cryo-FIB lift-out 新技術(shù)-- Kleindiek

低溫纖維提離技術(shù)可以在秀麗隱桿線蟲組織中實現(xiàn)分子級別分辨率的低溫電子斷層掃描，這一成果由馬克斯·普朗克生物化學(xué)研究所Juergen M. Plitzko和Miroslava Schaffer牽頭的聯(lián)合團隊取得。相關(guān)論文發(fā)表在2019年8月2日出版的《自然—方法學(xué)》上。

課題組人員描述了一種制備方法，該方法基于使用低溫夾具工具從高壓冷凍大塊樣品中有針對性地提取材料。這種提離技術(shù)使低溫電子斷層掃描可用于多細胞生物和組織，擴大了原位結(jié)構(gòu)生物學(xué)的應(yīng)用范圍。研究組開展了對秀麗隱桿線蟲（Caenorhabditis elegans）蠕蟲中細胞溶質(zhì)80S核糖體的結(jié)構(gòu)研究，從而證明了該提離技術(shù)的潛力。制備質(zhì)量允許亞圖分析，具有足夠的分辨率來區(qū)分單個核糖體易位狀態(tài)，并顯示核糖體結(jié)構(gòu)中細胞間的顯著變化。

據(jù)了解，低溫聚焦離子束銑削冷凍水化細胞技術(shù)，近為細胞內(nèi)部空間的研究提供了視角。結(jié)合低溫電子層析技術(shù)，這種方法可以進入細胞內(nèi)部天然結(jié)構(gòu)，從而實現(xiàn)對大分子的原位結(jié)構(gòu)研究。然而，這種方法主要局限于可以被*玻璃化冷凍保存的單個細胞。

附：英文原文

Title: A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue

Author: Miroslava Schaffer, Stefan Pfeffer, Julia Mahamid, Stephan Kleindiek, Tim Laugks, Sahradha Albert, Benjamin D. Engel, Andreas Rummel, Andrew J. Smith, Wolfgang Baumeister, Juergen M. Plitzko

Issue&Volume:  Volume 16 Issue 8

Abstract: Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

DOI: 10.1038/s41592-019-0497-5

 

期刊信息

Nature Methods：《自然—方法學(xué)》，創(chuàng)刊于2004年。隸屬于施普林格·自然出版集團，IF：28.467