Atomic Recombination in Dynamic Secondary Ion Mass Spectrometry Probes Distance in Lipid Assemblies: A Nanometer Chemical Ruler

Abstract

The lateral organization of biological membranes is thought to take place on the nanometer length scale. However, this length scale and the dynamic nature of small lipid and protein domains have made characterization of such organization in biological membranes and model systems difficult. Here we introduce a new method for measuring the colocalization of lipids in monolayers and bilayers using stable isotope labeling. We take advantage of a process that occurs in dynamic SIMS called atomic recombination, in which atoms on different molecules combine to form diatomic ions that are detected with a NanoSIMS instrument. This process is highly sensitive to the distance between molecules. By measuring the efficiency of the formation of ¹³C¹⁵N^- ions from ¹³C and ¹⁵N atoms on different lipid molecules, we measure variations in the lateral organization of bilayers even though these heterogeneities occur on a length scale of only a few nm, well below the diameter of the primary ion beam of the NanoSIMS instrument or even the best super-resolution fluorescence methods. Using this technique, we provide direct evidence for nanoscale phase separation in a model membrane, which may provide a better model for the organization of biological membranes than lipid mixtures with microscale phase separation. We expect this technique to be broadly applicable to any assembly where very short scale proximity is of interest or unknown, both in chemical and biological systems.

Figure 1

Schematic of the atomic recombination experiment and sitespecifically labeled lipids. (A) When the ¹³C and ¹⁵N labels are far apart during Cs⁺ bombardment (beam shown schematically—actual diameter on the order of 100 nm), ¹²C¹⁵N⁻ and ¹³C¹⁴N⁻ are primarily observed, with only natural abundance ¹³C¹⁵N⁻. (B) When the ¹³C and ¹⁵N labels are close together, higher counts of ¹³C¹⁵N⁻ are observed, with less ¹²C¹⁵N⁻ and ¹³C¹⁴N⁻. The ratio ℜ ≡ ¹³C¹⁵N⁻/(¹²C¹⁵N⁻ + ¹³C¹⁵N⁻) is, therefore, related to the average distance between ¹³C and ¹⁵N labels in the sample under Cs⁺ bombardment. (C) Isotope-labeled lipids used in this study, from left to right: ¹³C₃-DOPC, ¹⁵N-DOPC, 2,3,4-¹³C₃-CHOL, and 25,26,27-¹³C₃-CHOL and cartoon representations (no color = natural abundance). ¹³C₃- and ¹⁵N-labeled DSPC and POPC follow the same headgroup labeling pattern as labeled DOPC; DSPC is shown with straight fatty acid tails in the schematics.

Figure 2

Atomic recombination in ideally mixed monolayers of DOPC, ¹³C₃-DOPC, and ¹⁵N-DOPC. (A) Increasing the concentration of ¹³C₃-DOPC in monolayers with a fixed percent (10 mol %) of ¹⁵N-DOPC decreases the average distance between ¹³C and ¹⁵N labels. (B) The measured ℜ(¹³C¹⁵N⁻) increases as the mole fraction of ¹³C₃-DOPC increases (black). Simulated values for two limiting cases where there is no recombination (purple) and complete, distance-independent recombination (orange) are plotted. (C) Data for lipids in randomly mixed monolayers can be used as a standard for other compositions that might contain a nonrandom distribution of molecules. Note that the horizontal blue boundary is set by the natural abundance of ¹³C.

Figure 3

Systematic approaches to separating isotopic labels with leaflet asymmetry and site-specific cholesterol labeling. (A) Compositions (in mol %) are as follows: (1) 50:50 ¹⁵N-DOPC:¹³C₃-DOPC, (2) 50:50 2,3,4-¹³C₃-CHOL:¹⁵N-DOPC, (3) 50:50 25,26,27-¹³C₃-CHOL:¹⁵N-DOPC, (4) 50:50 ¹⁵N-DOPC:DOPC and 50:50 25,26,27-¹³C₃-CHOL:DOPC, (5) 50:50 ¹⁵N-DOPC:DOPC and 50:50 2,3,4-¹³C₃-CHOL:DOPC, (6) 50:50 ¹⁵N-DOPC:DOPC and 50:50 ¹³C₃-DOPC:DOPC. (B) Data for case 6 demonstrates that when ¹³C and ¹⁵N atoms are separated by approximately 5 nm ℜ(¹³C¹⁵N⁻) is much smaller. (C) ℜ(¹³C¹⁵N⁻) decreases as a function of the approximate distance between ¹³C and ¹⁵N atoms for all configurations shown in part A. Mean distances between labels were estimated from X-ray scattering and molecular dynamics models.^–

Figure 4

Atomic recombination in microscale and nanoscale phase separating bilayers. (A) An SLB patch of 20:20:25:35 ¹⁵N-DSPC:¹³C₃-DSPC:CHOL:DOPC formed from the fusion of a single giant unilamellar vesicle shows coexisting liquid phases. In the ¹⁵N-DSPC quantitative ion image of the SLB patch, the ordered phase with more ¹³C₃-DSPC and ¹⁵N-DSPC is clearly visible as a region of higher ¹⁵N (seen as ¹²C¹⁵N⁻). (B) An SLB patch of 20:20:25:35 ¹⁵N-DSPC:¹³C₃-DSPC:CHOL:POPC shows a uniform ¹²C¹⁵N⁻ signal in quantitative ion images. Scale bars are 5 µm. (C) When ℜ(¹³C¹⁵N⁻) within each phase or for the whole field of view is plotted vs the concentration of ¹³C₃-DSPC, deviations from random mixing are observed (solid black squares; note that the vertical axis is expanded compared with Figures 2 and 3). As described in the text, the composition of SLBs with nanoscale L_o/L_d domains and isotope labels in different phases is 20:20:25:20:15 ¹³C₃-DSPC:DSPC:CHOL:¹⁵N-POPC: POPC. The (dis)ordered domains refer to data from regions in panel A; the nanodomains refer to panel B. Error bars are the standard deviation of NanoSIMS measurements on three different SLB patches with very similar ¹³C₃-DSPC concentrations.

Figure 5

Comparison of recombination data from micro- and nanoscale phase-separating mixtures. (A) ℜ(¹³C¹⁵N⁻) values for SLBs of lipid compositions displaying microscale liquid/liquid phase coexistence, microscale liquid/gel phase coexistence, and nanoscale liquid/liquid phase coexistence with isotopic labels either both in the same phase or in different phases. Composition A (nanoscale L_o/L_d): 20:20:25:35 ¹³C₃-DSPC, ¹⁵N-DSPC, CHOL, POPC. Composition B (nanoscale L_o/L_d): 20:20:25:20:15 ¹³C₃-DSPC/DSPC/CHOL/¹⁵N-POPC/POPC. Composition C (microscale gel/L_d): 20:20:60 ¹³C₃-DSPC/¹⁵N-DSPC/DOPC. Composition D (microscale L_o/LS): 20:20:25:20:15 ¹³C₃-DSPC/DSPC/CHOL/¹⁵N-DOPC/DOPC. (B) ℜ(¹³C¹⁵N⁻) can be more easily visualized by computing the difference between the measured ℜ(¹³C¹⁵N⁻) and the fit to the line through the data for ideally mixed monolayers. The error bars represent the propagated uncertainty from the standard deviation of each measurement of a different SLB patch (at least 3 per composition) and the average uncertainty in the monolayer data points (3 per composition) used to calculate the linear fit (see text for more details).