Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS

Abstract

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.

Versatile Micropatterns of N‐Heterocyclic Carbenes on Gold Surfaces: Increased Thermal and Pattern Stability with Enhanced Conductivity

“…For this reason, as ample of printed BIMe@Au was subjected to 120 8 8Cf or 16 h. To our delight, the stripes of NHC remained almost unaltered as analyzed by ToF-SIMS,since this method offers high resolution and sensitivity towards all molecular fragments on as urface (Figure 2a,b). [16] This confirms the observation by Glorius,F uchs,a nd co-workers that NHCs grouped on as urface remain stationary. [8] High-resolution XPS of the N1sregion revealed ar etention of BIMe@Au of 94 % ( Figure 2c,s ee the Supporting Information for further details).…”

Distribution of salicifoline in freeze-fixed stems of Magnolia kobus as observed by cryo-TOF-SIMS

“…Phosphatidylcholine is a main component of biological membranes in plant cells 28 , and reports have shown that the m / z 184.07 ion can be detected as a phosphocholine ([C 5 H 15 NO 4 P] + ) fragment in TOF-SIMS measurements using dried biomaterials 29–31 . In the cryo-TOF-SIMS spectrum of phosphatidylcholine (Fig.…”

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution

“…Time-of-flight secondary ionization mass spectrometry (ToF-SIMS) can image biological samples with nanometerscale resolution [20] and is increasingly applied to numerous biological applications [21,22]. The detection of biomolecules such as phospholipids (PL) and small peptides at these small focal sizes can be fostered by using bismuth cluster ion sources as well as by mixing the compounds into MALDI matrices for matrix-enhanced SIMS (ME-SIMS [23,24]).…”