarged FOV size, the built-in Z-focusing, and the ability to shift FOVs systematically between sessions, were included in only a small subset of the present data (18 out of 212 recordings); importantly, however, all present recordings had the lowered weight and the thinner, more flexible connection cable of MINI2P, allowing for good spatial coverage and reliable classification of spatially modulated cell types (see below).…”

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

“…By keeping the craniotomy used for the helper virus injection free from cement and temporarily closing it with a silicone sealant, we could reopen it several weeks later to introduce the rabies virus carrying tdTomato (RABV-tdTomato). Once the rabies virus reached the MEC, we were able to identify a cell expressing both the calcium indicator and tdTomato in vivo with a two-6/30 photon miniscope (Figure 7B, Zong et al, 2017Zong et al, , 2021Obenhaus et al, 2021), demonstrating the feasibility of using this approach to characterise the spatial firing properties of input cells in freely moving mice.…”

“…Mice were imaged with a custom two-photon benchtop microscope (Femtonics, Hungary) or two-photon miniscope (Zong et al, 2017(Zong et al, , 2021Obenhaus et al, 2021). In both cases, the mice were head-fixed via their head bar and allowed to run freely on a black styrofoam wheel (approximately 85 mm radius and 70 mm width) with a metal shaft fixed through the centre.…”

“…ΔF/F was calculated by first multiplying the neuropil fluorescence of every cell with a neuropil coefficient (0.7) and subtracting the resultant trace from the raw fluorescence trace. The baseline for every cell was then subtracted and the result divided by baseline (Obenhaus et al, 2021;Zong et al, 2021). The baseline was calculated as the median of the first 20% of frames sorted ascendingly after smoothing the neuropil corrected trace with a hanning window.…”