Inductively coupled plasma-mass spectrometry (ICP-MS) is a common method for elemental quantification in many different applications. Also, in studies with biological cells, such as bacteria, plant or mammalian cells, total elemental levels per cell can be averaged over a cell population using ICP-MS. However, information about the heterogeneity of a cell population is lost, due to the required sample preparation. Single-cell ICP-MS (scICP-MS) opens a new perspective into variations within a cell population by analyzing one cell at a time. This allows new insights for cell studies, such as drug uptake, toxicity assessments, and in the presence and behavior of endogenous elements in cells. However, this raises the question of the interpretation of heterogeneous elemental distribution within a cell population, as well as the evaluation of biological variance and reproducibility. Here it is shown that the heterogeneity of a cell population can depend on the growth state and the growth condition.

For this purpose, the green alga Chlamydomonas reinhardtii was cultivated in two light modes, in 16 h light and 8 h darkness (16:8) as well as in 24 h light (24:0). Once the cultivation started, samples for scICP-MS analysis were harvested after 4 h, 8 h, 12 h, 24 h, 28 h, 32 h, 36 h, 48 h and 52 h. For scICP-MS, the endogenous element Mg is analyzed due to its high abundance in the alga as it is the central atom of chlorophyll. In addition, the average Mg content per cell was determined using classical ICP-MS. During the first 24 h of cell growth, the Mg content per cell is bimodally distributed in both lightning conditions. That suggests that two cell subpopulations are in two different growth phases, before and after mitosis. The number of cells with a high Mg content increases during that period. After 24 h, two subpopulations are still observed with a median Mg content at 15 and 125 fg/cell. In the following hours of cell growth, the influence of the different light conditions comes into play. After 48 h, the Mg content of the cells grown under continuous light (24:0) show a similar distribution as at the beginning of cultivation. After 52 h, the Mg content is still bimodally distributed, only the number of cells before or after mitosis varies. Both is not the case with cells grown under day-night conditions (16:8). Here, a gaussian distribution of the Mg content is observed after 48 h of growth with a median Mg content of 190 fg/cell. After 52 h, the Mg content is still gaussian-like distributed with a median of 200 fg/cell. This suggests that the growth of C. reinhardtii was synchronized by the day-night conditions, meaning that all cells are in the same growth stage. This finding has been supported by subsequent experiments. 

While the classical determination of the average Mg content per cell via ICP MS showed no significant differences between the 24:0 and 16:8 conditions, only scICP-MS resolved the influence of the light conditions to the cell growth and synchronization. In the case of C. reinhardtii, the latter is a crucial requirement for ensuring biological reproducibility in uptake studies.