自製梯度折射率透鏡固定器用以觀測活體小鼠中腦區鈣離子成像

Abstract

本研究聚焦於克服現有技術在觀測小鼠深層腦部影像時的挑戰，特別是在光學系統穩定性、重現性以及實驗流程簡化方面的困難。傳統的深層成像技術，雖然在橫向解析度與光路延伸上有所突破，但仍面臨樣本移動誤差導致無法觀測同一區域以及實驗耗時較長等問題。因此本研究設計並製作了一種自製GRIN rod holder與連接器，結合雙光子顯微鏡系統，目的是在快速、高效且穩定地對小鼠中腦黑質致密部（SNc）進行神經活動的觀測。 在系統性能測試中，使用標準螢光小球樣品進行解析度驗證，結果顯示本系統橫向解析度可穩定維持在1.40 ± 0.0025 微米。為了進一步驗證系統穩定性，我們設計了多種重現率測試，包括短時間內重接合、移動小鼠後的再連接影像分析，結果證明系統在這些情境下均能維持高準確性的影像重現，並且可以觀測到同一顆神經細胞。除此之外，利用活體小鼠的神經鈣離子指示劑（GCaMP6f），我們成功記錄了黑質致密部神經元的鈣離子濃度動態變化。 與其他文章內技術相比，本研究的新設計顯著縮短了實驗步驟以及所需時間，從原本的 30–60 分鐘減少至僅需 5–10 分鐘，極大提升了實驗效率，本系統不僅適用於觀測鈣離子濃度變化，亦具備延伸應用於記錄神經活動中的電位影像的潛力。 總而言之，本研究提出的雙光子顯微鏡結合梯度折射率透鏡系統，兼具高解析度、穩定性與操作便捷性，為未來神經科學研究中的深層腦部影像分析和動態活動觀測開啟了可能性。

This study focuses on overcoming the challenges in observing deep brain images of mice, particularly in terms of optical system stability, reproducibility, and the simplification of experimental procedures. While traditional deep imaging techniques have made breakthroughs in lateral resolution and optical path extension, they still face issues such as errors caused by sample movement, which prevent consistent observation of the same region, and prolonged experimental durations. Therefore, we designed and fabricated a custom-made GRIN rod holder and connector, combined with a two-photon microscope system, to achieve rapid, efficient, and stable observation of neural activity in the substantia nigra pars compacta (SNc) of the midbrain. In system performance testing, resolution verification using standard fluorescent microspheres demonstrated that the lateral resolution of the system could be stably maintained at 1.40 ± 0.0025 μm. To further validate system stability, we designed various reproducibility tests, including reassembly in a short time and reattachment after moving the mouse, confirming that the system could maintain highly accurate image reproduction under these conditions and observe the same neuron. Furthermore, using the neural calcium indicator GCaMP6f in live mice, we successfully recorded the dynamic changes in calcium ion concentrations in the neurons of the SNc. Compared to techniques discussed in other studies, the new design proposed in this study significantly reduces experimental steps and required time, from the original 30–60 minutes to just 5–10 minutes, greatly improving experimental efficiency. The system is not only suitable for observing calcium ion concentration changes but also has potential for extended applications in recording voltage images of neural activity. In summary, the two-photon microscope combined with a GRIN lens system proposed in this study offers high resolution, stability, and operational convenience, opening new possibilities for deep brain image analysis and dynamic activity observation in future neuroscience research.