C60‐ToF SIMS imaging of frozen hydrated HeLa cells

Piwowar, Alan M.; Keskin, Selda; Delgado, Melissa Ortiz; Shen, Kan; Hue, Jonathan J.; Lanekoff, Ingela; Ewing, Andrew G.; Winograd, Nicholas

doi:10.1002/sia.4882

Cited by 35 publications

(33 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…FH: The preparation routine was similar to that described by Piwowar et al 44 and according to Robinson and Castner. 18 First, the medium was removed.…”

Section: Frozen-hydrated Preparation Of Cellsmentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

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.

show abstract

“…FH: The preparation routine was similar to that described by Piwowar et al 44 and according to Robinson and Castner. 18 First, the medium was removed.…”

Section: Frozen-hydrated Preparation Of Cellsmentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Kulp et al demonstrated the potential of TOF-SIMS combined with principal components analysis to distinguish chemical differences in three closely related human breast cancer cell lines [47]. In 2013, the Ewing and Winograd groups showed the potential of TOF-IMS for frozen hydrated cells after ammonium formate washing [48]. Ide et al showed that changes in lipid composition on breast cancer cell lines can be studied using TOF-SIMS [49].…”

Section: Introductionmentioning

confidence: 99%

Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSI-TOF-SIMS

Vanbellingen

Castellanos

Rodriguez-Silva

et al. 2016

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

In the present work, we show the advantages of label-free, tridimensional mass spectrometry imaging using dual beam analysis (25 keV Bi3+) and depth profiling (20 keV with a distribution centered at Ar1500+) coupled to time of flight secondary ion mass spectrometry (3D-MSI-TOF-SIMS) for the study of A-172 human glioblastoma cell line treated with B-cell lymphoma 2 (Bcl-2) inhibitor ABT-737. The high spatial (~250 nm) and high mass resolution (m/Δm ~ 10,000) of TOF-SIMS permitted the localization and identification of the intact, unlabeled drug molecular ion (m/z 811.26 C42H44ClN6O5S2−[M-H]−) as well as characteristic fragment ions. We propose a novel approach based on the inspection of the drug secondary ion yield which showed a good correlation with the drug concentration during cell treatment at therapeutic dosages (0 – 200 μM with 4 h incubation). Chemical maps using endogenous molecular markers showed that the ABT-737 is mainly localized in subsurface regions and absent in the nucleus. A semi-quantitative workflow is proposed to account for the biological cell diversity based on the spatial distribution of endogenous molecular markers (e.g., nuclei and cytoplasm) and secondary ion confirmation based on the ratio of drug-specific fragments to molecular ion as a function of the therapeutic dosage.

show abstract

“…The high yield of lipid based ions, combined with the subcellular lateral resolution imaging capabilities of ToF-SIMS makes it an excellent tool for studies of lipids, 1-4 cells, [5][6][7][8][9][10][11] and tissues. [12][13][14][15][16] Studies have been carried out in a variety of areas including the analysis of cancer cells, [17][18][19] cancer tissues, 20,21 muscular dystrophy, 22,23 and drug permeation into skin.…”

Section: Introductionmentioning

confidence: 99%

ToF-SIMS of tissues: “Lessons learned” from mice and women

et al. 2015

View full text Add to dashboard Cite

The ability to image cells and tissues with chemical and molecular specificity could greatly expand our understanding of biological processes. The subcellular resolution mass spectral imaging capability of time of flight secondary ion mass spectrometry (ToF-SIMS) has the potential to acquire chemically detailed images. However, the complexities of biological systems combined with the sensitivity of ToF-SIMS require careful planning of experimental methods. Tissue sample preparation methods of formalin fixation followed by paraffin embedding (FFPE) and OCT embedding are compared. Results show that the FFPE can potentially be used as a tissue sample preparation protocol for ToF-SIMS analysis if a cluster ion presputter is used prior to analysis and if nonlipid related tissue features are the features of interest. In contrast, embedding tissue in OCT minimizes contamination and maintains lipid signals. Various data acquisition methodologies and analysis options are discussed and compared using mouse breast and diaphragm muscle tissue. Methodologies for acquiring ToF-SIMS 2D images are highlighted along with applications of multivariate analysis to better identify specific features in a tissue sections when compared to H&E images of serial sections. Identification of tissue features is necessary for researchers to visualize a molecular map that correlates with specific biological features or functions. Finally, lessons learned from sample preparation, data acquisition, and data analysis methods developed using mouse models are applied to a preliminary analysis of human breast tumor tissue sections.

show abstract

C60‐ToF SIMS imaging of frozen hydrated HeLa cells

Cited by 35 publications

References 10 publications

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

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

Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSI-TOF-SIMS

ToF-SIMS of tissues: “Lessons learned” from mice and women

Contact Info

Product

Resources

About