Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning

Abstract

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing

“…And finally, we applied this method to several relevant in-vivo dynamically varying samples such as cells, muscle mitochondrial activity, intravital imaging of blood flow in the skull, and neural firing in the barrel cortex. Liquid lens technology has recently attracted attention for applications such as headmountable brain neural activity measurement [39], or endoscopic imaging by providing both lateral [40] and axial [41,42] scanning. Due to their high potential for low cost and stability, many different methods of producing liquid lenses have been proposed.…”

Rodent models for psychiatric disorders: problems and promises

“…Thus, to observe the changes in detailed compartments of cells, further computational process is essential to reduce the noise in the data. To overcome the limitation of this head-mount single-photon micro-endoscopy, along with the development of two-photon head-mountable microscopes [36] new two-photon table-top microscopic approaches have developed to measure animal behavioral navigation with 3D virtual reality environments. For example, a 3D virtual reality behavioral platform for open field can provide a scene that can be changed by the paws' movements of awake and headfixed rodents to two-photon table-top microscope [37,38].…”

A minimally invasive lens-free computational microendoscope

“…A marked benefit of the lensless microendoscope system presented is the ability to computationally refocus on objects that are positioned at different depths without any actuated components and using only a single camera frame. Conventionally, optical endoscopes with depth-scanning capabilities require components capable of physically varying the focal plane, such as electrically tunable lens, which makes brain mounting of freely moving animals difficult due to increased distal footprint and weight (23)(24)(25)(26). In stark contrast to these bulky approaches, we can simply calibrate the system responses at different depths and reconstruct the scene volumetrically without actuation from a single camera snapshot.…”