Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Summary

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Abstract

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 µm thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Introduction

Radiation, including X-rays, gamma rays, and heavy-ion beam, is widely used for biological applications such as in cancer diagnosis and treatment, and for ion-beam breeding. Numerous studies and technical developments are currently focusing on the effects of radiation^1,2,3. Microbeam irradiation is a powerful means of identifying radiosensitive sites in living organisms⁴. The Takasaki Advanced Radiation Research Institute of National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki) has been developing a technology to irradi....

Protocol

1. Strains and maintenance

1. Select a suitable strain of C. elegans and Escherichia coli (food) depending on the purpose of the experiment.
   NOTE: In the present paper, wild-type N2¹⁰C. elegans (Figure 2A) is generally used, and HBR4:goeIs3[pmyo-3::GCamP3.35::unc-54 - 3'utr, unc-119(+)]V¹¹ is only employed for imaging assay. E. coli OP50 was used as food for C. elegans. Some mutants with abnormal body shape, such as the unc-119(e2498) III mutant with a coiled shape (Figure....

Results

Active C. elegans individuals could be immobilized successfully using an ultra-thin, wettable PDMS, microfluidic chip (worm sheet). We investigated the suitability of different cover films for sealing the worm sheet, as described in protocol section 3. To evaluate the sealing effects of the cover films, we determined the motility of animals 3 h after on-chip immobilization using cover glass (thickness: 130-170 µm), PET film (thickness: 125 µm), and PS film (thickness: ~130 µm), r.......

Discussion

On-chip immobilization of C. elegans under live conditions using a wettable PDMS microfluidic chip enables the efficient targeted microbeam irradiation of multiple animals. The ease of handling and features to prevent drying make this system suitable for applications not only in microbeam irradiation, but also in several behavioral assays. These worm sheets have already been commercialized and can be easily obtained. Conventional microfluidic chips, such as olfactory chips, are associated with problems including.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C. elegans wild-type strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	N2	Wild-type C. elegans strain generally used in this study
C. elegans unc-119(e2498) III mutant strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	CB4845	C. elegans strain only employed as an example of mutants with abnormal body shape
C. elegans transgenic strain HBR4	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	HBR4	The genotype of this transgenic C. elegans strain is HBR4:goeIs3[pmyo-3::GCamP3.35:: unc-54–3’utr, unc-119(+)]V. This strain was only employed for imaging observation.
E. coli strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	OP50	E. coli strain used as food for C. elegans
Worm Sheet IR (50/60)	Biocosm, Inc., Hyogo, Japan	BCM17-0001	Microfluidic chip with 25 straight 50/60-µm width channels used in all experiments and observation in this paper
Worm Sheet 60	Biocosm, Inc., Hyogo, Japan	BCM18-0001	Microfluidic chip with 20 straight 60 µm-width channels. This is sitable for adults 3-5 days after hatching at 20°C.
Worm Sheet 50	Biocosm, Inc., Hyogo, Japan	BCM18-0002	Microfluidic chip with 20 straight 50 µm-width channels. This is sitable for youg adults ~3 days after hatching at 20°C.
MICRO COVER GLASS	MATSUNAMI GLASS IND. LTD.	C030401	Cover glass (thickness: 130-170 µm) used in locomotion assays in Protocol 3
Polystyrene Film	Biocosm, Inc., Hyogo, Japan	BCM18-0001/ BCM18-0002	Bundled items of Worm Sheets. PS filim (thickness: ~130 µm) used in locomotion assays in Protocol 3.
Polyester Film Lumirror	TORAY INDUSTRIES, INC., Tokyo, Japan	Lumirror T60 (t 125 µm)	PET filim (thickness: 125 µm) used in locomotion assays in Protocol 3
IWAKI 60 mm/non-treated dish	AGC Techno Glass Co., Ltd., Shizuoka, Japan).	1010-060	Non-treated dish used in incuvation of C. elegans in Protocol 1
IWAKI 35 mm/non-treated dish	AGC Techno Glass Co., Ltd., Shizuoka, Japan).	1010-035	Non-treated dish used in locomotion assays in Protocol 3
Milli-Q	Merck, France		Ultrapure water
Kimwipe S-200	Nippon Paper Crecia Co., Ltd., Tokyo, Japan	62020	120 mm x 215 mm; 200 sheets/ box
WormStuff Worm Pick	Genesee Scientific Corporation, CA, USA)	59-AWP	Platina picker specilized for picking up C. elegans
Research Stereo Microscope System	OLYMPUS CORPORATION, Tokyo, Japan	SZX16	Micriscope used in all experiments and observation in this paper
Motorized Focus Stand for SZX16	OLYMPUS CORPORATION, Tokyo, Japan	SZX2-ILLB	This was used for bright field observation in Protocol 3-8.
Objective Lens (×1)	OLYMPUS CORPORATION, Tokyo, Japan	SDFPLAPO1×PF	NA: 0.15; W.D.: 60 mm. This lends was used for bright field observation in Protocol 3-8.
Objective Lens (×2)	OLYMPUS CORPORATION, Tokyo, Japan	SDFPLAPO2XPFC	NA: 0.3; W.D.: 20 mm. This  lends was used for imaging observations.
Mercury Light Source	OLYMPUS CORPORATION, Tokyo, Japan	U-LH100HG	The broad emission spectrum enables a range of fluorescence imaging experiments to be conducted using all common fluorophores.
SZX16 Fluorescent filter unit (High performance for YFP)	OLYMPUS CORPORATION, Tokyo, Japan	SZX2-FYFPHQ	Ex: 490-500 nm; Em: 510-560. This was used for imaging observation of HBR4 strain.
Digital Camera High Speed EXILIM	CASIO COMPUTER Co., Ltd, Tokyo, Japan		EX-F1 Figure 1B, 1E, 1F, Figure 2A-C, and Video 1 were obtained by using this digital camera.
Office 2013	Microsoft Co. Ltd, Redmond, WA, USA		EXCEL Software used for statistical analyses