Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

Abstract

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging. Video LinkThe video component of this article can be found at

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

“…Different substrates have been used for XFM analysis of mammalian cells, including 2 m thick polycarbonate foils/films (Bacquart et al ., ; Ortega et al ., ; Kosior et al ., ), 4 m thick prolene films (Matsuyama et al ., ), formvar coated TEM grids (Bohic et al ., ; Paunesku et al ., ; Harris et al ., ; Wagner et al ., ; Chen et al ., ; Paunesku et al ., ) and Si 3 N 4 windows (McRae et al ., ; Finney et al ., ; Matsuyama et al ., ; Wolford et al ., ; Marmorato et al ., ; Weekley et al ., ; Arora et al ., ; Marvin et al ., ; Yuan et al ., ; Grubman et al ., ). A discussion of the relative merits of some substrates is described elsewhere (Carter et al ., ; Finney & Jin, ). Si 3 N 4 windows are by far the most frequently used substrate for adherent cell XFM imaging.…”

“…). One possible approach is to use standard office tape (‘Scotch tape’) to affix the windows to the bottom of a cell culture vessel (Finney & Jin, ). Upon UV irradiation, cell suspensions can be added into the entire dish containing the windows (Fig.…”

“…NIH/3T3 mouse embryonic fibroblast cells, originally purchased from American Tissue Culture Collection (http://www.atcc.org, cat# CRL1658), were routinely grown as described before in complete culture medium (Finney & Jin, ; Jin et al ., ). At about 80% confluence, cells were washed with Dulbecco's phosphate buffer saline without calcium and magnesium (D‐PBS, Invitrogen Life Technologies, Waltham, Massachusetts, USA, cat#14190) and detached by incubation with 0.5 mL 0.05% Trypsin‐EDTA for about 2 min.…”

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

“…Samples for X-ray uorescence microscopy were prepared following the protocol outlined by Finney and Jin (Finney and Jin, 2015). Brie y SiN windows (Norcada, Edmonton, Alberta, Canada) were attached to the bottom of a 35mm tissue culture dish and HFFs were seeded overnight and infected with RH∆Ku80∆HXG (parental) parasites or ∆VIT::DHFR TS parasites at an MOI of 3.…”

Radiation Dose Limits for Bioanalytical X-ray Fluorescence Microscopy

“…Fixatives permeate samples to covalently bond and immobilize the major biochemical constituents (i.e., proteins, lipids, and carbohydrates). 5 However, the influence of chemical fixation (and any subsequent histological staining) on elemental distributions can be significant. 6 Perturbations may include element-specific changes in concentration and spatial location, either of which may compromise subsequent biological interpretation.…”