Single-Cell Physiology

Abstract

Single-cell techniques have a long history of unveiling fundamental paradigms in biology. Recent improvements in the throughput, resolution, and availability of microfluidics, computational power, and genetically encoded fluorescence have led to a modern renaissance in microbial physiology. This resurgence in research activity has offered new perspectives on physiological processes such as growth, cell cycle, and cell size of model organisms such as Escherichia coli. We expect these single-cell techniques, coupled with the molecular revolution of biology's recent half-century, to continue illuminating unforeseen processes and patterns in microorganisms, the bedrock of biological science. In this article we review major open questions in single-cell physiology, provide a brief introduction to the techniques for scientists of diverse backgrounds, and highlight some pervasive issues and their solutions.

Keywords: cell cycle; growth; microbiology; microfluidics; quantitative biology; systems biology.

Figure 1. Single-cell experiments of Escherichia coli reveal the size control mechanism by parameters that are not accessible in a population average experiment

(a) Generation time, τ, is negatively correlated with newborn size, s_b, ruling out the timer model. (b) Single-cell data show a systematic deviation from the growth law. The population average data (red lines and red symbols) confirm the classic work of Schaechter et al. (79), but single-cell data (black symbols) systematically deviate from the growth law. This deviation is related to how cells control their size in a steady-state growth condition. (c) Cells add a constant size, Δ, from birth to division, independent of their size at birth, s_b. Figure based on data from Wang et al. (98).

Figure 2. Cell cycle of Escherichia coli cells

(a) In slow-growth conditions (generation time larger than C and D periods combined), the cell cycle has three distinct steps: the time between birth and initiation of chromosome replication (B period, green arrow; length depends on generation time), the time of chromosome replication (C period, blue arrow; 40 min), and the gap between termination of chromosome replication and division (D period, orange arrow; 20 min). (b) In fast-growth conditions, multiple cell cycles must overlap because the period C + D is constant and longer than the generation time. The illustration shows chromosome replication spanning two generations. Under all growth conditions, one round of the replication cycle must be coupled to one round of the division cycle.

Figure 3. Shape changes of Escherichia coli cells during the transition from slow-growth to fast-growth conditions

(a) Anisotropic change of cell morphology observed by Woldringh et al. (104) leading to the conclusion that the old pole is metabolically inert in terms of the turnover rate of constituent materials relative to the sides of the cell wall. (b) A nutrient shift-up experiment in a microfluidic device consistent with and extending the anisotropic interpretation. The tapered pole was indeed the result of the old pole dimensions changing slower than the size of the adjacent side walls. The tapered old pole eventually regained normal shape and dimensions, indicating the cell pole is not completely inert. Panel a is reprinted from Reference , with permission from Elsevier.

Figure 4. Strong effect of fluorescence imaging on growth

(a) A phase contrast/GFP overlay of a single field of view of Escherichia coli cells expressing Ssb-GFP (a GFP fusion of a single-stranded binding protein) grown in steady-state conditions. Cells imaged approximately 20 times per cell cycle for approximately 3 days with a fluorescence excitation illumination gradient across the field of view (cells on the left-hand side experience minimal illumination, whereas the exposure is maximum on the right-hand side). (b) A slice from an image taken with the same optical alignment of an FITC (fluorescein isothiocyanate) solution showing the intensity gradient. (c) Co-plot of FITC fluorescence intensity (a.u.) and growth rate (1/h) versus position in the field of view. As fluorescence intensity increases, the growth rate decreases. The insets show the intensity profile of the fluorescence image along a line passing through cells from different channels, as they experience different excitation light intensity.