Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS

Abstract

In ToF-SIMS analysis, the experimental outcome from cell experiments is to a great extent influenced by the sample preparation routine. In order to better judge this critical influence in the case of lipid analysis, a detailed comparison of different sample preparation routines is performed—aiming at an optimized preparation routine for systematic lipid imaging of cell cultures. For this purpose, human mesenchymal stem cells were analyzed: (a) as chemically fixed, (b) freeze-dried, and (c) frozen-hydrated. For chemical fixation, different fixatives, i.e., glutaraldehyde, paraformaldehyde, and a mixture of both, were tested with different postfixative handling procedures like storage in phosphate buffered saline, water or critical point drying. Furthermore, secondary lipid fixation via osmium tetroxide was taken into account and the effect of an ascending alcohol series with and without this secondary lipid fixation was evaluated. Concerning freeze-drying, three different postprocessing possibilities were examined. One can be considered as a pure cryofixation technique while the other two routes were based on chemical fixation. Cryofixation methods known from literature, i.e., freeze-fracturing and simple frozen-hydrated preparation, were also evaluated to complete the comparison of sample preparation techniques. Subsequent data evaluation of SIMS spectra in both, positive and negative, ion mode was performed via principal component analysis by use of peak sets representative for lipids. For freeze-fracturing, these experiments revealed poor reproducibility making this preparation route unsuitable for systematic investigations and statistic data evaluation. Freeze-drying after cryofixation showed improved reproducibility and well preserved lipid contents while the other freeze-drying procedures showed drawbacks in one of these criteria. In comparison, chemical fixation techniques via glutar- and/or paraformaldehyde proved most suitable in terms of reproducibility and preserved lipid contents, while alcohol and osmium treatment led to the extraction of lipids and are therefore not recommended.

Functional link between plasma membrane spatiotemporal dynamics, cancer biology, and dietary membrane-altering agents

“…Fluorescent probe-free techniques such as mass spectrometry, Raman spectroscopy, and, as already mentioned, EM can be used to circumvent some of these issues. However, these techniques require cell fixation and longer acquisition times as well as a high degree of expertise and sophisticated equipment [73, 80, 81]. A different set of fluorescent ratiometric probes, which are sensitive to differences in physicochemical membrane properties and can distinguish between a spectrum of tightly packed raft and relatively fluid non-raft domains, include Laurdan and ANEP (e.g., di-4-ANEPPS, di-4-ANEPPDHQ, and di-8-ANEPPS) dyes.…”

Preparation of cellular samples using graphene cover and air-plasma treatment for time-of-flight secondary ion mass spectrometry imaging

“…3,4 Since ToF-SIMS operates under an ultrahigh vacuum environment, biological samples such as cells and tissues should be properly prepared to be compatible with vacuum conditions, such as by chemical xation followed by dehydration, freeze drying, or frozen hydration method. [5][6][7][8] Compared to chemical xation, frozen hydrated sample preparation has been more recognized for preserving cell morphology and native distribution of molecules including diffusible ions, as well as for enhancing molecular ion intensities for phospholipids for ToF-SIMS imaging of hydrated cells. 9,10 Despite the aforementioned advantages, a frozen hydration method requires laborious, painstaking procedures for reproducible results, as well as special equipment like a liquid nitrogen cooled stage to keep samples frozen during the analysis.…”

Storage of cell samples for ToF-SIMS experiments—How to maintain sample integrity

“…Previous studies on cell analysis have compared various preparation methods focusing on different aspects in order to maintain sample integrity and to reduce analytical artifacts. [2][3][4][5][6][7][8][9][10][11][12] Such studies are necessary to ensure that differences in measured spectra are due to true sample differences (e.g., "healthy" versus "diseased" cells) and not sample preparation-induced artifacts. It was inter alia revealed that frozen-hydrated analysis of freeze-fractured cells is the goldstandard for adequate detection of diffusible elements in cells.…”

“…6 Still, this method struggles with problems in terms of complexity, reproducibility, and applicability to a large a) Electronic mail: Marcus.Rohnke@phys.chemie.uni-giessen.de number of samples. 3,11,13 For these purposes, chemical fixation and freeze-drying have proven to be beneficial. 11,14,15 In addition, to obtain reliable results from biological samples, it is important to minimize their natural intrinsic variance by ensuring comparable conditions at all times prior and during measurement.…”