Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils
Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils
复制标题
DOI：
10.1093/femsec/fiv106
复制
发表时间：
2015-10-01
影响因子：
4.2
通讯作者：
Woebken, Dagmar
中科院分区：
文献类型：
--
作者：
Eichorst, Stephanie A.;Strasser, Florian;Woebken, Dagmar
将环境样品与稳定的同位素标记底物一起孵育，然后通过高分辨率二次离子质谱（NanoSIMS）或拉曼显微光谱进行单细胞分析的组合方法提供了对微生物原位功能的深入了解。这种方法在土壤中的应用有限，可能是由于微生物细胞分散在大的颗粒背景中。我们开发了一种管道，用于从土壤中有效制备细胞提取物，用于随后的单细胞方法，通过将细胞分离与细胞和土壤颗粒分离结合，然后进行细胞浓缩。该程序进行了评估，通过检查其对细胞回收率和微生物群落组成在两种土壤的影响。这种方法产生的细胞级分具有显著降低的土壤颗粒负载和足够小的尺寸，以允许通过NanoSIMS进行单细胞分析，如分别通过N-15（2）和C-13-UL-纤维素孵育检测活性N-2固定和纤维素响应微生物时所示。同样的程序也适用于土壤微生物的拉曼显微光谱分析，通过与C-13标记的碳源和氧化氘（D2 O，一般活动标记）的微宇宙孵育进行评估。所描述的样品制备程序能够使用NanoSIMS和拉曼显微光谱对土壤微生物进行单细胞分析，但也应该有利于单细胞分选和测序。
The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N-2-fixing and cellulose-responsive microorganisms via N-15(2) and C-13-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a C-13-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.