Impact of sample preparation methods on single cell X-ray microscopy and light elemental analysis evaluated by combined Low Energy X-ray Fluorescence, STXM and AFM

Background: X-ray Fluorescence (XRF) microscopy has become a widespread method to observe the location of elemental components within cell. Although it is becoming a widely used technique in many laboratories and synchrotron facilities, sample preparation for this microscopy remains one of the main concerns. Indeed, the information available to researchers on how to treat samples are insufficient and not clear, sometimes leading to waste of time and/or compromising the success of the experiment. It is indeed recognised that a correct sample preparation is the first important step in performing a successful and meaningful analysis, i.e. achieving a condition in which the cells are closer to their physiological state. The majority of the commonly used protocols of cell fixation for X-ray microscopy have been mainly imported and adapted from immunocytochemistry. Therefore, many fixation methods are described, but none of them have been tested in a systematic way and declared the most eligible for synchrotron-based X-ray microscopy. Methods: HEC-1-A endometrial cell line, human spermatozoa and Human Embryonic Kidney HEK-293 cell line were chosen as cellular models; all type of cells were fixed using both organic solvents and cross-linking methods, as follow: 70% ethanol, paraphormaldehyde at 3.7 % and 2%; in addition HEK-293 cells were also subjected to methanol/acetone treatment and to cryofixation. Comparison among the different fixation methods was conducted by coupling Low Energy X-ray fluorescence (LEXRF), with Scanning Transmission X-ray Microscopy (STXM) and Atomic Force Microscopy (AFM). Results: Organic solvents induce greater cellular dehydration, with the maximal impact on the distribution and depletion of the diffusive elements. Paraphormaldehyde seems to be able to provide robust and replicable data. Finally, the cryofixed cells give the best results both in terms of morphology and elemental content. Conclusion: Although cryofixation appears to be the most appropriate method as it allows the cell to be kept closer to physiological conditions, it has some technical limitations. PFA, when used at conventional concentration of 3.7%, is also an excellent alternative method for X-ray microscopy.