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.
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.
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 radiation1,2,3. 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 irradiate individual cells under microscopic observation using heavy-ion microbeams5, and has established methods to enable targeted microbeam irradiation of several model animals, such as the nematode Caenorhabditis elegans4,6, silkworms7, and Oryzias latipes (Japanese medaka)8. Targeted microbeam irradiation of the nematode C. elegans allows the effective knockdown of specific regions, such as the nerve ring in the head region, thus helping to identify the roles of these systems in processes such as locomotion.
A method for on-chip immobilization of C. elegans individuals without the need for anesthesia has been developed to allow for microbeam irradiation4. In addition, to improve microfluidic chips used in the previous study4, we have recently developed wettable, ion-penetrable, polydimethylsiloxane (PDMS) microfluidic chips, referred to as worm sheets (see Table of Materials), for immobilizing C. elegans individuals9. These comprise of ultra-thin soft sheets (thickness = 300 µm; width = 15 mm; length = 15 mm) with multiple (20 or 25) straight microfluidic channels (depth = 70 µm; width = 60 µm or 50 µm; length = 8 mm) at the surface (Figure 1A-D). The microfluidic channels are open and allow multiple animals to be enclosed in them simultaneously (Figure 1E). The sheets are resilient to allow the microfluidic channels to be expanded (by ~10%, Figure 1F), 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.
The channels do not hurt the worms when they are being enclosed or when they are collected. Furthermore, the sheets are made from PDMS, which is essentially hydrophobic, but water retention can be achieved by imparting hydrophilicity to the material. The water retention and thickness are favorable characteristics of the worm sheets. The water-retention capacity prevents dehydration of the animals after prolonged immobilization and enables long-term observations to be carried out.
In addition, as described previously9, the sheets are only 300 µm thick, allowing heavy ions such as carbon ions (with a range of about 1 mm in water) to pass through the sheet enclosing the animals. This allows the ion particles to be detected and the applied radiation dose to be measured accurately. Moreover, the worm sheets can be reused and are thus economical. With the conventional injection method, the animals enclosed are sometimes dead and they cannot be taken out of the channel; their eggs can also clog the channels. This makes the chip unusable. Chips are, therefore, basically disposable and the cost-benefit ratio is poor.
In the present paper, we describe in detail a series of methods for on-chip immobilization of live C. elegans individuals using worm sheets. Through locomotion assays of animals 3 h after on-chip immobilization, we evaluated the suitable cover film. In addition, we showed the examples of on-chip immobilization for both imaging observations and microbeam irradiation.
1. Strains and maintenance
2. Selection of buffer solution for on-chip immobilization
3. Selection of suitable cover film for on-chip immobilization
4. On-chip immobilization
NOTE: Disposable, sterile gloves should be worn to avoid contaminating the worm sheets.
5. Collection of animals from a worm sheet
6. Application of worm sheets for imaging observations
NOTE: Worm sheets can be widely used in microscopic observations. The chip can retain water and does not affect the motility of C. elegans individuals after 3 h of on-chip immobilization9. In addition, the chip itself has no autofluorescence, making it suitable for use in fluorescence imaging assays. A sample application for fluorescence imaging assay is given below.
7. Application of worm sheets for microbeam irradiation
NOTE: The collimating microbeam irradiation system5 can use several heavy-ion particles accelerated from the azimuthally varying field cyclotron installed at the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) facility of QST-Takasaki (Figure 4A). There is an automatic stage for irradiation under the beam exit (Figure 4B). The procedure for heavy-ion microbeam irradiation of C. elegans using this system is as follows.
8. Treatment of worm sheets for repeated use
NOTE: Worm sheets can be used repeatedly at least 10 times9 with no adverse effects on the animals if cleaned and sterilized properly after use as follows.
