Xi'an Institute of Optics and Fine Mechanics: New progress in large field two-photon scattering microscopy imaging technology

Adaptive optics is a technique that improves imaging quality by correcting wavefront distortion. Interference focus sensing (IFS), as a new method proposed in the field of adaptive optics in recent years, has been proven to have significant effects in correcting complex aberrations in deep tissue imaging. This technology is based on measuring a single location within the sample to determine the calibration mode. This article proposes an image-based interferometric focal sensing method (IBIFS), which uses conjugate adaptive optics configuration and feedback information from image quality indicators to progressively estimate and correct the wavefront throughout the entire field of view. The sample conjugate configuration achieves synchronous correction of multiple points within the entire field of view by measuring each position point by point and correcting the mode. We conducted experimental verification of the method using fluorescent microspheres and mouse brain slices as samples on our independently built two-photon microscope system. The results indicate that compared with methods based on regions of interest, this method not only has a larger effective field of view, but also achieves more stable optimization effects.

Recently, the research team led by Dr. Yao Baoli from the National Key Laboratory of Ultrafast Optics Science and Technology at the Xi'an Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, made progress in the field of large field two-photon scattering microscopy imaging. The related research results were published in Nanophotonics.

The most common AO method in the field of two-photon microscopy imaging is the Zernike mode decomposition method, which has a good effect on compensating for weaker aberrations. However, due to the limitations of the optical memory effect range, phase correction is only effective for a small field of view.

In response to the above issues, the research team proposed a large field wavefront correction method for deep tissue microscopy imaging - image-based interferometric focal sensing wavefront correction method (Figure 1). This method utilizes full field image information evaluation parameters as inputs for the interferometric focus induction method, achieving more stable correction effects while exhibiting high stability and anti-interference characteristics.

Figure 1. Schematic diagram of image-based interferometric focus sensing (IBIFS) method

In the resonance scanning galvanometer two-photon excitation fluorescence microscopy imaging system, researchers first performed large field wavefront correction on the fluorescent ball sample under the scatterer (Figure 2). The experimental results showed that the ROI based method only had good correction effect on the field of view near the reference point B1, while the IBIFS method (MHF based) can adjust the correction phase by using the image information feedback of the entire field of view, which has the correction effect of the entire field of view.

In the scattering correction experiment of mouse brain nerve slice samples, the experimental results (Figure 3) showed that the ROI based correction effect depends on the sample structure distribution in the reference area, with better local optimization effect and poorer global optimization effect. The total intensity enhancement factor of the image corrected by the IBIFS method is 37% higher than that based on small area signals, achieving more stable large field of view correction. This technology can be applied to high-speed resonance scanning two-photon microscopy, providing enhanced microscopy imaging tools for fields such as neuroscience and developmental biology.

Figure 3. Scattering correction experiment results of mouse brain slice samples

The research is supported by the National Natural Science Foundation of China's National Major Scientific Instrument Development Project, National Key R&D Program, and Shaanxi Province's Key Industrial Chain Project.
The first author of the paper is Yang Ruiwen, a doctoral student from Xi'an Institute of Optics and Fine Mechanics in 2021. The corresponding authors are Researcher Yao Baoli and Senior Experimenter Yang Yanlong. Xi'an Institute of Optics and Fine Mechanics is the first completion unit and the corresponding unit.

Source: opticsky