Highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry

To realise highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry (ICP-MS), we developed a modified high efficiency cell introduction system (HECIS), consisting of a large-bore high performance concentric nebulizer (LB-HPCN) with a centre capillary tube of 150 mu m inner diameter and a custom-made small-volume (15 cm(3)) on-axis spray chamber that uses a sheath gas flow near the chamber exit to suppress cell deposition. We also assembled an external ion pulse counting unit to directly read the ion pulse current from the electron multiplier of the ICP-MS via a function generator with no dead time, in order to obtain data with sufficiently high time resolution (i.e., 0.05-1 ms). As compared to a conventional ICP-MS working at its minimum integration time (10 ms), this assembly led to more than ca. 13-fold higher signal-to-background ratios for P-31, and made higher throughput of cells to the plasma more feasible. By using the modified HECIS and the external ion pulse counting unit for determination of the cell introduction efficiencies of different-sized unicellular microbes, including yeast (Saccharomyces cerevisiae), cyanobacterium (Synechocystis sp. PCC 6803), red algae (Cyanidioschyzon merolae 10D and Galdieria sulphuraria), and green alga (Chlamydomonas reinhardtii CC-125), it was revealed that their cell introduction efficiencies ranged from 86% (for C. reinhardtii CC-125 with a mean cell diameter of 6.4 mu m) to ca. 100% (for other microbes with mean cell diameters of 2.0-3.0 mu m), implying that by use of the ICP-MS system, the cell introduction efficiencies are able to reach approximately 100% and tend to decrease with increasing cell sizes (at least more than 3.1 mu m in mean diameter). A wide range of biologically important elements, such as C, Mg, Al, P. S, K, Ca, Cr, Mn, Fe, and Zn, were tested for reasonable detection using the ICP-MS system. Results likely corresponding to separate cell events were obtained for some elements present in each microbe.