In Situ Cell-by-Cell Imaging and Analysis of Small Cell Populations by Mass Spectrometry

Abstract

Molecular imaging by mass spectrometry (MS) is emerging as a tool to determine the distribution of proteins, lipids, and metabolites in tissues. The existing imaging methods, however, mostly rely on predefined rectangular grids for sampling that ignore the natural cellular organization of the tissue. Here we demonstrate that laser ablation electrospray ionization (LAESI) MS can be utilized for in situ cell-by-cell imaging of plant tissues. The cell-by-cell molecular image of the metabolite cyanidin, the ion responsible for purple pigmentation in onion (Allium cepa) epidermal cells, correlated well with the color of cells in the tissue. Chemical imaging using single-cells as voxels reflects the spatial distribution of biochemical differences within a tissue without the distortion stemming from sampling multiple cells within the laser focal spot. Microsampling by laser ablation also has the benefit of enabling the analysis of very small cell populations for biochemical heterogeneity. For example, with a ∼30 μm ablation spot we were able to analyze 3-4 achlorophyllous cells within an oil gland on a sour orange (Citrus aurantium) leaf. To explore cell-to-cell variations within and between tissues, multivariate statistical analysis on LAESI-MS data from epidermal cells of an A. cepa bulb and a C. aurantium leaf and from human buccal epithelial cell populations was performed using the method of orthogonal projections to latent structures discriminant analysis (OPLS-DA). The OPLS-DA analysis of mass spectra, containing over 300 peaks each, provided guidance in identifying a small number of metabolites most responsible for the variance between the cell populations. These metabolites can be viewed as promising candidates for biomarkers that, however, require further verification.

Surface analysis of lipids by mass spectrometry: More than just imaging

“…These techniques have been applied to the analysis of lipids (mostly PLs) from a variety of substrates including tissue [107,[123][124][125], cell pellets [126] and chicken egg yolk [119]. The LAESI approach has found application for single cell analysis [127,128]. Single cells (plant and human cheek cells)…”

Biomolecular Imaging with a C60-SIMS/MALDI Dual Ion Source Hybrid Mass Spectrometer: Instrumentation, Matrix Enhancement, and Single Cell Analysis

“…Nanostructure initiator MS is another laser microprobe-based approach and is capable of imaging with~20 μm resolution for smaller biomolecules (m/z G1500) in single cells, and can achieve yoctomole-range detection limits under ideal conditions [9]. Furthermore, the advent of ambient ion sources such as desorption electrospray ionization [10,11], laser ablation electrospray ionization [12][13][14], and liquid microjunctions [15,16] have recently extended microprobe MSI to ordinary environmental conditions, enabling in situ analysis of biological samples, including live organisms [16][17][18][19].…”

Abscisic Acid–Responsive Guard Cell Metabolomes of Arabidopsis Wild-Type and gpa1 G-Protein Mutants

“…For example, using a nanoelectrospray tip to sample individual geranium (Pelargonium zonale) petal, leaf, and stem cells, ;20 compounds, primarily alcohols and terpenoids, were identified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) (Lorenzo Tejedor et al, 2012); however, it is difficult to identify more metabolites and achieve quantitative analysis. In another approach, laser ablation ESI-MS was applied to onion (Allium cepa) epidermal cells and sour orange (Citrus aurantium) oil glands and ;10 metabolites were identified, primarily sugars and flavonoids (Shrestha et al, 2011). Because the reported sampling diameter and depth were both 30 µm, this approach would not be applicable for single-cell sampling of small cells such as guard cells.…”