低溫冷凍固定法於超解析顯微技術之效能評估與分析

Abstract

傳統光學顯微鏡受到繞射極限的限制，無法清晰觀察到 200 奈米以下的細微結構，電子顯微鏡(EM)及超解析顯微技術(SR)的出現突破了這個瓶頸，帶我們向下窺探奈米尺度的世界。近年來更是開發出冷凍電子顯微鏡(Cryo-EM)，解決了電子顯微鏡無法使用在生物樣本的困難。考慮到 Cryo-EM 的優異性，在初步的簡易實驗中，可以觀察到影像的品質在不同條件下呈現出不同的樣貌。為了進一步找到最佳的影像品質，我們參考 Cryo-EM 設備的運作程序，開發出簡易的操作機台，自動化的流程操作起來更為便利，減少了人為操作失誤的同時也能提高時間精度，對於實驗的可重複性極為重要。 最後，我們使用 Ex-dSTORM 來觀察超微結構上的影響。分析三組獨立實驗的統計結果，寬場顯微鏡(widefield microscopy)所拍攝的影像在冷凍時長 120 秒至 180 秒間獲得的影像平均亮度較高；實驗中我們也分析了每組的隨機光學重建顯微鏡(dSTORM)影像，注意到拉長冷凍時間的同時，會有螢光分子分布不均勻的現象。總結以上結果，我們認為 120 秒至 150 秒左右的冷凍時長能得到較亮的影像及較優的品質，是最適合拍攝單分子影像的條件。

Super-resolution microscopy circumvents the diffraction limit of light, about 200 nm, and produces images with enhanced resolution that well-surpassed the strength of traditional light microscopes. The imaging of subcellular architecture and detailed morphology used to frustrate the traditional light microscope is carried out effortlessly with super-resolution techniques. Recently, cryogenic electron microscopy (short as Cryo-EM) was brought to the world and received accolades for allowing electron microscopy to visualize biological specimens with preserved structural integrity. However, the connection between image quality and cryogenic condition has never been clearly clarified. In response, this research aims to explore the applicability of the cryo-method on single-molecule microscopy. Importantly, we disclose the relationship between the time length of cryogenic freezing (short as cryo-freezing) and the ultra-structure of cells and discover the optimal temporal condition for the optimal representation of cellular details. Time length of 120s to 180s cryo-freezing revealed higher average brightness in widefield microscopy. Taking it further to SMLM, the specific time length of 120s to 150s cryo-freezing outperforms other temporal conditions by brighter visual output and more saturated fluorophore distribution, making it an ideal condition for SMLM imaging to explore the subcellular details of intact cells. In addition, we developed a convenient and simplistic machine for automatic operation to bolster both the validity and reproducibility of the research with superior precision and accuracy.