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Examination of trafficking of phagocytosed colloid particles in neutrophils using synchrotron-based X-ray fluorescence microscopy (XFM)

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Abstract

Synchrotron-based X-ray fluorescence microscopy (XFM) can localise chemical elements at a subcellular level. 99mTechnetium stannous (TcSn) colloid is taken up by phagocytes via a Complement Receptor 3 mediated phagocytic process. In the current study, XFM was used to examine the intracellular trafficking of TcSn colloid in neutrophils. XFM was performed on TcSn colloid, and neutrophils labelled with TcSn colloid, in whole blood. We developed a set of pixel by pixel analysis and mapping techniques incorporating cluster analysis that allowed us to differentiate neutrophils and artefactual contaminants, and we examined the changes in element distribution that accompany neutrophil phagocytosis of TcSn colloid. Sn became associated with half the neutrophils. Within cells, Sn colocalised with iron (Fe) and sulphur (S), and was negatively associated with calcium (Ca). Despite the high sensitivity of XFM, Tc was not detected. XFM can help clarify the intracellular processes that accompany neutrophil phagocytosis. The subcellular colocalisation of Sn with Fe is consistent with fusion of the colloid-containing phagosome with neutrophil granules. The association of Sn with S suggests that proteins rich in S-containing amino acids are present in the phagosome. The negative colocalisation with Ca indicates that ongoing maturation of the TcSn colloid phagosome is no longer calcium dependent one hour after phagocytosis.

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Acknowledgements

Many thanks to Natasha Williams for technical assistance in preparing cell samples. The study was supported by Australian Synchrotron grant numbers P1371/AS092/XFM_QLD and P2428/AS102/XFM2428.

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Authors and Affiliations

  1. Queensland X-Ray PET/CT Centre, Level 3 Mater Medical Centre, 293 Vulture St, South Brisbane, QLD, 4101, Australia

    Stuart C. Ramsay

  2. Townsville Nuclear Medicine, Mater Hospital, Townsville, QLD, Australia

    Stuart C. Ramsay & Nathan Cassidy

  3. School of Medicine & Dentistry, James Cook University, Townsville, QLD, Australia

    Stuart C. Ramsay & Natkunam Ketheesan

  4. School of Veterinary & Biomedical Sciences, James Cook University, Townsville, QLD, Australia

    Natkunam Ketheesan

  5. Australian Synchrotron, 800 Blackburn Rd, Clayton, 3168, VIC, Australia

    Martin D. de Jonge, Daryl L. Howard & David Paterson

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  1. Stuart C. Ramsay
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  2. Nathan Cassidy
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Correspondence to Stuart C. Ramsay.

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Ramsay, S.C., Cassidy, N., de Jonge, M.D. et al. Examination of trafficking of phagocytosed colloid particles in neutrophils using synchrotron-based X-ray fluorescence microscopy (XFM). J Biol Phys 37, 493–506 (2011). https://doi.org/10.1007/s10867-011-9233-9

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