NanoSIMS - A method to study soil-plant-microbial interactions Email: daniel.murphy@uwa.edu.au NanoSIMS - A method to study soil-plant-microbial interactions Nano-scale secondary ion mass spectrometry (NanoSIMS) Daniel Murphy Chair in Soil Science (土壤学科带头人) Australian Research Council - Fellow The University of Western Australia Guest Professor (客座外聘教授) Chinese Academy of Agricultural Sciences

SEM showing bacteria on the surface of root Limitation of standard IRMS Traditional Isotope Ratio Mass Spectrometry (IRMS) requires a sample size (mg plant or soil) that does not enable micro-scale separation of isotope signals from individual soil minerals, plant cells or microbes. The structural arrangement between soil, plant and microbes is lost. 传统的同位素质谱仪所要求的样品尺寸（毫克植物或土壤），无法分离得到土壤矿物质、植物细胞或微生物各自的同位素信号。 Limitation of IRMS: In an IRMS sample the isotope signal is from both plant + microbe. 2 µm SEM showing bacteria on the surface of root 磨过的样品的同位素信号来自“植物+微生物”这个整体。

Why is micro-scale imaging important? A spoon of soil contains many million microbes. They have organized structure. plant root bacteria fungi nematode archaea Simultaneous analysis of identity (who are they?) and function (what are they doing?) of microbial populations with consideration for maintaining the soil structure (where are they located?) is one of our research interests. NanoSIMS is an instrument that works at a suitable scale for isotope studies.

Micro-scale interactions Our research interest is to: 植物-土壤-微生物相互关系 Describe spatial arrangement and interaction of soil minerals, plant roots, organic matter and microbial cells. 描述土壤矿物质、植物根系、土壤有机质以及微生物细胞的空间布局 Quantify carbon and nutrient flow and competition in the rhizosphere. 定量研究根际碳、氮和磷元素的流动 Understand soil-plant-microbial function ecosystem services. 理解“土壤-植物-微生物”功能 Micro scale Plot scale Catchment scale

Micro-scale interactions However: Soil-plant-microbial interactions occur at a scale smaller (micro- to nano-meters) than many methods of assessment. “土壤-植物-微生物”的相互作用在较小的尺度（纳米到微米范围）进行，常规方法很难观测 Many methods destroy the spatial arrangement in soil between biology, chemistry & physics. 很多实验方法会破坏土壤中生物、化学和物理性质的空间布局 Quantifying while simultaneously visualizing these interactions is therefore challenging. 定量并且可视化“土壤-植物-微生物”的相互作用是一项挑战

Biochemical processes occur at a micro-scale smaller than many methods NanoSIMS: Micro-scale interactions Biochemical processes occur at a micro-scale smaller than many methods Process Physical scale Method Gas fluxes m3 or hectares Greenhouse gas monitoring cm3 or grams soil SOM fractionation Microbial biomass Suction cups mm or <1 gram soil Inorganic nutrients Roots Micro-samplers Aggregates Molecular assays Faunal grazing Sand µm CAT scanning Rhizosphere Silt Mass spectrometer Clay Microbial growth Imaging 14C nm Microscopy Organic matter turnover Nutrient cycling NanoSIMS Herrmann et al. (2007) Soil Biology & Biochemistry 39: 1835-1850.

Nano-Secondary Ion Mass Spectrometry (纳米质谱仪简介) NanoSIMS is an Ion Microprobe linked to a Mass Spectrometer. We are using this to study Soil-Plant-Microbe Interactions at 100 nm resolution. NanoSIMS技术通过将离子微探针连接到质谱仪，来研究在100纳米解析度上的“土壤-植物-微生物”相互作用。

NanoSIMS (纳米质谱仪简介) NanoSIMS is an ion microprobe linked to a mass spectrometer. Element and isotopic analysis can be image mapped (100 nanometers). Concentrations of parts per million can be detected. mass analyzer magnet Electrostatic sector primary ion source focal plane primary ions secondary ions primary electrons collection & processing secondary electrons Herrmann A.M. et al. (2007) Nano-scale secondary ion mass spectrometry – A new analytical tool in biogeochemistry and soil ecology: A review article. Soil Biology and Biochemistry, 39, 1835-1850.

