Cell ushered in a qualitative leap!

A major goal of contemporary neuroscience at iNature is to identify the neural populations underlying complex mammalian brain function, a goal made possible by new techniques for large-scale neural recordings during behavior, optical imaging being one of these techniques
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For 20 years, researchers have been trying to develop tiny 2P microscopes -- 2P Microscopes -- that can be placed on the heads of freely moving rodents
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However, early 2P miniature microscopes faced significant challenges, including temporal dispersion in the excitation fiber, slow scanning speeds, heavy weight, and image distortion caused by inflexible fiber optic cables
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On March 18, 2022, Norwegian University of Science and Technology Edvard I.
Moser (2014 Nobel Laureate in Physiology or Medicine) and Zong Weijian published a joint communication titled "Large-scale two-photon calcium imaging in freely moving" in Cell Online mice" research paper, which developed a small two-photon microscope (MINI2P) for fast, high-resolution, multiplanar calcium imaging of more than 1,000 neurons at a time in freely moving mice
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With a microscope weight of less than 3 g and a highly flexible connecting cable, the MINI2P enables stable imaging in a variety of analyses without hindering its behavior compared to untethered, unimplanted animals
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Increased cell yield is achieved through an optical system design with an expanded field of view (FOV) and a micro-tunable lens with increased z-scan range and speed, allowing fast and stable imaging of multiple interleaved planes as well as 3D functional imaging
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Continuous imaging across multiple adjacent FOVs enables recordings from more than 10,000 neurons in the same animal
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Large-scale proof-of-principle data were obtained from cell populations in the visual cortex, medial entorhinal cortex, and hippocampus
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In addition, on January 6, 2021, Chen Liangyi, Wang Aimin and Wu Runlong of Peking University jointly communicated (Zhong Weijian and Wu Runlong, jointly trained by the Institute of Molecular Medicine of Peking University, are the first authors of the paper) published online in Nature Methods entitled "Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging” research paper, which developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long working distance miniature objective, capable of Multiplanar imaging was performed with a lateral resolution of ~1 μm over a volume of 420 × 420 × 180 μm
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Together with a detachable design that allows long-term repeat imaging, the study's miniature two-photon microscope could help decipher neuronal mechanisms in freely behaving animals (click to read)
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A major goal of contemporary neuroscience is to identify the neural populations underlying complex mammalian brain functions, a goal made possible by new techniques for large-scale neural recordings during behavior, optical imaging being one of these techniques
.

Genetically encoded indicators, such as GCaMPs, are used to optically monitor changes in intracellular free calcium, allowing simultaneous monitoring of the activity of large and broadly identifiable populations of individual neurons within the microscopic field of view (FOV)
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For imaging of behaving rodents, researchers rely on stationary benchtop two-photon (2P) microscopes, in which the animal performs tasks while its head is fixed under the objective
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Such methods are suboptimal for studying non-stationary behaviors, such as navigation, because they prevent the identification of cells that are inherently spatial during their firing
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So, for 20 years, researchers have been trying to develop tiny 2P microscopes -- the 2P Microscope -- that can be placed on the heads of freely moving rodents
.

However, early 2P miniature microscopes faced significant challenges, including temporal dispersion in the excitation fiber, slow scanning speeds, heavy weight, and image distortion caused by inflexible fiber optic cables
.

Conversely, calcium imaging in freely moving mice took off with the invention of the single-photon (1P) miniature microscope, in which the optical sectioning and 3D imaging capabilities of 2P excitation were sacrificed for size reduction and lightweight cable connections
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Using the 1P device, activity can be imaged at near-cellular resolution in sparsely active thin-layered brain regions or dense regions if the calcium indicator is only expressed in a small subset of neurons
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However, due to background fluorescence contamination, the inability to detect small calcium transients, and the lack of z-axis information, the 1P micromicroscope is not well suited for active and densely labeled areas
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These limitations have prompted attempts to develop a new generation of miniaturized 2P devices with similar resolution, speed, and z-scanning capabilities to 2P benchtop microscopes, as well as FOVs approaching that of 1P miniature microscopes
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Of particular interest is the development of a novel 2P microscopic microscope that includes (1) a hollow-core photonic crystal fiber (HC-920) for delivering 920 nm femtosecond laser pulses; (2) a fast microelectromechanical system ( MEMS) scanner for fast spot scanning; (3) soft fiber bundles for collecting fluorescence
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Schematic diagram of the article (image courtesy of Cell) The latest micromirrors have a FOV of over 400 × 400 μm2 and a 180 μm z-scan capability
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However, its weight (~5 g) and stiffness of the fiber optic bundle interfere with the animal's locomotion, impairing any experiment in which mice must navigate freely for extended periods of time
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Here, the study demonstrates a 2P micro-microscope, the MINI2P, that both increases cell yield by an order of magnitude and overcomes the limitations of previous versions by meeting the requirements for fatigue-free exploratory behavior without compromising the quality or stability of imaging
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In conclusion, this study designs a 2P miniature microscope (MINI2P) that can study neural activity in thousands of individually identifiable cells with high sampling rate and high spatial resolution and stability, while mice are exposed to various environments and Move freely in behavioral analysis
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By preserving the optical sectioning capability of 2P excitation, MINI2P can be used for calcium imaging in a variety of preparations with high signal-to-noise ratios, regardless of somatic or neuronal labeling density or neuronal activity levels
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Despite the added hardware, the MINI2P reduces head piece weight and increases flexibility due to the tapering of the collection fiber bundles, allowing animals to perform at the same level of flexibility as the 1P microscope
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The recordings remained stable over several weeks with minimal motion artifacts, even during strenuous behaviors such as climbing, jumping, and threat-induced escapes
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