Abstract
Current analytical methods are not capable of providing rapid, sensitive, and comprehensive chemical analysis of a wide range of cellular constitutes of single cells (e.g., lipids, metabolites, proteins, etc.) from dispersed cell suspensions and thin tissues. This capability is important for a number of critical applications, including discovery of cellular mechanisms for coping with chemical or environmental stress and cellular response to drug treatment, to name a few. Here we introduce an optically guided platform and methodology for rapid, automated recognition, sampling, and chemical analysis of surface confined individual cells utilizing a novel hybrid laser capture microdissection/liquid vortex capture/mass spectrometry system. The system enabled automated analysis of single cells by reliably detecting and sampling them either through laser ablation from a glass microscope slide or by cutting the entire cell out of a poly(ethylene naphthalate)-coated membrane substrate that the cellular sample is deposited on. Proof of principle experiments were performed using thin tissues of Allium cepa and cultured Euglena gracilis and Phacus cell suspensions as model systems for single cell analysis using the developed method. Reliable, hands-off laser ablation sampling coupled to liquid vortex capture/mass spectrometry analysis was conducted for hundreds of individual Allium cepa cells in connected tissue. In addition, more than 300 individual Euglena gracilis and Phacus cells were analyzed automatically and sampled using laser microdissection sampling with the same liquid vortex capture/mass spectrometry analysis system. Principal component analysis-linear discriminant analysis, applied to each mass spectral dataset, was used to determine the accuracy of differentiation of the different algae cell lines.
Highlights
Cell-to-cell variation is inherent to multi-cellular organisms
The results reported here serve as the foundation for future studies using LMD-LVC/ESI-MS for robust, high throughput automated chemical analysis of single cells deposited on a surface
A glass microscope slide with Allium cepa tissue or a polyethylene naphthalate (PEN) slide with algae cells deposited on it (Figure 1A) was placed in the regular microscope slide holder of the LMD system
Summary
Cell-to-cell variation is inherent to multi-cellular organisms. Even starting from a single genetic precursor, natural stochastic cellular processes create variation in their chemistries over time (Chung et al, 2014). Using laser ablation electrospray ionization-mass spectrometry (LAESI-MS), metabolic analysis was only achieved for major chemical constituents from 70 μm × 400 μm onion single cells (Shrestha and Vertes, 2009). Another MS-based technique, matrix assisted laser desorption/ionization-MS (MALDI-MS) has been used for single cell analysis from tissue (Li et al, 2000; Ong et al, 2015; Jansson et al, 2016) and, less commonly, from cellular suspensions (Urban et al, 2010). Sensitive and rapid chemical analysis of individual cells of dispersed cell populations and of thin tissues remains a challenge that, if solved, could provide solutions for a number of important biomedical problems
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