Imaging trace element distributions in single organelles and subcellular features

Abstract

The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron‐based x‐ray fluorescence microscopy

“…Cryogenic sample preparation has not yet emerged as the primary means to prepare adherent mammalian cells for XFM studies. Of approximately two dozen recent publications on adherent mammalian cells analysed by XFM, half or so used plunge freezing to prepare their biological specimens (Bohic et al, 2001;Ilinski et al, 2003;Harris et al, 2005;Bacquart et al, 2007;Matsuyama et al, 2010;Kosior et al, 2012;Yuan et al, 2013;Chen et al, 2014;Kashiv et al, 2016). Very recently, high-pressure freezing followed by freeze substitution and sectioning has been also applied to adherent mammalian cells in order to reveal metal distribution at single organelle level (Kashiv et al, 2016).…”

“…Of approximately two dozen recent publications on adherent mammalian cells analysed by XFM, half or so used plunge freezing to prepare their biological specimens (Bohic et al, 2001;Ilinski et al, 2003;Harris et al, 2005;Bacquart et al, 2007;Matsuyama et al, 2010;Kosior et al, 2012;Yuan et al, 2013;Chen et al, 2014;Kashiv et al, 2016). Very recently, high-pressure freezing followed by freeze substitution and sectioning has been also applied to adherent mammalian cells in order to reveal metal distribution at single organelle level (Kashiv et al, 2016). Nevertheless, the other dozen studies employed chemical fixation either by aldehyde-based chemicals or organic solvents such as ethanol or methanol (Paunesku et al, 2003;Wagner et al, 2005;Yang et al, 2005;McRae et al, 2006;Finney et al, 2007;Corezzi et al, 2009;Matsuyama et al, 2009;Wolford et al, 2010;Marmorato et al, 2011;Weekley et al, 2011;Marvin et al, 2012;McRae et al, 2013).…”

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis

“…Prior to cryo-fixation, the microalgal cultures were concentrated by gentle centrifugation for 10 min. This cryo-preparation is recognized as the most suitable method to preserve the native chemistry of cells, including highly diffusible elements and trace elements [63][64][65]. For the freeze substitution (FS), a mixture of dried acetone and 1% osmium tetroxide was used.…”

“…Resin sections were rapidly collected on the water (< 30 s) of the diamond knife. Because it has been shown that some diffusible molecules and elements can be lost at this step [70,71], we visualized the distribution of highly-diffusible elements (chlorine, potassium and calcium) with the synchrotron X-ray fluorescence. The presence of these mobile elements is a rule-of-thumb criterion to assess the chemical preservation of cells during the sample preparation [72,73].…”

The emerging role of zinc transporters in cellular homeostasis and cancer

“…Newer micro-analytical techniques employing synchrotron X-ray fluorescence chemical imaging allow detection of both loosely and tightly bound metals with high spatial resolution and have demonstrated zinc pools in the nucleus, mitochondria and vesicles. 20 , 21 This intracellular compartmentalization of zinc is one of the mechanisms by which cells maintain zinc homeostasis and zinc regulates cellular functions. Cellular zinc homeostasis, both total intracellular zinc and compartmentalized zinc, is tightly controlled by the concerted action of proteins that transport, sense, store and release zinc.…”