Large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging

Research Background and Problem Statement

Multiphoton microscopy (MPM) is a powerful tool for deep tissue imaging, especially in the study of brain function in vivo. However, while traditional two-photon microscopy (2PM) can achieve a larger imaging field of view (FOV), its imaging depth is usually limited to superficial cortical areas and cannot penetrate into deeper brain structures. Meanwhile, although three-photon microscopy (3PM) can image at greater depths, thermal damage limits laser repetition rates, resulting in smaller FOVs and lower imaging throughput. Therefore, achieving large FOV (LFOV) and deep imaging while maintaining high resolution has become an urgent problem to be solved in the field of multiphoton microscopy.

To address this issue, Aaron T. Mok et al. developed a new multiphoton microscopy system—DeepScope—that optimizes fluorescence signal generation efficiency through a series of innovative techniques, enabling LFOV and single-cell resolution deep imaging. This research aims to break through the technical bottlenecks of traditional multiphoton microscopes and provide new tools for system-level neural circuit research.

Source of Paper and Author Information

This paper was written by researchers including Aaron T. Mok, Tianyu Wang, and Chris Xu. The first author, Aaron T. Mok, and the corresponding author, Chris Xu, are both from the School of Applied and Engineering Physics at Cornell University, USA. Other authors are from renowned institutions such as Boston University, Harvard University, and MIT. The paper was published in the open-access journal eLight in 2024, with the title “A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging.”

Research Methods and Experimental Design

a) Research Process and Experimental Details

1. Development of the DeepScope System

DeepScope is a dual excitation adaptive polygon-scanning multiphoton microscope (Dual Excitation with Adaptive Excitation Polygon-Scanning Multiphoton Microscope). Its core innovations include the following: - Adaptive Excitation: Utilizes electro-optic modulators (EOMs) to dynamically adjust laser power, reducing power in vascular shadow regions and thereby increasing effective power in regions of interest. - Beamlet Scanning Scheme: Uses a beamlet delay line to split a single laser pulse into two beamlets with a time interval of approximately 20 nanoseconds, effectively increasing the laser repetition rate and enhancing scanning speed. - Polygon Scanner: Employs a large-aperture (9.5 mm) polygon scanner, achieving a line scan rate of up to 6 kHz, significantly surpassing traditional galvanometer scanners.

2. Experimental Subjects and Sample Processing

The study primarily used transgenic mice and adult zebrafish as experimental subjects. During the experiments, chronic cranial window surgery was performed on the mice, and neurons were labeled using the GCaMP6s calcium indicator. For zebrafish, whole-brain imaging was conducted after anesthesia and fixation.

3. Experimental Procedures and Test Content

• Deep Imaging of Mouse Brain: Structural and functional imaging of Layer 6 (L6) and the Cornu Ammonis 1 (CA1) region of the hippocampus in the mouse brain was performed using the DeepScope system to verify the system’s deep imaging capabilities.
• Simultaneous Two-Photon and Three-Photon Imaging: Two-photon and three-photon imaging of superficial and deep cortical regions within the same field of view was conducted to demonstrate the system’s versatility.
• Whole-Brain Imaging of Zebrafish: Whole-brain structural imaging of adult zebrafish was performed to further validate the system’s wide-field imaging capabilities.

4. Data Analysis Algorithms

The researchers developed a set of MATLAB-based image processing scripts to separate two-photon and three-photon signals. They also used Suite2P software for motion correction, neuron segmentation, and fluorescence signal extraction of calcium activity data.

b) Main Results and Data Analysis

1. Deep Imaging of Mouse Brain

DeepScope successfully achieved large FOV imaging with a diameter of 3.5 mm, covering the deepest cortical regions of the mouse brain. Experimental data showed that at a depth of 600 microns, the system could record spontaneous activities of 917 neurons at a frame rate of 4 Hz. Additionally, imaging of the CA1 region of the hippocampus verified the system’s ability to image subcortical areas.

2. Simultaneous Two-Photon and Three-Photon Imaging

Experiments demonstrated that DeepScope could simultaneously record neural activities in superficial and deep cortical regions within the same field of view. For example, within the depth range of 320 to 600 microns, the system recorded calcium activities of 4,523 neurons at a frame rate of 11 Hz.

3. Whole-Brain Imaging of Zebrafish

DeepScope also demonstrated the ability to perform whole-brain structural imaging of adult zebrafish, with imaging depths exceeding 1 mm and FOVs greater than 3 mm. The results clearly displayed GCaMP6s-labeled nuclei in the telencephalon, optic tectum, and cerebellar regions, as well as third harmonic generation (THG) signals of bone structures and fiber tracts.

c) Research Conclusions and Value

The successful development of DeepScope provides new solutions for multiphoton microscopy, with its main contributions including: - Scientific Value: Achieving LFOV and single-cell resolution deep imaging, breaking through the technical limitations of traditional multiphoton microscopes. - Application Value: Can be widely applied in neuroscience, immunology, oncology, and other fields, providing essential tools for system-level neural circuit research and disease model analysis.

d) Research Highlights

• Technological Innovation: Adaptive excitation and beamlet scanning schemes significantly enhance fluorescence signal generation efficiency.
• Versatility: Supports simultaneous two-photon and three-photon imaging, meeting research needs for different depth regions.
• Wide-Field Imaging Capability: First achieved high-resolution whole-brain structural imaging of adult zebrafish.

e) Other Valuable Information

The design of DeepScope is simple and compact, making it easy to integrate into existing multiphoton microscopes, providing a practical solution for biomedical research laboratories.

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Summary and Significance

Aaron T. Mok et al.’s research not only addresses key issues in multiphoton microscopy but also provides powerful tools for future neuroscience research. The development of DeepScope marks a new phase in multiphoton microscopy, with its innovation and practicality making it a milestone achievement in the field.