Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)
利用纳米SIMS同位素成像和扫描电子显微镜（SEM）测量蓝藻生物膜中的代谢
发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2263 浏览次数: 9338
评审: Claudia CatalanottiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May/Jun 2016
Abstract
To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13C) EOM by two groups, cyanobacteria and associated heterotrophic microbes, are quantified. Methods for generating, preparing, and analyzing samples for quantifying uptake of stable isotope-labeled EOM in the biofilm are described.
Keywords: Stable isotopes (稳定同位素)
Extracellular matrix (细胞外基质)
Extracellular organic matter (细胞外有机物)
Microbial ecology (微生物生态学)
Substrate incorporation (底物掺入)
NanoSIP (NanoSIP)
Background
Stable isotope labeling combined with NanoSIMS (‘NanoSIP’) is an established method to quantify incorporation of stable isotope labeled substrates into individual microbial cells, which can then be extrapolated to estimate incorporation for a population of cells (for example, Lechene et al., 2006 and Woebken et al., 2012). Tracing multiple stable isotope labels (e.g., 13C and 15N) into individual cells can be used to examine differential incorporation between treatments over time (for example, Popa et al., 2007 and Stuart et al., 2016a). Biofilms present specific challenges to quantifying incorporation of label. Since individual organisms are embedded in an extracellular matrix and have a diverse range of cell sizes and shapes, cell counts and biomass calculations are difficult. Additionally, polymeric labeled substrates, such as EOM, can adhere to the matrix and cell surfaces, so unincorporated label needs to be accounted for. Imaging-based methods such as NanoSIMS, paired with SEM and fluorescence microscopy, are well-suited to address these challenges because cell sizes and unincorporated label can be identified. Here, we describe methods to address these challenges in order to quantify the incorporation of labels (13C and 15N) from a polymeric substrate (EOM) into a photosynthetic biofilm. EOM is extracellular material that is loosely associated with cells, and is separated from the cells in the biofilm. One drawback of this method is that biofilm spatial structure (the extracellular matrix) is not preserved. If the examination of spatial arrangements is desired, embedding and sectioning of the biofilm samples may be necessary (for example, Lechene et al., 2006).
Materials and Reagents
- Sterile cell scrapers (VWR, catalog number: 89260-224 or 89260-222 )
Note: The product “ 89260-224 ” has been discontinued. - 0.2 µm syringe filters (polyethersulfone membranes, e.g., Pall, catalog number: 4652 )
- Pipet tip
- 1.7 ml microcentrifuge tubes
- Sterile syringes
- Silicon (Si) wafers, sized to fit NanoSIMS holder (e.g., 5 x 5 mm, Ted Pella, catalog number: 16008 )
- Sterile tissue grinder (Fisher Scientific, catalog number: 02-542-08 )
- Biofilm culture
Note: We examined a unicyanobacterial mixed species biofilm, however, the protocol is suitable for analysis of most biofilm types, provided that an aqueous substrate can be added to the sample, with even distribution. A detailed description of our biofilms and culturing procedures can be found in Stuart et al., 2016a. - Stable isotope labeled compound appropriate to support growth, or metabolism, of organism(s) of interest (e.g., 99 atom percent excess [atm%] 13C-NaHCO3, Cambridge Isotope Laboratories, catalog number: CLM-441-1 )
- Appropriate defined medium for growth (e.g., modified artificial seawater base [ASN] medium, described in Stuart et al., 2016a)
- 50% ethanol (EtOH)
- 37% formaldehyde solution (Sigma-Aldrich, catalog number: 252549 )
- 10x phosphate buffered saline (Sigma-Aldrich, catalog number: P5493 )
- Sodium chloride (NaCl)
- Sterile MilliQ water
- 4% PFA (see Recipes)
- Sterile 10% sodium chloride (NaCl) solution (see Recipes)
- Modified artificial seawater base (ASN) medium recipe (with nitrate) (see Recipes)
Equipment
- Dounce homogenizer (WHEATON, catalog number: 357546 )
- Microcentrifuge, capable of variable rpm generating up to 15,000 x g
- Isotope-ratio mass spectrometer (e.g., ANCA-IRMS, PDZE Europa Limited, Crewe, England)
- Chambers for biofilm cultivation (e.g., Pyrex sealable flasks, Corning, PYREX®, catalog number: 4985-1L )
- Incubator (as appropriate for cultivating the organism of interest)
- Dissecting microscope (e.g., Fisher Scientific, model: Fisher ScientificTM 420 Series , catalog number: 11-350-124)
- Desiccator cabinet (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5317-0070 )
- Epifluorescence light microscope (e.g., Leica Microsystems, model: Leica DMI600B )
- Diamond or carbide scribe (e.g., Ted Pella, catalog number: 54412 )
- Gold (or other conductive metal) sputter coater (e.g., Cressington, model: Sputter Coater 108 )
- NanoSIMS 50 or 50 L (AMETEK, Cameca, model: NanoSIMS 50 or NanoSIMS 50 L )
- Scanning electron microscope (SEM) with better than 50 nm resolution and micrograph recording capability (e.g., FEI, model: FEI Inspect F SEM )
Software
- Ion image data processing software (e.g., LIMAGE, L. Nittler, Carnegie Institution of Washington, Washington, DC, USA; ImageJ with MIMS plugin; Look@NanoSIMS [Polerecky et al., 2012] or similar)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Stuart, R. K., Mayali, X., Thelen, M. P., Pett-Ridge, J. and Weber, P. K. (2017). Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM). Bio-protocol 7(9): e2263. DOI: 10.21769/BioProtoc.2263.
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分类
微生物学 > 微生物新陈代谢 > 其它化合物
细胞生物学 > 细胞成像 > 电子显微镜
生物化学 > 其它化合物 > 碳酸氢盐
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