Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis

Michael A Robinson,David G Castner

doi:10.1186/1559-4106-8-15

Michael A Robinson, David G Castner

Open Access

https://doi.org/10.1186/1559-4106-8-15

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Journal: Biointerphases	Publication Date: Jul 5, 2013
Citations: 74	License type: cc-by

Affiliation: University of Washington

Abstract

The information that is obtained from single cells during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is influenced by the method that was used to prepare the cells. The removal of extracellular media before analysis is necessary, but the rinsing technique should not damage the plasma membrane of the cell. The presence of intracellular salts reduced the secondary ion yield an average of 2.6-fold during Bi3+/C60++ depth profiles. Chemical fixation followed by rinsing removed a majority of the intracellular salts, “recovering” the positive secondary ion yields. The formaldehyde-fixation process removed a majority of the intracellular Cl-, but other key anions were not removed in significant amounts. The data presented here is consistent the anion neutralization mechanism largely responsible for the lower ion yields. All of the organic secondary ions that were detected in the freeze-dried cells were also detected in the formaldehyde-fixed cells, suggesting that the fixation process did not remove any molecular species to an extent that is detectable by ToF-SIMS. Compared to freeze dried cells, well preserved, frozen-hydrated cells showed little increase, or a decreased yield, for most low mass ions, but an increased yield for larger mass fragments. This is consistent with a reduced damage cross section at cryogenic analysis temperatures, although proton donation from water and reduction the salt effects in the presence of water likely also play roles. Numerous ions detected from the frozen-hydrated cells were not detected from the freeze dried cells, however many of these ions were attributed to chemical combinations of water, salts and the ammonium acetate rinsing solution.

Highlights

Time-of-flight secondary ion mass spectrometry (ToFSIMS) is a powerful tool that has been used to explore a wide range of biologically relevant samples including: cells and tissues [1,2,3,4], lipids [5], proteins on surfaces [6,7], DNA [8,9], drug eluting stents [10,11], explanted biomaterials [12] and decellularized matrix [13]
Salt and salt adduct peaks were excluded from the peak lists. The purpose of these experiments was to examine how various methods of sample preparation affected the chemical information obtained from single cells, with an emphasis on SI yields from depth profiles
Sample preparation plays a key role in determining the information that is obtained from single cells with ToFSIMS

Summary

Introduction

Time-of-flight secondary ion mass spectrometry (ToFSIMS) is a powerful tool that has been used to explore a wide range of biologically relevant samples including: cells and tissues [1,2,3,4], lipids [5], proteins on surfaces [6,7], DNA [8,9], drug eluting stents [10,11], explanted biomaterials [12] and decellularized matrix [13]. The unique abilities of ToF-SIMS to acquire a mass spectrum with high mass resolution, as well as produce chemical maps with submicron spatial resolution [14] provides an effective method to probe biological cells and tissues. These strengths, along with the capacity to sputter etch organic material [15,16], may enable the 3D visualization of sub-cellular features including drug, metabolite or. They can be separated into one of two fundamental categories: either the cells are dehydrated prior to analysis, or the intracellular water is conserved and the cells are analyzed frozen-hydrated. Either the media is removed with an isotonic rinsing solution, or the cells are freeze-fractured, removing a portion of the cell and all of the media above it, creating a pristine surface for analysis [18,19]

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Conclusion