Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy

Nolin, Frédérique; Ploton, Dominique; Wortham, Laurence; Tchelidze, Pavel; Balossier, G.; Banchet, Vincent; Bobichon, Hélène; Lalun, Nathalie; Terryn, Christine; Michel, Jean

doi:10.1016/j.jsb.2012.08.011

Cited by 7 publications

(12 citation statements)

References 19 publications

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“…We then investigated changes in the water and element/ion contents of the cells during the various stages of apoptosis, with our cryo-correlative imaging technique [ 9 , 10 , 17 ]. We treated cells with AMD for 7h to induce apoptosis and we then cryofixed the cells directly in liquid ethane.…”

Section: Resultsmentioning

confidence: 99%

“…We then used chromatin fluorescence on ultrathin cryo-sections to identify each stage of apoptosis. We then carried out correlative cryo-imaging [ 9 , 10 ], in a targeted ultrastructural cryo-analysis of the water and element/ion contents of the main compartments of apoptotic cells.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies determining cell volume and water content by light microscopy [ 3 , 7 ] during apoptosis obtained conflicting results [ 8 ]. Our direct quantification of dry mass and water content through the application of dark-field imaging to ultrathin cryosections [ 9 ] showed that water content was 10 to 15% higher in all compartments of apoptotic cells than in control cells. However, as the water content of cells not engaged in apoptosis (ST 0) was similar to that of apoptotic cells, an increase in water content is clearly not sufficient to induce apoptosis.…”

Section: Discussionmentioning

confidence: 99%

“…We quantified water and elements/ions with our recently developed cryo-correlative microscopic approach coupling fluorescence imaging for the identification of H2B-GFP-tagged chromatin and scanning transmission electron microscopy (STEM) imaging. STEM imaging was used for the nano-quantification of water by dark-field imaging and of elements by energy dispersive X-ray spectroscopy [ 9 , 10 , 17 ] . A detailed description of our method can be found in [ 17 ], and we provide a brief description here.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, as these methods have produced divergent results [ 8 ], hydration of apoptotic cells is still an open field. We recently developed a correlative light and cryo-scanning transmission electron microscopy (cryo-STEM) method [ 9 , 10 ] for the simultaneous quantification of water and ions at the nanoscale, within cell compartments. We used this method to study the changes in water and ion contents in the various organelles of cancerous cells during apoptosis induced with actinomycin D (AMD).…”

Section: Introductionmentioning

confidence: 99%

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Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach

et al. 2016

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Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach

et al. 2016

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Targeted Nano Analysis of Water and Ions in the Nucleus Using Cryo-Correlative Microscopy

et al. 2014

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The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.

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Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach

Nolin

Michel

Wortham

et al. 2013

Cell. Mol. Life Sci.

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The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128-462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.

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Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy

Cited by 7 publications

References 19 publications

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach

Targeted Nano Analysis of Water and Ions in the Nucleus Using Cryo-Correlative Microscopy

Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach

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