SCA can describe cell-to-cell heterogeneities in cell populations that are not accessible with bulk methods. Some of the applications of SCA include medical diagnostics, basic research, and even bioprocess development in which heterogeneity within cell populations seems to play a major role. Understanding population dynamics might also be the key for greater productivity and product quality in large-scale production in biotechnology3, 6, 38,39. FC is probably the most commonly used single-cell analytical technology, because numerous commercial and user‑friendly devices are available on the market. In the present study, the population dynamics in synchronous algal cultures, which have not been well characterized, were investigated as model processes in more detail by FC to reveal the potential of FC for label‑free bioprocess monitoring. In addition to the experimental data, a theoretical approach was included to aid in interpretating the SSC signal in relation to cellular ingredients or structural parts of the cell.
Bulk dynamics in synchronized photoautotrophic cultures as a model process
FC was applied to a light–dark synchronized strain (CC1690) of Chlamydomonas reinhardtii. Photoautotrophic eukaryotic cells can often be synchronized by systematic application of light–dark periods and dilutions to equal cell density at the end of the dark phase (Banfalvi 2011)40. Under the described experimental conditions, the cells showed a characteristic change in cell number, mean cellular diameter, and chlorophyll accumulation over time. These general developmental stages are typical for light–dark synchronized algal cultures29,30,41.
Validity of the chlorophyll fluorescence signal measured by FC and interpretation of the SSC signal in algal cells
In the past, it was shown that quantification of the chlorophyll amount per cell is technically feasible by using the intensity of the emitted chlorophyll fluorescence per cell as a quantitative measure16,17,18,42. The linearity of the FC data is not always guaranteed. The most likely explanation is an unfavorable combination of cell size and geometry of the laser beam within the FC device used when using pulse height to measure the fluorescence intensity24,25. This leads to an underestimation of the FC signal. For polydisperse cell size distributions, this ultimately will lead to an underestimation of cell-to-cell heterogeneity by the FC device. Under the specific conditions used in this study, systematic underestimation of the detected chlorophyll fluorescence intensity by the FC device was described in relation to the reference analysis (Figure 1). The underestimation was much higher than expected and already affected relatively small algal cells after 3 h of illumination. Nevertheless, the mean of the cell size distribution as determined by Coulter Counter measurements never exceeded the laser beam diameter of 15 µm (Figure 1). Even during the latest time points within the light phase, only a small proportion of cells exceeded the laser diameter (Figure S2). In addition to the cell size changing over time, another specific property of Chlamydomonas cultures might cause the unexpected deviation from reference techniques. During most of the developmental cycle, cells of Chlamydomonas have a typical elliptical shape31, but regardless of the “real” cell shape, the Coulter Counter device displays the determined cell sizes as the equivalent sphere diameter, assuming a spherical shape of the particles after quantifying the particle volume42. In addition to this, the movement of the cells through the flow cuvette is supported in FC devices with a laminar flow pattern within the carrier liquid enabling hydrodynamic fixation of the cells. Thus, elliptical cells will be aligned with their longer diameter parallel to the flow direction while moving through the flow cuvette. This virtually increases the diameter of the cells during their interaction with the laser beam. This very likely leads to the pronounced underestimation of the chlorophyll content that was observed even with relatively small cells in synchronized cell cultures of Chlamydomonas.
The measured SSC intensity derived from a single cell is based on the interplay between different parameters11,12,13,14,15. Briefly, a single cell can be understood as a particle with different constituents in different volume fractions. The different constituents can be approximated as granular structures with given RI and different diameters. It is well accepted that the SSC signal depends on the cellular complexity of the single cells in which different small subcellular granular structures, such as mitochondria, contribute strongly to the SSC intensity11,12,13,14,15. To the best of our knowledge, this interpretation has so far been restricted to non-photosynthetic cells. Recent calculations based on the Lorenz-Mie theory of light scattering revealed that most likely lipid bodies and starch granules contribute to this signal within algal cells, with the starch granules having the highest SSC intensity (Figure 2, Table 1, Figure S4). Calculations were made with the freeware tool MiePlot, which was originally created for quantitative analysis of scattering phenomena in the atmosphere32. The large difference between the reference RI (approximated as RI from cytoplasm) and RI of starch granules and lipid droplets causes the high contribution of both storage compounds to the SSC signal in algal cells. This contribution is greater than that of mitochondria and much greater than the contribution of a ribosome.
The described nonlinearity in the chlorophyll fluorescence signal will also be present in other commercially available FC devices that share a similar laser beam geometry25, making it of great interest for the scientific community or in industrial applications. Questions arise about how the nonlinearity may affect the SSC signal and how the performance of the FC device is disrupted for quantitative cell characterization. The nonlinearity and thus the underestimation of cell-to-cell heterogeneity will also be present in the SSC signal, because emitted light and the SSC intensity are gathered in the same optical path (in 90° direction) and are subsequently separated into different spectral channels through optical components. Thus, the underestimation should be identical for SSC and chlorophyll fluorescence within each distinct cell. The data generated by the FC device is still of high value for bioprocess monitoring and can be applied in various situations. First, cell-to-cell heterogeneity is underestimated for larger and elliptical cells but it can still be determined and used for semi‑quantitative culture characterization or quality controls in biotechnological production processes. Second, subpopulations can still be visualized and identified under the described circumstances. Third, as SSC and chlorophyll fluorescence are gathered within the same optical path of the device, the specific relationship between both parameters can still be used as a quantitative measure for bioprocess monitoring. Such possible sensitive measures include determination of covariance and correlation coefficients over the processing time. These considerations have been further applied on the fast-growing model culture.
