Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques

Abstract

Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.

Introduction

As early as 1935, Schoenheimer et al. [1], [2] started using stable isotopes to study intermediary metabolism. Since then, stable isotope labelling has been used extensively to explore biological processes, often combined with mass spectrometry [3], [4] and Nuclear Magnetic Resonance [5], [6]. One advantage of using stable isotope labelling is that isotopic labels have little or no effect on the biochemical properties of the target molecules, so one can presume that stable isotope-labelled molecules behave in the same way as non-labelled molecules. The ability to carry out quantitative analysis with stable isotope labelling is also useful for fields like proteomics [7], [8]. However, most quantitative measurements of stable isotope ratios use bulk samples that have been extracted from whole tissues, making it impossible to draw conclusions about subcellular compartments or contributions of different cell types. Imaging the precise location of stable isotopes within cells by NanoSIMS can provide direct evidence of the distribution of labelled molecules and allow us to track molecules quantitatively as they interact with specific cells and tissues.

The CAMECA NanoSIMS 50 is a high-resolution secondary ion mass spectrometry (SIMS) instrument which can detect chemical and isotopic distributions with a lateral resolution of 50 nm. The instrument uses a 16 keV primary Cs⁺ beam to bombard the sample surface and collects at least five selected negative secondary ions using a Mauttach–Herzog mass analyzer with electrostatic sector and asymmetric magnet configuration [9]. The design of the NanoSIMS simultaneously optimizes high spatial resolution, high mass resolution, and high sensitivity, which makes it a powerful tool for studying many types of samples. The sensitivity can allow excellent analysis of species at concentrations of parts per billion for some analytes, which makes it particularly useful in imaging trace elements in cosmochemistry [10], [11], geology [12], [13], and plant science [14], [15]. The excellent mass resolution, m/Δm > 4500 with 80% transmission [16], enables quantitative analysis of stable isotopes even when there are close isobaric interferences, such as ¹²C¹H⁻ with ¹³C⁻, ¹²C¹⁵N⁻ with ¹²C¹⁴N¹H⁻, and ¹²C¹³C¹H₂⁻ with ¹³C¹⁴N⁻ [17]. Combining these instrumental advantages with stable isotope labelling, NanoSIMS analysis has proven useful for studying biological samples in the last decade. For instance, Thompson et al. [18] identified a symbiosis model between a cyanobacterium and unicellular alga by imaging the exchange of fixed ¹³C and ¹⁵N. Kraft et al. [19] used a NanoSIMS to observe, for the first time, phase separation in lipid mixtures. Lechene et al. [20], [21] studied the fixed nitrogen exchange between host and bacterial cells and quantitatively measured mammalian cardiomyocyte renewal. In biological studies, the most common stable isotopes used to label molecules are ²H, ¹³C, ¹⁵N, and ¹⁸O; all are readily imaged by the NanoSIMS [22], [23], [24], [25].

Due to the complexity of most biological problems, correlative techniques have been developed to extract as much information as possible from the samples. For instance, instruments have been designed to allow correlative fluorescence microscopy and electron microscopy [26], [27]. Some groups studying biological samples have used NanoSIMS in combination with fluorescence microscopy [28], in situ hybridization techniques [29], [30], transmission electron microscopy (TEM) [20], [31], atomic force microscopy (AFM) [19], [32], synchrotron techniques [14], ICP-MS [31], and electron probe microanalysis [15]. All of these techniques have provided useful information to understand complex biological mechanisms, such as phylogenetic identification of individual cells from fluorescence in situ hybridization [33], extremely sensitive and rapid imaging of trace elements over large areas from synchrotron X-ray fluorescence techniques [34], and subcellular morphology from TEM [20]. The techniques of backscattered electron (BSE) imaging in a scanning electron microscope (SEM) and AFM have been extensively developed in recent years so that they now have new capabilities that can be applied to studies with biological samples. For instance, low-voltage BSE imaging has been applied to study both the morphology of biological materials and to reconstruct 3-D images by tomography [35], [36], [37]. The excellent spatial resolution of AFM makes it easy to visualize single molecules [38], [39] and interactions between molecules [40], [41].

This article reports three recent investigations combining analysis in the NanoSIMS with site-specific correlative AFM and BSE imaging. We discuss the critical sample preparation protocols and experimental methods developed for imaging at the molecular, cellular, and tissue scale.