Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification

Kosior, Ewelina; Bohic, Sylvain; Suhonen, Heikki; Ortega, Richard; Devès, Guillaume; Carmona, Asunción; Marchi, Florence; Guillet, Jean Francois; Cloetens, Peter

doi:10.1016/j.jsb.2011.12.005

Cited by 96 publications

(67 citation statements)

References 23 publications

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“…However, XFM does not usually show much cellular ultrastructure, because the light elements (such as H, C, N, and O, which are the main constituents of biological materials) have low fluorescence yield (4). At the multi-keV X-ray energies needed to excite most X-ray fluorescence lines of interest, these light elements show little absorption contrast, but phase contrast can be used to image cellular structure (5,6) and this can be combined with scanned-beam XFM (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Deng

Vine

Chen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

164

131

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Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolution beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ∼90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context. ptychography | X-ray fluorescence microscopy | cryogenic biological samples X -ray fluorescence microscopy (XFM) offers unparalleled sensitivity for quantitative mapping of elements, especially trace metals which play a critical role in many biological processes (1-3). It is complementary to light microscopy, which can study some elemental content in live cells (with superresolution techniques possible) but which is more difficult to quantitate because it depends on the binding affinities of fluorophores. However, XFM does not usually show much cellular ultrastructure, because the light elements (such as H, C, N, and O, which are the main constituents of biological materials) have low fluorescence yield (4). At the multi-keV X-ray energies needed to excite most X-ray fluorescence lines of interest, these light elements show little absorption contrast, but phase contrast can be used to image cellular structure (5, 6) and this can be combined with scanned-beam XFM (7-11).One can also acquire phase-contrast X-ray images with a resolution beyond X-ray lens limits by recording the diffraction pattern from a coherently illuminated, noncrystalline sample in an approach called coherent diffraction imaging (CDI) (12). This approach has been used to image isolated dried cells (13-15), and 3-nm resolution has been achieved when imaging silver nanocubes (16). The traditional CDI approach requires that samples meet a so-called "finite support" (17) requirement with no observable scattering outside of a defined region; although some limited success has been obtained (18,19), this finite support condition has proven difficult to achieve with single cells surrounded by ice layers. Ptychography (20-22) is a recently realized CDI method [with an older history (23)] that circumvents this isolated cell requirement by instead scanning a limitedsize coherent illumination spot across the sample. Ptychography has been used to image freeze-dried diatoms at 30-nm resolution (24) and bacter...

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mentioning

confidence: 99%

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Deng

Vine

Chen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

164

131

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“…Historically, different substrates, ranging from thin polycarbonate foils/films [35][36][37] , formvar coated gold transmission electron microscopy (TEM) grids [38][39][40][41][42] and silicon nitride windows 5,17,[43][44][45][46] , have been used to grow mammalian cells and used for X-ray fluorescence imaging. In comparison among the existing and potential substrates for imaging adherent mammalian cells by XFM, silicon nitride membrane (Si 3 N 4 ) windows are most suitable due to their low fluorescence background and relatively simple elemental composition [4][5][6]47,48 .…”

Section: Discussionmentioning

confidence: 99%

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Finney

Jin²

2015

JoVE

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X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging. Video LinkThe video component of this article can be found at

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“…This information was used in combination with XFI to provide quantitative estimates of subcellular concentration for a variety of trace elements. 54 …”

Section: Experimental Methods and Strategiesmentioning

confidence: 99%

Elemental and Chemically Specific X-ray Fluorescence Imaging of Biological Systems

et al. 2014

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Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification

Cited by 96 publications

References 23 publications

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Elemental and Chemically Specific X-ray Fluorescence Imaging of Biological Systems

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