A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice

Abstract

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

Three-photon excited fluorescence imaging in neuroscience: From principles to applications

“…In another work, only the beam at 910 nm for 2PM is remotely tunable by using an electrically tunable lens ( Takasaki et al, 2020 ). Recently, remote focusing was implanted into a miniature 3PM so that the imaging plane could be adjusted without disturbing the animals behavior ( Klioutchnikov et al, 2022 ).…”

“…It also enables recording the calcium activity of the neuronal populations at 950 μm below the cortical surface when the animal is freely moving, with a FOV of 140 × 140 μm (120 × 120 pixels/frame) and an imaging speed of 28 Hz. Recently, the same group built 2-g miniature 3PM that is bearable for freely behaved mice ( Klioutchnikov et al, 2022 ). With the 3PM, neurons and vascular structures were observed in mice from layer 1 to the corpus callosum.…”

“…Combined with cutting-edge optical technologies, such as adaptive excitation sources (AES) ( Li et al, 2020 ), adaptive optics (AO) ( Rodríguez et al, 2021 ; Streich et al, 2021 ; Qin et al, 2022 ; Sinefeld et al, 2022 ), and Bessel beam ( Chen et al, 2018 ; Rodríguez et al, 2018 ), 3PM has made significant advances in imaging performance. In application scenarios, increasing research groups have demonstrated the capabilities of 3PM imaging for probing neural structural and functional dynamics beyond the depth of 2PM, including the applications of 3PM in imaging rodents ( Horton et al, 2013 ; Ouzounov et al, 2017 ; Wang T. et al, 2018 ; Liu et al, 2019a ; Weisenburger et al, 2019 ; Klioutchnikov et al, 2020 , 2022 ; Wang et al, 2021 ; Choe et al, 2022 ), fishes ( Chow et al, 2020 ; Rodríguez et al, 2021 ; Akbari et al, 2022a ), flies ( Tao et al, 2017 ; Hsu et al, 2019 ; Aragon et al, 2022 ), and other kinds of tissues ( He et al, 2019 ; Yildirim et al, 2020 ; Wang et al, 2021 ).…”

Imaging the brain in action: a motorized optical rotary joint for wide field fibroscopy in freely moving animals

“…In parallel with the development of miniaturized one-photon microscopes, efforts have been made to assemble mini-multiphoton microscopes that can be mounted on the head of freelymoving mice. However, in addition to electrical connections, these systems also require the use of an optical fiber to bring the excitation laser beam to the imaged surface, 28,29 and, for one of them, an image guide (tapered fiber-bundle) to collect the emitted photons. 29 These optical cables constitute an extra difficulty in allowing the mice to navigate and rotate freely in their environment.…”

Multi-beam miniaturized volumetric scanning microscopy with a single 1-dimensional actuation

“…Such endoscopes may be significantly more compact and can be integrated with a variety of imaging modalities that provide valuable functional, molecular or sub-surface structural information. Miniature optical imaging is also of great interest in neuroscience as head-mounted systems for real-time monitoring of brain activity [5,6] Miniature laser scanning mechanisms are typically achieved by microelectromechanical systems (MEMS) mirrors/actuators [7,8] or resonant fibre scanners [9][10][11][12]. While these mechanisms enable compact high speed laser scanners for endoscopy, high driving voltages and complex fabrication are required, thus limiting the affordability and accessibility of such devices.…”