Single cell dynamics determined by FC measurements
For decades, it has been known that synchronization of cell cultures increases homogeneity of the cells compared to unsynchronized bulk cultures. Results from this study show that synchronized Chlamydomonas cells possess unexpected cell-to-cell diversity both in chlorophyll content (RCCC) and SSC (Figure S2 and S3, Figure 3). The observed heterogeneity was not diminished after “re-cloning” the cells before preparation of the pre‑culture. This means that the cell-to-cell heterogeneity was not based on genetic diversity in the cell culture. A high correlation between RCCC and SSC was found throughout the developmental cycle of the cells, regarding the means of the distributions (Figure 4). This matches the interpretation of both parameters for photosynthetic cells, which covers the cellular complexity, including storage compounds (starch and lipid droplets) in particular, within the parameter SSC, and the chlorophyll quantity (RCCC), which relates to the size of the photosynthetic apparatus within each cell. From a naive perspective, a cell containing a large amount of chlorophyll might also exhibit a high photosynthetic capability and thus should be able to accumulate a large quantity of storage compounds, coded by the SSC. Surprisingly, a relatively low correlation between both parameters was described at the single-cell level at each distinct time point (Figure 3 and Figure 4). Over time, the correlation changed, which implies nonparallel development of the cells. A very detailed analysis of starch-related single-cell dynamics in synchronous algal cultures was carried out by Garz and co-workers using an advanced microscopic imaging approach, based on Second Harmonic Generation microscopy29. They described a low and dynamic correlation between single-cell starch content and cellular volume in synchronized Chlamydomonas cells. Additionally, the former study also analyzed single-cell starch degradation rates during the night phase, indicating a complex mode of starch degradation that requires largely unknown regulatory mechanisms. The cells in the present study have been grown under identical conditions but the focus was more on development of the cells during the light phase. A high cell-to-cell heterogeneity for the starch content has been also shown in microscopic images with other strains of Chlamydomonas30. Additionally, the recent study described a remarkable differentiation of the cell population into two subpopulations during the time of culture. Both subpopulations possess highly different properties in terms of RCCC and SSC, but the exact biological implications remain unclear (Figure 3, e.g., 10 h light, 11 h light).
Finally, the question arises about what drives the deviating developmental paths and the high cell‑to-cell heterogeneity. In the past, many studies tried to identify the reason for heterogeneity in different cell cultures. For example, detailed analyses of bacterial cells clearly revealed that isogenic DC exhibit significant heterogeneity for copy numbers of many proteins and transcripts43,44. It is believed that stochasticity of gene transcription or within other highly regulated cellular events is the key driver43. One of these highly regulated cellular events might be the physiological response of synchronized algal cells to the applied light–dark cycle, altering the photosynthetic apparatus. Pigment composition measurements and functional analysis of the photosynthetic apparatus of two synchronized strains of Chlamydomonas (CC125 and CC3491) implied non-equal accumulation over time of major multiprotein complexes of the photosynthetic apparatus, such as light harvesting and core complexes30. More detailed analysis of molecular events in synchronous cultures was carried out using two very advanced systems biology approaches that indicated complex physiological responses by the synchronized Chlamydomonas strains to the diurnal rhythm2,27. It was shown, for example, that around 85% of the genes were differentially expressed during different phases of the light–dark cycle, which illustrates concerted alteration within the cellular physiology2. The three studies illustrate numerous physiological alterations of Chlamydomonas due to the light–dark cycle that are ultimately linked at the molecular level to highly stochastic events43. Additionally, asymmetric partitioning of storage compounds and asymmetric cell division were considered in a theoretical approach as the reason for cell‑to‑cell heterogeneity, and it was concluded that a complex combination of different phenomena is necessary to explain the heterogeneity in synchronous cultures41. It appears that the cell-to-cell heterogeneity inside synchronized Chlamydomonas cultures is intrinsically driven and seems to be rather common and not an unusual trait.
Combined with these internal factors, external factors affecting the cell population might also be important. For algal cells, homogenous illumination and supply of nutrients including CO2 inside the culture vessel might be of great importance. To understand the external factors in more detail, a systematic analysis of mixing times, mass transfer, and light distribution should be performed in future for the photobioreactors used (devices used to culture the photoautotrophic cells).
The current study has shown that FC can be used for label-free process monitoring. The knowledge gained about the strong contribution of starch granules and lipid bodies inside the algal cells to the SSC signal in particular opens new paths for bioprocess monitoring in algal cultures, an as yet unexplored topic, especially in industrial production processes. Recent studies have shown the potential of analysis of distinct light-scattering properties of cell cultures using a novel technique called Photon Density Wave spectroscopy as a valuable tool for bioprocess monitoring45,46.
Another clear improvement to classical FC is the use of on-line FC in which the FC device is capable of automated sample analysis with very high time resolution7,47. Independently of the extensive usage of FC in different scientific disciplines and its huge potential, single-cell analysis has rarely been used to quantitatively study dynamics in synchronous algal cultures31.