Submicron 3-D mass spectrometry imaging reveals an asymmetric molecular distribution on chemotaxing cells

Introduction

Mass spectrometry techniques are immensely useful for the identification and characterization of molecular components in biological samples.^1–3 For instance, mass spectrometry imaging (MSI) permits the characterization of molecular components with high sensitivity and without the need for labels or pre-selection of molecules of interest.⁴ Using MSI, molecules can be detected and localized simultaneously.⁵ The spatial resolution of MSI is ultimately determined by the dimensions of the desorption probe (from tens of nanometers to hundreds of micrometers).^6–8 One such technique, Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS), particularly excels in the ability to provide good spatial resolution. Recent developments of surface probes for the analysis of biological samples have been based on the search for higher molecular desorption yields; for example, the introduction of cluster and nanoparticle probes for surface interrogation of biological surfaces with enhanced secondary ion yield and reduced damage cross section has permitted the detection of a broad range of chemical classes.^9–15 In addition, the combination of high spatial resolution cluster ion probes with reduced damage sputtering sources permits a 3D characterization of biological samples.

D. discoideum are ~10 μm diameter motile eukaryotic cells that during starvation aggregate into dendritic streams to form a fruiting body as a mechanism to disperse spores to start new colonies. During aggregation, relayed pulses of cyclic adenosine monophosphate (cAMP) activate signal transduction pathways that cause cells to move toward the source of cAMP.¹⁶ The cells form dendritic aggregation streams, and because when the streams start forming, the single cells outside aggregation streams are consistently observed moving toward the streams, one can assign a front and back to a cell near a stream. Although there is some understanding of the distribution of selected cytoskeleton and signal transduction components in the moving cells, little is known about the general distribution of molecules in these cells.

In this report, we examine the distribution of chemical species associated with D. discoideum chemotaxis using a dual beam interrogation probe based on a 25 keV Bi₃⁺ imaging probe combined with a 20keV Ar₁₅₀₀⁺ sputtering beam in spectral, imaging, and delayed-extraction imaging TOF-SIMS modes for high spatial resolution. We observe an asymmetric distribution of three prominent molecules or molecule fragments in the aggregating cells, illustrating the advantages of 3D-MSI.

Methods

Results

Visual inspection of D. discoideum cells developed on the Au/Si chips showed that cells were aggregating (Figure 1A and B). Closer inspection showed a recurrent trend of single cells moving towards a recently formed stream. Cell integrity and morphology was preserved during the drying process, burst cells were not observed, and sample reproducibility was very high. A good correlation was observed between the optical and the TOF-SIMS images using HCBU, BA and BA-DE modes (see for example Figure 1B and C). Summed spectra of the TOF-SIMS images in HCBU mode were used to detect characteristic molecular ion signals from the single cells and from the streams in the process of aggregation. The m/z = 0-230 region of the spectra showed ions at m/z of 139.00, 160.98, 166.03, 184.07, 224.10 detected on the cells, and the characteristic substrate signal of Au⁺ ion at 196.97 detected in regions where no cells were present (Figure 2A). The m/z = 500-950 region showed ions at m/z of 518.30, 520.32, 740.50, 742.51, and 908.58 detected on the cells, as well as characteristic Au/Si chip substrate gold cluster ions at m/z 590.90 (Au₃⁺), 787.87 (Au₄⁺), and 418.94 (Au₂C₂H⁺) detected in regions where no cells were present (Figure 2B). These observations are in good agreement with our previous FT-ICR SIMS analysis of D. discoideum cells developed on Au/Si chips.¹⁷ There were no obvious differences in the HCBU mode m/z signals from single cells and from streams, and the ~1.2 μm resolution m/z signals showed an apparently homogenous distribution on the ~10 μm diameter cells and the streams of cells.

Figure 1. Optical (A, 4500×4500 μm and B, 120×120 μm field of view) and secondary ion total images (C, 120×120 μm field of view) of aggregating D. discoideum cells on a Au/Si substrate.

Data are representative of 3 independent experiments.

Figure 2. Representative TOF-SIMS positive ion HCBU mode spectrum of D. discoideum cells developed on a Au/Si substrate (250×250 μm field of view).

A and B show selected regions of the spectrum; insert shows the entire spectrum. Data are representative of 3 independent experiments.

Higher spatial resolution imaging (~300 nm resolution) was performed over smaller fields of view (40×40 μm; dashed squares in Figure 1B and C) using BA and BA-DE modes to study the distribution of molecular components in the cells and in the streams. The analysis has the added benefit that multiple 2-dimensional scans can be added, thus increasing the signal to noise and increasing the probability of observing lower abundance components.²⁴ There appeared to be asymmetric distributions on cells and streams of ions at m/z = 221, 236 and 240, while all the other signals were assigned to either Au/Si chip substrate peaks or components evenly distributed across the cells and the streams (Figures 3, 4, and S1). The signals at m/z = 221 and 236 were concentrated at the leading edge and sides of cells moving toward streams and at the borders/edges of streams, while the signal at m/z = 240 was concentrated at the edges of cells and the sides of streams (Figures 3, 4, and S1).

Figure 3. A) Optical image of aggregating D. discoideum cells (40×40 μm field of view). B) TOF-SIMS BA mode total secondary ion image of the same field. C-I) TOF-SIMS BA mode images pf this field at m/z C) 591 (Au₃⁺ substrate, light grey), D) 184, (Blue), E) 518 (Blue), F) 740 (Blue), G) 221 (Red), H) 236 (Red), and I) 240 (Red). J-L) Overlays of 591 (Au₃⁺ substrate, light grey), 184 (Blue), and J) 221 (Red), K) 236 (Red), and L) 240 (Red).

Data are representative of 3 independent experiments.

Figure 4. Distribution of additional ions in the 40×40 μm field of view in Figure 3 from the tridimensional analysis using BA mode at m/z A) 125, B) 147, C) 166, D) 184, E) 224, F) 518, G) 520, H) 740, I) 742, and J) 908.

Images F-J are binned x4 for clarity. Data are representative of 3 independent experiments.

Discussion

In this report, we observed asymmetric distributions of molecular components at m/z = 221, 236 and 240 on chemotaxing cells using 3D-MSI. We also observed several other molecular components that showed an even distribution on cells. Beam effects related to the incident angle of the primary beam during TOF-SIMS analysis (e.g., shadow effects) were not the cause of the asymmetric distributions, since they are expected for all the observed secondary ion signals and not a subset of them.

A challenge during 3D-MSI is the high diversity of biological molecules responsible for the ion signals, and complementary mass spectrometry tools would be needed for candidate assignments. One possibility is to perform tandem mass spectrometry for structural identification; however, due to the low secondary ion yields observed at the m/z of interest using TOF-SIMS, this approach is almost unpractical at the spatial resolution level required despite recent advances in MS/MS SIMS instrumentation.^14,25 Alternatively, ultrahigh resolution mass spectrometry might permit assignments based on accurate mass measurements.^17,26

Ethical approval

Dictyostelium discoideum is a unicellular invertebrate eukaryotic microbe, so no human subjects or vertebrate animal ethical approval was needed for these studies. Dictyostelium discoideum is a BSL-1 organism, and all work with live cells was done under a Texas A&M Institutional Biosafety Committee-approved BSL-1 protocol (IBC2018-155, with the most recent re-approval on March 22, 2022).

Data availability