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- Fletcher, John, 1978, et al.
(author)
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Uncovering new challenges in bio-analysis with ToF-SIMS
- 2008
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In: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 255:4, s. 1264-1270
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Conference paper (peer-reviewed)abstract
- The introduction of cluster ion beams for routine SIMS analysis has greatly improved the prospects for characterising biological samples. The ultimate quality of the secondary ion image remains limited by the efficiency of the primary beam. Without overcoming the low ionisation probabilities associated with SIMS, the highest lateral resolution available for molecular imaging becomes limited by the statistical probability of any ions being generated from the area of the pixel. C(60)(+) primary ions are currently the most efficient available for routine analysis but although commercial systems have been demonstrated to produce spot sizes under 200 nm, focusing the beam produced in such electron impact sources results in rather low ion currents. The time scale for such high lateral resolution analysis can become impractical on conventional time-of-flight instruments. Molecular depth pro. ling capability has been revealed using SF(5)(+) and C(60)(+) ion beams and recent work has advanced the technique by combining the pro. ling and imaging abilities of these high efficiency projectiles to generate 3D molecular maps of biological systems. In this paper we discuss the challenges associated with 2D and 3D bio-analysis with emphasis on how instrumental advances aid such investigations yet demonstrating the obstacles that need to be overcome using a range of model and real world biological samples. We discuss complications with the biological matrix, challenges in manipulating and visualising the data and look at how instrumental advantages might aid the routine generation of these 3D molecular maps. (C) 2008 Elsevier B. V. All rights reserved.
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- Vaidyanathan, Seetharaman, et al.
(author)
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Exploratory analysis of TOF-SIMS data from biological surfaces
- 2008
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In: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 255:4, s. 1599-1602
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Conference paper (peer-reviewed)abstract
- The application of multivariate analytical tools enables simplification of TOF-SIMS datasets so that useful information can be extracted from complex spectra and images, especially those that do not give readily interpretable results. There is however a challenge in understanding the outputs from such analyses. The problem is complicated when analysing images, given the additional dimensions in the dataset. Here we demonstrate how the application of simple pre-processing routines can enable the interpretation of TOF-SIMS spectra and images. For the spectral data, TOF-SIMS spectra used to discriminate bacterial isolates associated with urinary tract infection were studied. Using different criteria for picking peaks before carrying out PC-DFA enabled identification of the discriminatory information with greater certainty. For the image data, an air-dried salt stressed bacterial sample, discussed in another paper by us in this issue, was studied. Exploration of the image datasets with and without normalisation prior to multivariate analysis by PCA or MAF resulted in different regions of the image being highlighted by the techniques. (C) 2008 Elsevier B. V. All rights reserved.
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| 5. |
- Vaidyanathan, Seetharaman, et al.
(author)
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Subsurface Biomolecular Imaging of Streptomyces coelicolor Using Secondary Ion Mass Spectrometry
- 2008
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In: Analytical Chemistry. - 0003-2700 .- 1520-6882. ; 80:6, s. 1942-1951
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Journal article (peer-reviewed)abstract
- Imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) with buckministerfullerene (C60) primary ions offers the possibility of mapping the chemical distribution of molecular species from biological surfaces. Here we demonstrate the capability of the technique to provide biomolecular information from the cell surface as well as from within the surface, as illustrated with the distribution of two antibiotics in Streptomyces coelicolor (a mycelial bacterium). Differential production of the two pigmented antibiotics under salt-stressed and normal conditions in submerged cultivations could be detected from the TOF-SIMS spectra of the bacteria, demonstrating the potential of the technique in studying microbial physiology. Although both the antibiotics were detected on the cell surface, sputter etching with C60+ revealed the spectral features of only one of the antibiotics within the cells. Exploratory analysis of the images using principal component analysis assisted in analyzing the spectral information with respect to peak contributions and their spatial distributions. The technique allows the study of not only lateral but also the depthwise distribution of biomolecules, uniquely enabling exploration of the processes within biological systems with minimal system intervention and with little a priori biochemical knowledge of relevance.
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| 6. |
- Vaidyanathan, Seetharaman, et al.
(author)
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TOF-SIMS investigation of Streptomyces coelicolor, a mycelial bacterium
- 2008
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In: Applied Surface Science. - 0169-4332. ; 288:4, s. 922-925
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Journal article (peer-reviewed)abstract
- Streptomyces coelicolor is a mycelial microorganism that produces several secondary metabolites, including antibiotics. The physiology of the organism has largely been investigated in liquid cultures due to ease of monitoring different physiological parameters and more homogeneous culture conditions. However, solid cultures reflect the natural physiology of the microorganism better, given that in its natural state it grows in the soil. Imaging mass spectrometry with TOF-SIMS and C60+ primary ion beams offers a potential route to studying chemical changes at the molecular level, both intracellular and extracellular that can help in understanding the natural physiology of the microorganism. Here, we report the application of the technique for studying the lateral distribution of the chemical species detected in a population, grown in both liquid and solid cultures. The capability of the technique for studying biological systems with minimal system intervention is demonstrated.
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