Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging

Summary

An innovative method for fabricating microfluidic devices using polyethylene terephthalate (PET) lamination significantly reduces the cost and complexity of entrapping and imaging multiple live zebrafish embryos.

Abstract

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calcium signal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

Introduction

The ability to respond adequately to injury is critical to the survival of every organism at every scale, from a single cell to multiple tissues. Wounding and its associated responses, such as phagocyte recruitment to damaged areas¹ are, therefore, significant topics in cell and tissue biology. Wounds are sensed immediately after the tissue barrier is breached, causing a tissue gradient response involving wound contraction^2,3 that coordinates wound healing and subsequent regrowth⁴. Due to the mechanical nature of this contraction, tools used during experimentatio....

Protocol

This study used 3 days post fertilization (dpf) embryos but can be designed to use 2-14 dpf embryos. The zebrafish experiment was conducted by internationally accepted standards. The Animal Care and Use Protocol was approved by The Purdue Animal Care and Use Committee (PACUC), adhering to the Guidelines for using Zebrafish in the NIH Intramural Research Program (protocol number: 1401001018).

1. Assembly of the PET microfluidics device

NOTE: Any part of this protocol may be paused at any time except for steps 1.8 and 1.9, which are sensitive to the drying time of cyanoacrylate.

1. Design ....

Results

For comparison of image quality produced by a laser scanning confocal with and without the aid of the RADISH, embryos expressing the intensiometric calcium biosensor GCaMP6f^22,24 in the outer epithelial layer were stained using MemGlow 560 and imaged with an inverted laser scanning confocal covering the entire thickness of the tail fin fold at a time interval of 1 min per frame (Video 2). Without the device (Figure .......

Discussion

The core premise of the PET lamination method involves cost reduction and minimization of technical barriers to entry compared to traditional means of creating microfluidic devices, such as soft lithography or PDMS molding. As such, the only critical steps in this protocol are the accurate alignment of PET layers during lamination and waterproofing of the device edges after construction. All other parts of the method may be modified according to experimental needs (e.g., if immobilization of younger or older embryos is r.......

Materials

Name	Company	Catalog Number	Comments
25 mm diameter round coverglass #1 thickness	Chemglass Life Sciences	CLS-1760-025	Base mounting material. Thickness was chosen based on the specifications of the microscope lens.
Adobe Illustrator v28.5	Adobe	N/A	Vector editor for designing the device pattern.
Calcium Chloride Dihydrate	Fisher	C79	For making E3 medium.
Design PNG Files (Online Mirror)	N/A	N/A	https://i.ibb.co/QPYj7BL/multipositionalv7-1.png https://i.ibb.co/FDpQYXc/multipositionalv7-2.png https://i.ibb.co/5TG4SB6/multipositionalv7-3.png
Design Space for Desktop v8.39	Cricut	N/A	Proprietary software to drive the craft cutter.
Fusion Plus 7000L	GBC	1703098	Thermal laminator. This specific model was selected for quality of life features, such as maximum sheet stack size.
German Carbide Premium Blade	Cricut	10396595	Replacement blade for craft cutter.
Gray Basic Tool Set	Cricut	10307854	Tool set for weeding cuts, cleaning mats, and mounting PET sheets. Optional.
Magnesium Chloride Hexahydrate	Acros Organic	223211000	For making E3 medium.
Maker 3	Cricut	10669040	Craft cutter. The Cricut Maker 3 was selected over the Silhouette Cameo 4 for software user friendliness, but any craft cutter capable of reading black and white images will work.
MemGlow 560	Cytoskeleton	MG02-10	Red live cell dye for cell membranes. No washing needed. Used in Figure 3.
No. 22 Carbon Scalpel Blade	Surgical Design	22-079-697	Scalpel blade for manual cut adjustments. Any similar craft knife (e.g., X-acto #2 knife) will also work.
Potassium Chloride	Fisher	BP366	For making E3 medium.
Sodium Chloride	Fisher	BP358	For making E3 medium.
StandardGrip Adhesive Cutting Mat	Cricut	10138842	Mounting mat for craft cutter. The LightGrip cutting mat will also work, but avoid the StrongGrip and FabricGrip cutting mats as the strength of the adhesive may warp the final cut during weeding and removal. Adhesive on mats will eventually wear out with continued use; replace mats as needed.
Stationery Tape 12 mm	Deli	30014	Office tape. Deli brand was chosen via testing for ease of removal after lamination.
Super glue, liquid	Loctite	1364076	Cyanoacrylate glue. Glue used must be liquid. Gel formulations will not sufficiently seal or waterproof device edges.
PET Thermal Laminating Pouches, 3 mil	Scotch	TP3854	PET sheets with thermal adhesive. Sheets arrive as sandwiched pairs and should be separated before use. Cut with adhesive side facing down.
PET Thermal Laminating Pouches, 5 mil	Scotch	TP5854	PET sheets with thermal adhesive. Sheets arrive as sandwiched pairs and should be separated before use. Cut with adhesive side facing down.
Tricaine (MS-222)	Syndel	IC10310680	Fish anaesthetic.