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

Abstract

Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment’s success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.

Методы Рентгеновского Флуоресцентного Микроанализа И Спектроскопии Комбинационного Рассеяния Света Для Исследования Гистологических Срезов Мышечных Тканей

“…Необходимо отметить, что многих проблем, связанных с химической фиксацией и вымыванием легких элементов можно было бы избежать при использовании техники криофиксации с быстрой заморозкой [13]. К сожалению, полная цепочка криогенных процессов " приготовление образцов -хранение -измерение" в рамках нашего исследования невозможна.…”

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

“…Cryo-fixation can be performed relatively easily and is widely used on small samples, such as cells, 6,12–15 viruses, proteins etc. , or porous specimens such as plants; especially for the latter, cryo-fixation is currently the most popular sample preparation protocol.…”

“…Yet, cryo-xation and sectioning of human or animal so tissues have remained a challenge. 7,8 Cryo-xation can be performed relatively easily and is widely used on small samples, such as cells, 6,[12][13][14][15] viruses, proteins etc., or porous specimens such as plants; especially for the latter, cryo-xation is currently the most popular sample preparation protocol. [16][17][18][19][20][21] A proper vitrication 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.…”