Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Summary

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Abstract

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Introduction

The sense of touch provides animals with crucial information about their environment. Depending on the applied force, touch is perceived as innocuous, pleasurable, or painful. The tissue deformation during touch is detected by specialized mechanoreceptor cells embedded in the skin that express receptor proteins, most commonly ion channels. The steps linking force perception to ion channel activation during touch and pain are not fully understood. Even less is known about how the skin tissue filters mechanical deformation and whether mechanoreceptors detect changes in strain or stress^1,2,

Protocol

1. Device Fabrication

1. Download the attached mask file (Supplemental File 1) and generate a chrome mask using a commercial service or in-house facility. As the smallest dimension on the device is 10 µm (actuator membrane thickness), ensure that the mask has sufficiently high resolution, within ± 0.25 µm, to reliably produce the features.
2. Follow standard SU-8 photolithography methods (e.g., references^24,25,26) to fabricate the mold for subsequent production of PDMS devices; a summary of th....

Results

SU-8 Lithography and Chip Bonding
The lithography protocol and PDMS molding follow standard procedures. Details can be found elsewhere^23,24,25,26. The PDMS should peel off the wafer without problems after curing. If the SU-8 features rip off during PDMS peeling, either the SU-8 adhesion layer or the silanization was insufficient. If plasma-a.......

Discussion

This protocol demonstrates a method for delivering precise mechanical stimulation to the skin of a roundworm trapped in a microfluidic chip. It is intended to facilitate the integration of physical stimuli for answering biological questions and aims to streamline mechanobiology research in biological labs. This method extends previous assays to assess the function of mechanosensory neurons in C. elegans. Previous quantitative and semi-quantitative techniques measured forces^1,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chrome mask	Compugraphics (http://www.compugraphics-photomasks.com/)		5'', designed in AutoCAD (Autodesk, Inc.)
Chrome mask	Mitani-Micronics (http://www.mitani-micro.co.jp/en/)		5'', designed in AutoCAD (Autodesk, Inc.)
Chrome mask	Kuroda-Electric (http://www.kuroda-electric.eu/		5'', designed in AutoCAD (Autodesk, Inc.)
4'' Silicon wafer (B-test)	Stanford Nanofabrication Facility
SU-8 2002	MicroChem
SU-8 2050	MicroChem
Spin-coater	Laurell Technologies	WS-400BZ-6NPP/LITE
Exposure timer	Optical Associates, Inc	OAI 150
Illumination controller	Optical Associates, Inc	2105C2
SU-8 developer	MicroChem
2-Propanol	Fisher Scientific	A426F-1GAL
Acetone	Fisher Scientific	A18-4
Trichloromethylsilane (TCMS)	Sigma-Aldrich	92361-500ML	Caution: TCMS is toxic and water-reactive
Sylgard 184 Elastomer Kit	Dow Corning		PDMS prepolymer
Biopsy punch, 1 mm	VWR	95039-090
Oxygen Plasma Asher	Branson/IPC
Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW	New England Small Tube Corporation	NE-1300-01
Nalgene syringe filter, 0.22 μm	Thermo Scientific	725-2520	to filter all solution, small particles would clog the chip
Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID	Solomon Scientific	BPE-T50
Syringe, 1 ml	BD Scientific	309628	for worm trapping and release
Syringe, 20 ml	BD Scientific	309661	for gravity-based flow
Gilson Minipuls 3, Peristaltic pump	Gilson		to suck solutions and worms out of the chip
Microfluidic flow controller, equipped with 0–800 kPa pressure channel	Elveflow	OB1 MK3	pressure delivery
Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD	McMaster-Carr	5195 T52	connection from house air to pressure pump
Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD	McMaster-Carr	5195 T51	connect pressure pump to small tubng
Push-to-Connect Tube Fitting for Air	McMaster-Carr	5111K468	metric - imperial converter
Straight Connector for 6 mm × 1/4″ Tube OD	McMaster-Carr	5779 K258
Leica DMI 4000 B microscopy system	Leica
63×/1.32 NA HCX PL APO oil objective	Leica	506081
Hamamatsu Orca-Flash 4.0LT digital CMOS camera	Hamamatsu	C11440-42U
Lumencor Spectra X light engine	Lumencor		With cyan and green/yellow light source
Excitation beam splitter	Chroma	59022bs	in the microscope
Hamamatsu W-view Gemini Image splitting optics	Hamamatsu	A12801-01	to split green and red emission and project them on different areas on the camera chip
Emission beam splitter	Chroma	T570lpxr	in the image splitter
Emission filters GCamp6s	Chroma	ET525/50m	in the image splitter
Emission filters mCherry	Chroma	ET632/60m	in the image splitter