SiCTeC: An inexpensive, easily assembled Peltier device for rapid temperature shifting during single-cell imaging.

Benjamin D Knapp,Kerwyn Casey Huang,Lillian Zhu,Melanie Blokesch

doi:10.1371/journal.pbio.3000786

Benjamin D Knapp, Kerwyn Casey Huang + Show 2 more

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https://doi.org/10.1371/journal.pbio.3000786

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Abstract

Single-cell imaging, combined with recent advances in image analysis and microfluidic technologies, have enabled fundamental discoveries of cellular responses to chemical perturbations that are often obscured by traditional liquid-culture experiments. Temperature is an environmental variable well known to impact growth and to elicit specific stress responses at extreme values; it is often used as a genetic tool to interrogate essential genes. However, the dynamic effects of temperature shifts have remained mostly unstudied at the single-cell level, due largely to engineering challenges related to sample stability, heatsink considerations, and temperature measurement and feedback. Additionally, the few commercially available temperature-control platforms are costly. Here, we report an inexpensive (<$110) and modular Single-Cell Temperature Controller (SiCTeC) device for microbial imaging—based on straightforward modifications of the typical slide-sample-coverslip approach to microbial imaging—that controls temperature using a ring-shaped Peltier module and microcontroller feedback. Through stable and precise (±0.15°C) temperature control, SiCTeC achieves reproducible and fast (1–2 min) temperature transitions with programmable waveforms between room temperature and 45°C with an air objective. At the device’s maximum temperature of 89°C, SiCTeC revealed that Escherichia coli cells progressively shrink and lose cellular contents. During oscillations between 30°C and 37°C, cells rapidly adapted their response to temperature upshifts. Furthermore, SiCTeC enabled the discovery of rapid morphological changes and enhanced sensitivity to substrate stiffness during upshifts to nonpermissive temperatures in temperature-sensitive mutants of cell-wall synthesis enzymes. Overall, the simplicity and affordability of SiCTeC empowers future studies of the temperature dependence of single-cell physiology.

Highlights

While chemical perturbations during single-cell imaging experiments have been made possible by microfluidic technologies [1, 2], other environmental variables such as temperature have been more difficult to precisely and rapidly manipulate during an experiment
Single-Cell Temperature Controller (SiCTeC) consists of a ring-shaped Peltier module that permits illumination of samples during microscopy—which is crucial for single-cell investigations (Fig 1A and 1B)—and ensures that heating is uniform across the sample
We focused on temperature-sensitive mutants of two penicillin-binding proteins (PBPs) in E. coli

Summary

Introduction

While chemical perturbations during single-cell imaging experiments have been made possible by microfluidic technologies [1, 2], other environmental variables such as temperature have been more difficult to precisely and rapidly manipulate during an experiment. How cells respond to temperature fluctuations, such as during transitions into and out of a host, remains understudied; in E. coli, the few studies of temperature shifts have shown long timescales for growth-rate equilibration [12] and transient changes to the synthesis rate of tRNA synthetases [13]. While intriguing, these limited results highlight the need for a device that enables single-cell imaging to investigate how individual cells respond to temperature fluctuations at high spatiotemporal resolution

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