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Visualisation and profiling of lipids in single biological cells using Time-of-Flight Secondary Ion Mass Spectrometry

Tian, Hua

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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Abstract

Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote.With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Visualisation and profiling of lipids in single biological cells using Time-of-Flight Secondary Ion Mass Spectrometry
Degree type:
Doctor of Philosophy
Degree programme:
PhD Chemical Engineering & Analytical Science (42 month)
Publication date:
2012-02-24T11:21:38
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
155
Abstract:
Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote.With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.
Keyword(s):
Thesis main supervisor(s):
Funder(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:156421
Created by:
Tian, Hua
Created:
24th February, 2012, 11:21:38
Last modified by:
Tian, Hua
Last modified:
21st December, 2012, 19:34:36

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