Miniature three-photon microscopy maximized for scattered fluorescence collection

Abstract

In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.

Full text

Nature Methods | Volume 20 | April 2023 | 617–622 617
nature methods
https://doi.org/10.1038/s41592-023-01777-3
Article
Miniature three-photon microscopy
maximized for scattered fluorescence
collection
Chunzhu Zhao 1,10 , Shiyuan Chen1,10, Lifeng Zhang2,10, Dong Zhang3,
Runlong Wu1, Yanhui Hu4, Fengqingyang Zeng5, Yijun Li4, Dakun Wu6, Fei Yu6,7,
Yunfeng Zhang8, Jue Zhang3,5, Liangyi Chen1, Aimin Wang 8,9 &
Heping Cheng 1,2
In deep-tissue multiphoton microscopy, diusion and scattering of
uorescent photons, rather than ballistic emanation from the focal point,
have been a confounding factor. Here we report on a 2.17-g miniature
three-photon microscope (m3PM) with a conguration that maximizes
uorescence collection when imaging in highly scattering regimes. We
demonstrate its capability by imaging calcium activity throughout the
entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser
power. It also enables the detection of sensorimotor behavior-correlated
activities of layer 6 neurons in the posterior parietal cortex in freely moving
mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging
allows the study of neural mechanisms in deep cortex and subcortical
structures, like the dorsal hippocampus and dorsal striatum, in freely
behaving animals.
Advances in miniaturized multiphoton microscopy have enabled
high-resolution brain imaging in freely behaving rodents that are
engaged in self-determined behaviors
1–7
. For instance, using a miniature
two-photon microscope (m2PM) that is capable of volumetric imaging
at single-spine and single-neuron resolutions
3,4,6,7
, researchers have
mapped the functional network topography of the medial entorhinal
cortex
8
, deciphered microcircuit dynamics in the dorsomedial prefron-
tal cortex during social competition9, and unraveled itch-signal process-
ing in the primary somatosensory cortex
10
. However, minimally invasive
m2PM brain imaging is limited to the upper cortical layers in rodents.
Owing to reduced scattering at longer laser wavelengths and improved
signal-to-background contrast due to its fifth-order nonlinear effect,
three-photon excitation (3PE) can substantially increase penetration
depth11–14. Benchtop three-photon microscopes can record neuronal
activity in the intact brain, with single-cell resolution in the mouse
hippocampus12. A miniature three-photon microscope (m3PM) has
been developed, which has attained stable imaging of layer 5 neuronal
activity at >1.1 mm depth in the visual cortex of freely moving rats
5
.
The laser power required (100 mW at a wavelength of 1,320 nm after
the objective) was close to the optical-damage threshold
15
. However,
for smaller animals, like mice, the headpiece is too heavy (5 g), and the
miniature objective is too large (7 mm in diameter). Moreover, in mice,
Received: 1 February 2022
Accepted: 13 January 2023
Published online: 23 February 2023
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1National Biomedical Imaging Center, State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life
Sciences, College of Future Technology, Peking University, Beijing, China. 2Research Unit of Mitochondria in Brain Diseases, Chinese Academy of Medical
Sciences, PKU-Nanjing Institute of Translational Medicine, Nanjing Raygen Health, Nanjing, China. 3Academy of Advanced Interdisciplinary Study, Peking
University, Beijing, China. 4Beijing Transcend Vivoscope Biotech, Beijing, China. 5College of Engineering, Peking University, Beijing, China. 6Hangzhou
Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China. 7Key Laboratory of Materials for High Power Laser, Shanghai
Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai, China. 8School of Electronics, Peking University, Beijing, China. 9State
Key Laboratory of Advanced Optical Communication System and Networks, Peking University, Beijing, China. 10These authors contributed equally:
Chunzhu Zhao, Shiyuan Chen, Lifeng Zhang. e-mail: czzhao@pku.edu.cn; wangaimin@pku.edu.cn; chengp@pku.edu.cn
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