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

Abstract

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.

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

“…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.…”

“…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.…”

“…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. HeLa cells stably expressing histone H2B tagged with GFP (H2B-GFP) were used to seed 25 cm 2 Nunc flasks at a density of 100 000 cells/mL.…”

Leucine-Based Pseudo-Proteins (LPPs) as Promising Biomaterials: A Study of Cell-Supporting Properties

“…The murine macrophage cell line, RAW264.7 (ATTC TIB-71), was purchased from the American Type Cell Culture Collection (ATCC, Manassas, VA, USA). HeLa cells stably expressing histone H2B-GFP were kindly provided by Prof. P.Tchelidze, who had used them in his previous research [12][13][14][15][16]. Primary mouse skin fibroblasts (pMSFs) had been previously prepared at our laboratory according to the protocol described in [17], and the early passages of pMSFs were stored at −86 • C. These cells were thawed and passaged for the current experiments.…”

Femtosecond responses of hydrated DNA irradiated by ionizing rays focus on the sugar-phosphate part

“…The respective importance of one effect over the other is still debated [3], but both undoubtedly contribute to cellular metabolism malfunctions. In fact, the relative importance of one effect over the other is likely to be dependent on the hydrometric content of cellular compartments [4].…”