Cell Biology

The sensing of and response to ambient chemical gradients by microorganisms via chemotaxis regulates many microbial processes fundamental to ecosystem function, human health, and disease. Microfluidics has emerged as an indispensable tool for the study of microbial chemotaxis, enabling precise, robust, and reproducible control of spatiotemporal chemical conditions. Previous techniques include combining laminar flow patterning and stop-flow diffusion to produce quasi-steady chemical gradients to directly probe single-cell responses or loading micro-wells to entice and ensnare chemotactic bacteria in quasi-steady chemical conditions. Such microfluidic approaches exemplify a trade-off between high spatiotemporal resolution of cell behavior and high-throughput screening of concentration-specific chemotactic responses. However, both aspects are necessary to disentangle how a diverse range of chemical compounds and concentrations mediate microbial processes such as nutrient uptake, reproduction, and chemorepulsion from toxins. Here, we present a protocol for the multiplexed chemotaxis device (MCD), a parallelized microfluidic platform for efficient, high-throughput, and high-resolution chemotaxis screening of swimming microbes across a range of chemical concentrations. The first layer of the two-layer polydimethylsiloxane (PDMS) device comprises a serial dilution network designed to produce five logarithmically diluted chemostimulus concentrations plus a control from a single chemical solution input. Laminar flow in the second device layer brings a cell suspension and buffer solution into contact with the chemostimuli solutions in each of six separate chemotaxis assays, in which microbial responses are imaged simultaneously over time. The MCD is produced via standard photography and soft lithography techniques and provides robust, repeatable chemostimulus concentrations across each assay in the device. This microfluidic platform provides a chemotaxis assay that blends high-throughput screening approaches with single-cell resolution to achieve a more comprehensive understanding of chemotaxis-mediated microbial processes.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress–signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual‐flow‐RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved.

Key features

• Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments.

Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 μm width, and 500 μm height.

Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines.

• Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC.

Graphical overview

Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis. (a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

For several decades, aging in Saccharomyces cerevisiae has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and high-throughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy.

Key features

• This protocol requires access to a photolithography lab in a cleanroom facility.

• Photolithography process for patterning photoresist on silicon wafers with multiple different feature heights.

• Soft lithography process for making PDMS microfluidic devices from silicon wafer templates.

The measurement of single cell size remains an obstacle towards a deeper understanding of cell growth control, tissue homeostasis, organogenesis, and a wide range of pathologies. Recent advances have placed a spotlight on the importance of cell volume in the regulation of fundamental cell signaling pathways including those known to orchestrate progression through the cell cycle. Here we provide our protocol for the Fluorescence Exclusion Method (FXm); references to the development of FXm; and a brief outlook on future advances in image analysis which may expand the range of problems studied utilizing FXm as well as lower the barrier to entry for groups interested in adding cell volume measurements into their experimental repertoire.

Microfluidic devices have become an integral method of cardiovascular research as they enable the study of shear force in biological processes, such as platelet function and thrombus formation. Furthermore, microfluidic chips offer the benefits of ex vivo testing of platelet adhesion using small amounts of blood or purified platelets. Microfluidic chips comprise flow channels of varying dimensions and geometries which are connected to a syringe pump. The pump draws blood or platelet suspensions through the channel(s) allowing for imaging of platelet adhesion and thrombus formation by fluorescence microscopy. The chips can be fabricated from various blood-compatible materials. The current protocol uses commercial plastic or in-house polydimethylsiloxane (PDMS) chips. Commercial biochips offer the advantage of standardization whereas in-house chips offer the advantage of decreased cost and flexibility in design. Microfluidic devices are a powerful tool to study the biorheology of platelets and other cell types with the potential of a diagnostic and monitoring tool for cardiovascular diseases.

This protocol provides a detailed description of how to fabricate and use the dual-flow-RootChip (dfRootChip), a novel microfluidic platform for investigating root nutrition, root-microbe interactions and signaling and development in controlled asymmetric conditions. The dfRootChip was developed primarily to investigate how plants roots interact with their environment by simulating environmental heterogeneity. The goal of this protocol is to provide a detailed resource for researchers in the biological sciences wishing to employ the dfRootChip in particular, or microfluidic devices in general, in their laboratory.

Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging. multFYLM captures approximately one thousand rod-shaped fission yeast cells from up to six different genetic backgrounds or treatment regimens. The immobilized cells are fluorescently imaged for over a week, while the progeny cells are removed from the device. The resulting datasets yield high-resolution multi-channel images that record each cell’s replicative lifespan. We anticipate that the multFYLM will be broadly applicable for single-cell whole-lifespan studies in the fission yeast (Schizosaccharomyces pombe) and other symmetrically-dividing unicellular organisms.

In many environments, bacteria favor a sessile, surface-attached community lifestyle. These communities, termed biofilms, are ubiquitous among many species of bacteria. In some cases, biofilms form under flow conditions. Flow chambers, and in particular microfluidic channels, can be used to observe biofilm development and physiological effects while varying nutrient conditions, flow velocities, or introducing antimicrobials to the biofilm in real time. Here, we describe a microfluidic-based kill-kinetics assay for the observation of antimicrobial effects on biofilms under flowing conditions.

Calcium is one of the most important intracellular messengers in biological systems. Ca²⁺ microfluorimetry is a valuable tool to assess information about mechanisms involved in the regulation of intracellular Ca²⁺ levels in research on cells and in living tissues. In essence, the use of a dye that fluoresces in the presence of a target substance allows the detection of changes in the concentration of this molecule by determining the changes in the fluorescence of the probe (increases or decreases, depending on the nature of the dye used; for a review see Tsien et al. 1985). In this regard, there have been developed two different methodologies to assess intracellular Ca²⁺ measurements. On the one hand, ratiometric methods are based on the use of a ratio between two fluorescence intensities linked to the physicochemical properties of the probe. This allows correction of artifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated since they require more expensive equipment with the possibility to change the wavelength emission/detection in a rapid way. Some ratiometric Ca²⁺ indicators are Fura-2 and Indo-1. On the other hand, on binding to Ca²⁺, indicators used for non-ratiometric measurements show a shift in their fluorescence intensity (the free indicator has usually a very weak fluorescence). Therefore, although an increase in fluorescence signal can be related directly to an increase in Ca²⁺ concentration, the fluorescence intensity depends on many factors such as acquisition conditions, probe concentration, optical path length, balance between the affinity constants of proteins binding Ca²⁺, among others. However, the fluxes of Ca²⁺ are of such a magnitude that these interferences are minor contributors to biases in the measurements. There are many non-ratiometric calcium indicators, some of which are Fluo-3, Fluo-4 and Calcium-Green-3. Consequently, the most suitable Ca²⁺-probe for each experiment will depend on the range of Ca²⁺ concentration that has to be evaluated, instrumentation, loading requirements, etc. In the present report we describe the protocol employed to quantify intracellular Ca²⁺ changes in peritoneal macrophages using Fura-2 as a fluorimetric probe and a microfluorimetric protocol that allows quantification of responding cells to a given stimulus, localization of the main intracellular domains sensing Ca²⁺ changes and a time-resolved analysis of the Fura-2 fluorescence that reflects the intracellular dynamics of Ca²⁺ in these cells (Través et al., 2013).