NanoSIMS (纳米质谱仪简介) Benefits: Stable isotope tracer measurements Isotopic mapping at the ~100 nm scale Solid materials including biological samples Imaging isotopic tracers (e.g., 13C, 15N) in biological materials at sub-cellular level Stable isotope tracer measurements 13C, 15N, 18O, 34S, 2D …….etc Uptake and assimilation in biological systems, in situ. Hybridisation experiments, microbial tagging. Elemental mapping Co-location of elements in complex systems. For example soil, rhizosphere. Isotopic mapping at parts per million 13C/12C in organic materials. 15N/14N uptake in microbial and plant cells. 18O tracing for P18O4 Herrmann et al. (2007) Soil Biology & Biochemistry

Why use NanoSIMS? (为何使用纳米质谱仪) Full periodic table H-U ppm detection 5-7 atomic mass units measured simultaneously This enables biological studies of for example 12C, 13C, 14N, 15N, 16O, 18O Very good separation of similar atomic mass units. 相似原子物质单位实现较好的分离 J.-L. Guerquin-Kern et al. 2005 Biochimica et Biophysica Acta 1724:228-238

NanoSIMS - Types of data collection 纳米质谱仪–收集数据的类型 Sample under vacuum Ion sources Primary ion column Secondary ion Collection Optics Energy Analyser Detector Mass Analyser Imaging Depth profile Mass spectrum Isotopic ratios Line scan 18 16 O/ 17 O 扫描 同位素比例 质谱 层次 绘图

Why use NanoSIMS? Mass spectrometer data is from each pixel 可从每一质谱像素中获取数据 Colour bar = 15N/14N Within the NanoSIMS image each pixel contains mass spectrometer data (256 x 256 pixels). Red = 15N/14N ratio in microbial cells Blue = Silica (mass 28) = Quartz soil Green = Carbon (mass 12) = same location on the 2 images: Top image = 15/14N ratio in bacteria Bottom image illustrates the location of the bacteria in the soil matrix alongside Silica (28Si) to identify quartz and 12C to identify carbon. 256 x 256 pixels = 65,536 data points per image 在纳米质谱仪图像中，每一像素都包括质谱数据（256×256像素）

纳米质谱图像 Mass spectrometer data is from each pixel 可从每一质谱像素中获取数据 Superimposed NanoSIMS images where Blue = 28Si-, Green = 12C14N- (represents organic matter) and Red = 15/14N ratio of 15N enriched P. fluorescens 蓝： 28Si- 红 ： 15N /14N 绿： 12C14N-（表示有机质） Herrmann et al. (2007) Soil Biology & Biochemistry 39, 1835-1850

Line-scan of 15N/14N across bacterial cells Why use NanoSIMS? Isotope data can be extracted from line scans 同位素数据可以从每一像素提取得到 Line-scan of 15N/14N across bacterial cells SEM image SEM image NanoSIMS Microns TEM image NanoSIMS 2 micron Clode et al. (2009) Plant Physiology 151:1751-1757. Microns

Why use NanoSIMS? Isotope data can be extracted from line scans 同位素数据可以从每一像素提取得到 Plant root cell wall Bacteria that have taken up Nitrogen (15N) (red) Bacteria Bacteria Plant cell Plant root cell wall Plant cell Bacteria Bacteria that have not taken up N (blue) Extracellular organic-C Natural abundance 15N enriched 技术突破: 在国际上率先应用纳米稳定性同位 素质谱技术(NanoSIMS)研究土壤养分吸收 Clode et al. (2009) Plant Physiology 151:1751-1757.

