Abstract
A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.
Highlights
Microscopy has transformed our understanding of biology and medicine
Compared with super-resolution fluorescence microscopy (Huang et al, 2009), which allows for the selection of the fluorescently labelled molecules or molecular assemblies, Coherent diffractive imaging (CDI) is based on the intrinsic mass density variations of biological specimens and enables quantitative three-dimensional imaging of the entire contents of cells, cellular organelles and biomaterials in their natural contrast (Miao et al, 2003; Shapiro et al, 2005; Jiang et al, 2008, 2010; Song et al, 2008; Nishino et al, 2009; Huang et al, 2009; Lima et al, 2009; de la Cuesta et al, 2009; Nelson et al, 2010; Giewekemeyer et al, 2010; Nam et al, 2013; Kimura et al, 2014; Gallagher-Jones et al, 2014; Bergh et al, 2008; Seibert et al, 2011; Schlichting & Miao, 2012)
Compared with zone-plate X-ray microscopy (Sakdinawat & Attwood, 2010; Weiß et al, 2000; Le Gros et al, 2005; Schneider et al, 2010; Meirer et al, 2011), CDI avoids the use of X-ray lenses and its resolution is only limited by the radiation damage imparted on biological specimens
Summary
Microscopy has transformed our understanding of biology and medicine. By using novel imaging technologies and labelling techniques, optical microscopy can routinely study dynamic processes in living cells (Stephens & Allan, 2003). Compared with super-resolution fluorescence microscopy (Huang et al, 2009), which allows for the selection of the fluorescently labelled molecules or molecular assemblies, CDI is based on the intrinsic mass density variations of biological specimens and enables quantitative three-dimensional imaging of the entire contents of cells, cellular organelles and biomaterials in their natural contrast (Miao et al, 2003; Shapiro et al, 2005; Jiang et al, 2008, 2010; Song et al, 2008; Nishino et al, 2009; Huang et al, 2009; Lima et al, 2009; de la Cuesta et al, 2009; Nelson et al, 2010; Giewekemeyer et al, 2010; Nam et al, 2013; Kimura et al, 2014; Gallagher-Jones et al, 2014; Bergh et al, 2008; Seibert et al, 2011; Schlichting & Miao, 2012). Compared with zone-plate X-ray microscopy (Sakdinawat & Attwood, 2010; Weiß et al, 2000; Le Gros et al, 2005; Schneider et al, 2010; Meirer et al, 2011), CDI avoids the use of X-ray lenses and its resolution is only limited by the radiation damage imparted on biological specimens
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