Optics Express︱World’s First Millimeter Field-of-View Miniature Two-Photon Microscope Released




Recently, a research paper titled "Millimeter field-of-view miniature two-photon microscopy for brain imaging in freely moving mice" was published online by the team led by Cheng Heping and Wang Aimin from Peking University in the journal Optics Express. This paper has revealed the design principles and outstanding imaging performance of the FHIRM-LF miniature two-photon microscope, the latest release from Transcend Vivoscope.
This miniature two-photon microscope headpiece integrates features such as a low-magnification miniature objective lens, a large field-of-view scanning mirror, and a small MEMS tilt angle. It weighs only 2.5 grams and provides an imaging field of view of 1000×788 μm². This tool offers powerful observation capabilities for studying the dynamic functional properties of neuronal populations in the brains of freely moving small animals.
Zhao Chunzhu, assistant researcher at the College of Future Technology of Peking University, is the first author of the paper, while Wang Aimin and Wu Runlong, both from Peking University, are the co-corresponding authors of the paper.

Screenshot online

High-quality data within millimeter-scale field of view is readily available
A typical miniature single-photon microscope is renowned for its millimeter-scale field of view, while miniature two-photon microscopes excel in imaging resolution, depth, and sectioning capabilities. The latest FHIRM-LF miniature two-photon microscope achieves all these advantages. By designing a low-magnification objective lens and a large field-of-view scanning mirror, and reducing MEMS tilt angles, the FHIRM-LF achieves an imaging field of view of 1000×788 μm². Using this microscope headpiece, the authors obtained high-resolution images of neurons, dendrites, and dendritic spines in the mouse cortex within a 1000×788 μm² field of view (Figure 1).


Fig. 1 a) FHIRM-LF headpiece; b) image of neurons labeled with GCaMP6s in the cortex of an awake mouse with a 1000 × 788 µm² FOV; (c) Left, image of dendrites labeled with YFP in the superficial layer of the cortex of an awake Thy1-YFPH transgenic mouse with a 1000 × 788 µm² FOV; right, images of dendrites and spines labeled with YFP in the superficial layer of the cortex of an awake Thy1-YFPH transgenic mouse with a 550 × 433 µm² FOV.


Wide-field, high-quality imaging under GRIN Lens
It is well-known that the imaging quality can be significantly reduced due to aberrations introduced by the GRIN Lens in relayed microscopy. To test the performance of FHIRM-LF with GRIN lens, the authors conducted observations of freely moving mouse hippocampal neurons through GRIN Lens relayed imaging (Fig. 2). The imaging FOV is 1000x 788 um² and imaging frame rate is 9Hz at 600 x512 pixels. Due to vignetting by the GRIN lens itself, the effective FOV is about 900 x788 μm². Through imaging the dorsal CA1 region of the hippocampus in freely moving mice using the GRIN Lens, the FHIRM-LF imaging headpiece achieved high-quality collection of calcium transient data from approximately 400 neurons in total. The mean signal-to-noise ratio (SNR) of this raw data was 9.2, with 98% of neuronal calcium transient had a SNR greater than 3.

Fig. 2 Dorsal hippocampal CA1 imaging using FHIRM-LF via a GRIN Lens with a diameter of 1 mm relay in a freely moving mouse. Continuous imaging calcium traces recordings of freely moving mice were conducted for 18.3 minutes with FHIRM-LF headpiece. The figure displays segmented images and calcium traces of the recorded 395 neurons, along with the signal-to-noise ratios of these neuronal calcium transients.


Z scanning range up to 500 μm within a millimeter-scale field of view
Transend Vivoscope's miniature two-photon microscope system is equipped with an advanced axial zooming ETL module (Electrically Tunable Lens). By integrating the z-scanning module, this miniature two-photon microscope achieves simultaneous imaging and recording of neurons in different functional layers of the cerebral cortex. Combined with the detachable ETL module, the axial zoom range of FHIRM-LF headpiece extends to 500 μm, allowing for the collection of neuronal images in a volume of 1000 × 788 × 500 μm³ during imaging of the mouse cerebral cortex (Figure 3). Furthermore, even with the axial zooming ETL module, two-photon microscopy imaging maintains its axis-resolution capability close to the theoretical diffraction limit, ensuring consistent imaging resolution.

Fig. 3 3D volume imaging of 1000 × 788 × 500 µm³ in the cortex of an awake mouse by FHIRM-LF integrating with a detachable ZSM


Easy to use, easy to upgrade
FHIRM-LF, as one of the three latest miniature two-photon imaging headpieces recently introduced by Transcend Vivoscope, can be seamlessly interchanged with other imaging headpieces within the same system. It is also compatible with various series of Transcend Vivoscope’s miniature two-photon imaging systems. With its standardized design and a straightforward headpiece replacement process, upgrading becomes hassle-free, allowing users to expand more possibilities while retaining the excellent performance of the existing miniature two-photon microscope.

For Miniature two-photon microscope, SUPERNOVA-100, there are three headpiece options available: FHIRM-HR, FHIRM-U, FHIRM-LF.