Two-Photon Image-Guided Automated 3D Locating of Micropipette Tip

The 21st century has been universally recognized by the scientific community as the era of neuroscience, with the patch clamp technique serving as a key to unraveling the intricate mysteries of brain science. Due to its advantages such as reduced sample damage and enhanced penetration capability, the application of two-photon microscope in deep imaging of live animals has become prevalent, making it a preferred choice over confocal microscope. At present, there exist a few near complete automation techniques of image-guided patching in vivo, which typically start from the micropipette tip appearing in the imaging field of view. The operational workflow for the step involving the installation of the micropipette until the micropipette tip becomes visible within the field of view has regrettably not been provided. However, it is crucial information that is of utmost importance to experimental personnel. In this paper, we present a comprehensive workflow for 3D locating of micropipette tip in a self-built two-photon microscope tailored for patch clamp experiments. Moreover, by fully incorporating the distinctive features of two-photon imaging, the study applies statistical methods to achieve automated execution of the corresponding workflow. The experimental results indicate that the process of micropipette tip locating takes an average of 58.4 seconds. Furthermore, the method is not sensitive to parameter selection, allowing experimenters to obtain suitable parameters for different systems through simple testing. The automated 3D micropipette tip locating method proposed in this paper, with its versatility, holds great potential for seamless integration into commercial two-photon microscope systems. It significantly contributes to reducing the work burden on experimenters and minimizing the time required for experimental preparation.