Why use NanoSIMS? Micro-site arrangement of soil pores and microbes can be studied 可对土壤孔隙和微生物的空间排列进行研究 Scanning electron microscope (SEM) NanoSIMS showing silica (28Si) and pore geometry NanoSIMS showing micro-site location 30 µm 10 µm Before doing any NanoSIMS analysis we used scanning electron microscope to identify a potentially suitable areas. This image gives the silica distribution using EDS X-ray microanalysis. The different colors are representing different silica concentrations. We than performed NanoSIMS analysis at the same spot. However, due to aberrations at the image edges we were restricted to a field of view of 30 um for each individual image. The images were stitched together. You probably can’t see it, but within the red square there are five red points, indicating that there is 15N enrichment. We zoomed down to a FOV = 10 x 10 um. In the right hand image, the 15N/14N ratio is giving. Green = Carbon (12C) Blue = Silica (28Si) Red = bacteria (12C15N) 10 x 10 µm 185 x 140 µm X-ray analysis of silica 75 x 50 µm (6 separate images) Herrmann et al. (2007) A novel method for the study of the biophysical interface in soils using nano-scale secondary ion mass spectrometry. Rapid Communication in Mass Spectrometry, 21, pp. 29-34.

Why use NanoSIMS? Micro-site arrangement of soil pores and microbes can be studied 可对土壤孔隙和微生物的空间排列进行研究 Scanning electron microscope (SEM) NanoSIMS showing silica (28Si) and pore geometry NanoSIMS showing 15N/14N ratio of bacteria 10 x 10 µm 30 µm 10 µm Before doing any NanoSIMS analysis we used scanning electron microscope to identify a potentially suitable areas. This image gives the silica distribution using EDS X-ray microanalysis. The different colors are representing different silica concentrations. We than performed NanoSIMS analysis at the same spot. However, due to aberrations at the image edges we were restricted to a field of view of 30 um for each individual image. The images were stitched together. You probably can’t see it, but within the red square there are five red points, indicating that there is 15N enrichment. We zoomed down to a FOV = 10 x 10 um. In the right hand image, the 15N/14N ratio is giving. 185 x 140 µm X-ray analysis of silica 75 x 50 µm (6 separate images) Herrmann et al. (2007) A novel method for the study of the biophysical interface in soils using nano-scale secondary ion mass spectrometry. Rapid Communication in Mass Spectrometry, 21, pp. 29-34.

Why use NanoSIMS? Area 15/14N ratio of bacteria 1 0.424 ±0.002 2 Compare levels of enrichment in target regions of interest (e.g. cells) Area 15/14N ratio of bacteria 1 0.424 ±0.002 2 0.486 ±0.003 3 0.548 ±0.006 4 0.543 ±0.018 5 0.632 ±0.017 Aresin 0.006 ±0.001 Bresin 0.009 ±0.001 Natural 0.003 1 3 4 5 2 B A Herrmann et al (2007) Rapid Communications in Mass Spectrometry 21, 29-34

Example 1: Plant-bacterial competition for N in wheat 植物和细菌对氮的竞争 Amino acids are an important source of organic N for plants and microbes. 氨基酸是植物和微生物的重要有机 氮 来源 NanoSIMS used to study plant-microbial competition for N in the rhizosphere. NanoSIMS 用以研究根际周围植物和 微生物对氮的竞争 Wheat grown in soil in a rhizo-tube. 15N labeled ammonium or amino-acid (glutamate) injected into rhizo-tube. Plant roots and soil fixed in resin after 5, 30, 90 min, 6 hr and 24 hr. Root and soil in resin Jones D.L., Clode P.L., Kilburn M.R., Stockdale E.A. and Murphy D.V. (2013). Competition between plant and bacterial cells at the microscale regulates the dynamics of nitrogen acquisition in wheat. New Phytologist 200: 796-807.

Example 1: Plant-bacterial competition for N NanoSIMS used to image-map for 15N/14N in metabolically active microbes and plant root cells using a 100 nm ion beam. NanoSIMS使用100纳米离子束来建立代谢活跃的微生物和植物根细胞的15N/14N的图像映射 Jones et al. (2013). New Phytologist 200: 796-807.

Example 1: Plant-bacterial competition for N Plant Root Sand grain Bacteria in rhizosphere Time = 0 30 minutes 24 hours 15/14N ratio images showing 15N enrichment over time Natural 15N/14N Enriched 5um 100um Plant Root Cells Jones et al. (2013). New Phytologist 200: 796-807.

