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Millimeter field of view miniaturized two-photon microscope released
Date: 2025-05-28Read: 1
On September 20, 2023, the team led by Cheng Heping and Wang Aimin from Peking University published a research paper titled "Millimeter field of view miniature 2-photon microscopy for brain imaging in freely moving mice" online in the academic journal Optics Express, revealing the design principle and imaging performance of the latest millimeter imaging field of view miniaturized two-photon microscope FHIRM-LF for ultra dimensional imaging. This miniaturized two-photon microscope probe, which integrates low magnification micro objectives, large field of view scanning mirrors, and small MEMS tilt angles, weighs only 2.5 grams and has an imaging field of view of 1000 × 788 μ m2. It provides a powerful observation tool for studying the dynamic function of brain neuron populations in the free behavior paradigm of small animals. Zhao Chunzhu, an assistant researcher at the School of Future Technology at Peking University, is the first author of the paper, while Wang Aimin and Wu Runlong from Peking University are the co corresponding authors of the paper.
Screenshot of the online publication of the paper
High quality data is readily available in millimeter level field of view
A typical miniaturized single photon microscope is known for its millimeter level field of view, while a miniaturized two-photon microscope has more advantages in imaging resolution, depth, and slicing ability. The latest millimeter level field of view miniaturized two-photon microscope FHIRM-LF from Super Vision has achieved the fusion of the advantages of the two. By designing a low magnification objective lens, a large field of view scanning mirror, and reducing the MEMS tilt angle, FHIRM-LF achieved 1000 × 788 μ m2The imaging field of view. Using this microscope probe, the author measured 1000 × 788 μ m2High resolution images of neurons, dendrites, and dendritic spines in the mouse cerebral cortex were observed under the field of view (Figure 1).
Figure 1. a) FHIRM-LF probe image; b) At 1000 × 788 μ m2Imaging of GCaMP6s expression neurons in the cerebral cortex of conscious mice under visual field；c) Imaging of cortical dendrites and dendritic spines in Thy1 YFPH transgenic mice with wakefulness.
High quality imaging with large field of view under GRIN Lens
In microscopy imaging relayed through gradient refractive index lenses (GRIN lenses), the aberrations introduced by GRIN lenses can greatly reduce the imaging quality. To test the effectiveness of the FHIRM-LF probe under GRIN Lens, the authors observed hippocampal neurons in freely moving mice using GRIN Lens relay imaging (Figure 2). Under the conditions of a field of view of 1000 × 788 μ m2, an imaging frame rate of 9 Hz, and 600 × 512 pixels, although the imaging field of view is slightly affected by the vignetting effect of GRIN Lens itself, its effective field of view still reaches about 900 × 788 μ m2. By using GRIN Lens to image and record the CA1 subregion on the dorsal side of the hippocampus in freely moving mice, the FHIRM-LF imaging probe achieved high-quality acquisition of approximately 400 neuronal calcium transient data. The average signal-to-noise ratio of these raw data was 9.2, and 98% of neurons had a calcium transient signal-to-noise ratio greater than 3.
Figure 2. Relay imaging of freely moving mouse hippocampal dorsal CA1 using a GRIN Lens with a diameter of 1mm. Using FHIRM-LF for continuous 18.3 minute imaging recording of freely moving mice, the figure shows the segmentation maps of 395 neurons recordedCa2+Trace and display the signal-to-noise ratio of calcium transients in these neurons.
Free zoom up to 500 μ m deep in millimeter level field of view
The ultra dimensional miniaturized two-photon microscope system is equipped with an advanced axial zoom ETL module. Using the principle of liquid lens, a miniaturized two-photon microscope is used to simultaneously image and record neurons in different functional layers of the cerebral cortex. Combined with a detachable zoom ETL module, the millimeter field of view probe FHIRM-LF expands the axial zoom range to 500 μ m, achieving the acquisition of 1000 × 788 × 500 μ m in imaging the mouse cerebral cortex3Neuronal image of volume (Figure 3). And after being equipped with a zoom ETL module, two-photon microscopy imaging still maintains a constant imaging resolution with the ability to approach the theoretical diffraction limit.
Figure 3. FHIRM-LF combined with detachable zoom ETL module for clear mouse cortex 1000 × 788 × 500 μ m3Three dimensional volume imaging.