Two-photon microscopy (TPM) enables the observation of cellular and subcellular dynamics and functions in deep nervous tissues, providing critical in situ and in vivo information for understanding neurological mechanisms.  

However, conventional TPM retains cellular resolution imaging over only a restricted field-of-view (FOV) (usually 0.5 × 0.5 mm², depending on the optical system), impeding the simultaneous visualization of large area phenomena such as the multi-region neuronal activity in mammalian brain.  

Recently, several TPM systems have been reported with custom design objectives to achieve large FOV with high-resolution imaging. However, these systems entail a sophisticated design and assembly of customized optical components, which significantly limits their acceptance and use by biologists. A commercial objective with a high numerical aperture (NA) that could provide a large FOV would be desirable, such that a standard TPM system could be used without significant changes.  

In a study published in the journal Optics Letters on Feb. 15, Prof. ZHENG Wei’s group from the Shenzhen Institute of Advanced Technology (SIAT) of the Chinese Academy of Sciences reported a novel method to extend the FOV of objectives and achieve high-resolution and large-FOV two-photon imaging. 

"We proposed a simple and effective method to extend the FOV of a commercial objective using adaptive optics (AO)," said Prof. YE Shiwei, the co-first author of this study, "this method enabled an imaging FOV diameter up to 3.46 mm wide while maintaining a lateral resolution of ~840 nm by using a commercial objective with a nominal FOV diameter of 1.8 mm."

The nominal FOV of objectives is recommended by the objective vendor and reflects the maximum imaging area where the optical aberration is considerably corrected.  

The team found that the incident light still can reach the area outside the nominal FOV of objectives (the extended FOV). However, when the signals from the extended FOV are used for imaging, the images are extremely blurred and distorted. This is because the large aberrations will lead to distorted point spread function and poor imaging quality. Furthermore, to utilize this property of objectives, they proposed and implemented a segment-scanning sensorless adaptive optics method to correct the large aberrations and extend the FOV of commercial objectives.  

Using this technology, the researchers clearly observed the cerebral nerve cells of Thy1-GFP-M mice brain slice, which revealed almost 1/4 of the entire mouse brain area. Additionally, in vivo imaging of the large-scale mouse cerebral microglia and vasculature were also presented. 

"The proposed method is compatible with any scanning optical microscope and does not require sophisticated design or assembly of customized optical components. It is believed that this work will motivate large-scale exploration of brain networks and neural phenomena," said Prof. ZHENG.  

Figure. (a) Schematic diagram of the proposed method. (b) Large FOV imaging of the Thy1-GFP-M mice brain slice. (Image by SIAT)

Media Contact:
ZHANG Xiaomin
Email:xm.zhang@siat.ac.cn