SEM showing bacteria attached to the root Example 1: Plant-bacterial competition for N Traditional mass spectrometry results indicate that plant roots take up ammonium and amino acid at the same rate. However isotope signal is from plant root + surface attached bacteria. 传统质谱结果表明植物根系吸收铵态氮和氨基酸的速率相同； 但其同位素信号来自“植物根系+表面所附细菌”这一整体。 SEM showing bacteria attached to the root 2 µm NH4 = inorganic ammonium sulphate AA = amino acid glutamate Jones et al. (2013). New Phytologist 200: 796-807.

Example 1: Plant-bacterial competition for N Plant competition for amino acid was in fact low. Bacteria on the surface hid this difference. Plant roots were poor competitors for 15N-amino acid and took up N mainly as 15NH4+. 15N-amino acid was rapidly used by bacteria. 15N from amino acid 15N from NH4+ 植物根系对15N氨基酸竞争处于劣势，因此主要吸收15N铵态氮 15N氨基酸被细菌迅速利用 2 Traditional IRMS data could not detect the difference between N sources Jones et al. (2013). New Phytologist 200: 796-807.

Example 1: Plant-bacterial competition for N Results from NanoSIMS analysis of the plant root cells showed a difference in NH4+ versus amino acid uptake. This could not be detected by traditional mass spectrometry due to the interference in isotope signal from the attached bacteria. 纳米质谱仪分析显示植物根系细胞对铵态氮和氨基酸的吸收速率不同； 而这一结果传统质谱无法分析得到，因为受到根系表面细菌的同位素信号的干扰。 NanoSIMS 15N/14N vascular cell wall data Traditional IRMS data could not detect the difference between N sources NH4 = inorganic ammonium sulphate AA = amino acid glutamate

Membrane and air-gap. Only mycorrhizal hyphae can cross the membrane. Example 2: Plant-fungal C and N transfer Wheat plants grown in 13CO2 enriched air. 15N labeled source added to soil. Membrane separated 15N from plant. 15N transfer via mycorrhizal fungi. 植物-真菌的碳、氮传输 小麦在13CO2富集的环境中生长； 添加15N标记的成分到土壤； 生物膜将15N从植物分离出来； 15N通过菌根真菌传输。 Root Side No Root Membrane and air-gap. Only mycorrhizal hyphae can cross the membrane. 13CO2 15N C. Kaiser et al. (2014). New Phytologist.

No direct access to 15N by root Example 2: Plant-fungal C and N transfer Tracing 13C via plant photosynthate (13CO2). Tracing 15N via mycorrhiza (AMF). 通过植物光合产物（13CO2)对13C进行示踪，通过菌根真菌对15N进行示踪。 13C 31P 15N 15N No direct access to 15N by root 13CO2 Xylem cell Endodermis Phloem cells Cortex with Mycorrhiza C. Kaiser et al. (2014). New Phytologist.

No direct access to 15N by root NanoSIMS example: Plant-fungal C and N transfer Tracing 13C via plant photosynthate (13CO2). Tracing 15N via mycorrhiza (AMF). Phloem cell is highly enriched in 13C from photosynthesis. 15N No direct access to 15N by root 13CO2 Xylem cell Endodermis Phloem cells Cortex with Mycorrhiza 13C/12C Xylem cell is highly enriched in 15N from mycorrhiza. 15N/14N C. Kaiser….. and D.V. Murphy. (2014). New Phytologist

合作前景 – Conclusions NanoSIMS enables high spatial resolution of isotopic data between plant, bacterial and fungal cells. 纳米二次离子质谱技术能对植物、细菌和真菌细胞的同位素数据进行高度空 间解析。 This is not possible with traditional mass spectrometry where soil+plant+microbes = one isotopic measurement. 传统质谱技术仅能得到“土壤+植物+微生物”整体的单次同位素观测结果。 NanoSIMS method offers exciting opportunities to study soil-plant-microbe interactions at a previously unconsidered scale. NanoSIMS为在微观尺度上研究“土壤-植物-微生物-真菌”相互作用提供 了前所未有的机遇。

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