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Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.

ABSTRACT: Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging subcellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With TOF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows comparison of pixel-to-pixel intensity and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated by imaging the cell membrane of resting RBL-2H3 mast cells.

SUBMITTER: Piehowski PD

PROVIDER: S-EPMC2758657 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.

Piehowski Paul D PD Davey Angel M AM Kurczy Michael E ME Sheets Erin D ED Winograd Nicholas N Ewing Andrew G AG Heien Michael L ML

Analytical chemistry 20090701 14

Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statist ...[more]

PMID: 19530687

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Project description:BackgroundTime-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components.ResultsThe present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins.ConclusionsThese new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments.

Dataset Information

Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.

Publications

Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.

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