Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

Abstract

Time of flight secondary ion mass spectrometry (ToF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single-cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in lipid composition between the plasma membranes of two cells. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative ToF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. Insitu fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single-cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

“…A differential interference contrast image of paired cells is shown in Fig 2A, (m∕z 69), a hydrocarbon fragment ubiquitous to the acyl chains found in phospholipids (4,20,21). This chemical image shows that the membranes of the two cell are in contact because the signal is continuous through the junction.…”

“…In fact, the membranes are fused, as evidenced by the cells' resistance to separation following trituration. The ion image also serves as an internal standard, which is used to compare ion signals from other molecules from the membrane surface (4,20,21). The C 5 H þ 9 signal is consistent throughout the junction, and thus fluctuations in the lipid signal were taken to reflect changes in concentration across the membrane surface.…”

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

“…In addition to the MSI experiments, it is capable of conducting near real-time in-situ SCMS to elucidate detailed chemical information from live eukaryotic cells 16 , which is a major advantage compared with other vacuum based SCMS techniques (such as MALDI 10 and SIMS 21 ). The small size of the probe tip provides the ability to be inserted into a live eukaryotic cell and to extract and ionize the intracellular compounds for immediate MS analysis.…”

Preparation of Single Cells for Imaging Mass Spectrometry

“…Static SIMS has also seen wide application in cellular imaging. Interesting recent examples include three dimensional imaging of oocytes [14], relative quantification of cholesterol in cell membranes [15], and distinguishing cancerous cells of differing breast cancer phenotypes [16]. Cellular imaging has also been shown by a variety of other mass spectrometric techniques, including desorption/ionization on silicon [17] and laser post-ionization secondary neutral mass spectrometry [18].…”

“…Currently, the vast majority of cellular MS imaging reports utilize some variation on this method. Improvements to Chandra's technique, most notably maintenance of the sample at cryogenic temperatures throughout the fracturing and analysis [22], have resulted in improvements to the cellular imaging analysis [15].…”