Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Abstract

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although Xray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells. Video LinkThe video component of this article can be found at https://www.jove.com/video/60461/ 4 , but suffers from radiation damage and lack of cryogenic capabilities to study frozen-hydrated cells. All these analytical techniques complement each another in the elemental imaging of cells, but for all techniques the sample preparation procedure is a crucial step. It should be kept simple to limit possible contamination as well as elemental redistribution and/or leakage to obtain meaningful results. As demonstrated in electron microscopy, a cryogenic workflow, including cryo-immobilization of the cell and cryotransfer to a cryoscanning stage, allows an optimal elemental preservation at subcellular levels as close as possible to the native state 5,6,7,8,9,10 . This understanding has been successfully implemented into the development of synchrotron cryo-soft X-ray microscopy (e.g., full field microscopes and scanning microscopes) to produce ultrastructural imaging of entire frozen-hydrated cells in 2D or 3D. Various cryogenic workflows were developed 11 for soft X-ray microscopes at Beamline 2.1 (XM-2) of the Advanced Light Source at Lawrence Berkeley National Laboratory 12 , beamline U41-XM at the electron storage ring BESSY II (Germany)

Correlative single-cell hard X-ray tomography and X-ray fluorescence imaging

“…1c ). The vitrified cells were then freeze-dried 21 to retain cell structure while gradually depleting water through sublimation. ( Fig.…”

“…Seeding cells on Si 3 N 4 membranes were adapted from the reported procedure 21 . Si 3 N 4 membranes (Cat# NX5200E, Norcada) were glow-discharged using a PELCO easiGlow Glow Discharge Cleaning System (Tedpella) operated at 30 mA for 30 seconds.…”

“…The chemical fixation procedure was modified from 21,25 . Cell culture media was removed from the wells containing the Si 3 N 4 membrane with attached cells.…”

Difficulties and artefacts in cryo-fixation of ovarian tissues for X-ray fluorescence analyses

“…, or porous specimens such as plants; especially for the latter, cryo-fixation is currently the most popular sample preparation protocol. 16–21 A proper vitrification implies the formation of amorphous ice in the sample, which is crucial for its preservation, while an incomplete process would cause formation of the ice crystals leading to the breaking of membranes and distortion of the sample morphology. A complete vitrification can be easily achieved with only small samples (<1 mm 3 ).…”