Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

The Lawrence Livermore National Laboratory Cryo-NanoSIMS: The Next Generation for High Spatial Resolution Functional Analysis

Authors:

Peter K. Weber1* (weber21@llnl.gov), Xavier Mayali1, Rachel Hestrin2, Megan Morris1,6, Melanie A. Brunet3, Mary Kraft3, Danielle Jorgensen4, Laurent Arnoldi5, Marc Debliqui5, Céline Defouilloy5, Jérôme Farcy5, Sarah Vitcher Fichou5, Ludovic Renaud5, Nicolas Saquet5, Aurelien Thomen5, Jennifer Pett-Ridge1, Rhona Stuart1

Institutions:

1Lawrence Livermore National Laboratory; 2University of Massachusetts–Amhurst; 3University of Illinois Urbana–Champaign; 4University of California–Berkeley; 5CAMECA Instruments; 6Oak Ridge Institute for Science and Education

Goals

Algal and plant systems have the unrivaled advantage of converting solar energy and CO2 into useful organic molecules. Their growth and efficiency are largely shaped by the microbial communities in and around them. The μBiospheres Science Focus Area seeks to understand phototroph-heterotroph interactions that shape productivity, robustness, the balance of resource fluxes, and the functionality of the surrounding microbiome. We hypothesize that different microbial associates not only have differential effects on host productivity but can change an entire system’s resource economy. Our approach encompasses single cell analyses, quantitative isotope tracing of elemental exchanges, omics measurements, and multi-scale modeling to characterize micro-scale impacts on system-scale processes. We aim to uncover cross-cutting principles that regulate these interactions and their resource allocation consequences to develop a general predictive framework for system-level impacts of microbial partnerships.

Abstract

In 2002, Lawrence Livermore National Laboratory (LLNL) installed a NanoSIMS 50 for the study of microbial ecology. NanoSIMS was unproven technology at the time, but it was rapidly adopted for microbial ecology because of its ability to trace isotopically labeled substrates into microbial communities. One limitation, however, has been that samples have to be prepared for high vacuum, which results in the loss of soluble species and can cause significant sample alteration. To overcome this limitation, LLNL worked with the manufacturer, CAMECA Instruments, to develop the next-generation NanoSIMS with a cryogenic stage. This winter at LLNL, CAMECA began the process of installing the prototype product of that collaboration: a first-of-its-kind cryo-NanoSIMS. This unique instrument has an analysis stage that can be cooled to liquid nitrogen temperatures and a cryogenic system for sample handling and introduction. With this cryogenic capability, we will be able to analyze frozen hydrated samples, which will capture soluble molecules that were previously lost during room temperature sample preparation methods. Sample freezing can also maintain spatial relationships among organisms without the addition of embedding resins that remove soluble molecules and mask the organic molecules that we seek to detect.

Another remarkable feature of the new cryo-NanoSIMS is its new 10 nm cesium ion source, which is used to image and analyze samples. This new cesium ion source generates an ion beam with unprecedented intensity, allowing the LLNL prototype cryo-NanoSIMS to achieve 10 nm spatial resolution (see Figure). This new cesium ion source allows us to resolve structures down to the size of a single phage.

In addition, the prototype cryo-NanoSIMS has been upgraded to improve useability and throughput, including redesigned electronics, sample stage, optical imaging, and sample introduction systems. The new electronics allow low energy sample analysis, improving the ability to resolve small structures by increasing depth resolution. The stage now has optical encoding and achieves better than 500 nm reproducibility, allowing 2 to 100 times faster automated analysis of selected targets, depending on the target size and analysis duration. The optical imaging system can now resolve micron-scale features and be used for automated sample registration, easing navigation. An automated sample introduction further increases ease of use and productivity.

We will be using our cryo-NanoSIMS for a wide range of applications, including host-microbe interactions in bioenergy relevant systems and soil carbon cycling. We will presen tperformance data, including initial analyses of microbial associations in perennial grasses and microalgae.

Image

Llnl Cryosims

Funding Information

This work was performed under the auspices of the U.S. DOE at Lawrence Livermore National Laboratory (LLNL) under Contract DE-AC52- 07NA27344 and supported by GSP, BER program under the LLNL Biofuels Science Focus Area, FWP SCW1039