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), respectively. As shown in Figure 5, there was no significant difference in motility (body bends) between control animals allowed to move freely for 3 h and animals enclosed in the worm sheet with PS film. In contrast, motility was significantly reduced in animals enclosed under a cover glass. Some animals appeared to have dried out, suggesting that the cover glass repelled the droplet, preventing a close seal and allowing the animals to partially dry out, resulting in reduced motility. The motility of animals enclosed using a PET film was also significantly decreased; although no drying was observed, the animals' motility tended to decrease uniformly, suggesting that the low oxygen transmission rate (~30 mL/[24 h·m²·MPa]), which is about 100 times lower than that of PS, caused the animals to suffocate. These results suggest that PS cover films should be used to enclose animals in the worm sheet.
We also applied the worm sheet technique for imaging observations and to perform region-specific microbeam irradiation. On-chip immobilization using a worm sheet with water retention and no autofluorescence was suitable for microscopic observation under live conditions. For example, we applied the technique to the HBR4 strain11 of C. elegans, in which a reporter gene expressed the calcium indicator GCaMP3.35 in all body-wall muscle cells. We observed the activities of all body-wall muscle cells in young adult animals at ≤3 days post-hatching in worm sheets with 50 µm-wide microfluidic channels, which allowed the animals space to bend slightly. The GCaMP3.35 signal intensity in the HBR4 strain corresponds to the contraction of the body-wall muscle cells. The Ca2+ wave propagation corresponding to the muscular activity was clearly observed (Video 1). Additionally, we confirmed that the worm sheet had no autofluorescence as shown in the last stage (last ~10 s) of Video 1. In this way, the lack of need for anesthesia allowed the physiological activities of the muscle cells to be observed under live conditions.
Furthermore, we applied the worm sheet for region-specific microbeam irradiation of C. elegans individuals. The multiple straight microfluidic channels on the worm sheet allowed multiple animals to be immobilized simultaneously, without the need for anesthesia, thus allowing sequential irradiation of ≥20 animals (enough for a group assay) in a short time (30 min for 20 individuals).
Figure 1: Schematic of a worm sheet. (A) Overview of a worm sheet with an American 1 cent coin for scale. The worm sheet was 300 µm thick, 15 mm wide, and 15 mm long. (B) The surface of the worm sheet contained 25 straight microfluidic channels (depth = 70 µm; width = 60 µm; length = 8 mm). (C) Schematic of the samples consisting of the bottom cover film, the worm sheet, and the cover film. (D) The worm sheet is a soft, ultra-thin sheet made from PDMS, and can be bent by pinching with flat tweezers. (E) Example of multiple animals enclosed in multiple channels.(F) Expansion of a microfluidic channel by pushing with a platina picker. The elasticity of the channel allows animals to be enveloped gently. Please click here to view a larger version of this figure.
Figure 2: Body form of C. elegans. (A) Wild-type (N2) C. elegans on an NGM plate. (B) An unc-119 mutant with abnormal shape on an NGM plate. (C) The unc-119 mutants enclosed in the microfluidic channels of the worm sheet. Please click here to view a larger version of this figure.
Figure 3: Schematics of microscope observation of live C. elegans individuals enclosed in the worm sheet. (A) Schematic of the stereomicroscope system for imaging observations. (B) Schematic of microscope observation of the worm sheet enclosing live C. elegans individuals. (C) Sectional view of the worm sheet enclosing live C. elegans individuals placed on the microscope stage. W.D. indicates the working distance of microscope. Please click here to view a larger version of this figure.
Figure 4: Schematic of collimating microbeam system at QST-Takasaki and targeted microbeam irradiation procedure for live C. elegans individuals. (A) Overview of the collimating microbeam irradiation system5, which can use several heavy-ion particles accelerated from the azimuthally varying field cyclotron installed at the TIARA of QST-Takasaki. (B) Overview of the beam exit and the automatic stage for irradiation. (C) Sample setting on the automatic stage of the collimating microbeam system. (D) Vertical positioning of the irradiation sample conducted in the irradiation room. (E) Fine-tuning of irradiation area conducted in the irradiation-control room. (F) Targeted microbeam irradiation of live C. elegans. The pharynx was clicked-on as the targeted position and irradiated by pushing the irradiation button. Please click here to view a larger version of this figure.
Figure 5: Motility of C. elegans after on-chip immobilization using cover glass, polyester (PET) film, and polystyrene (PS) film. Bars indicate mean body bends of animals 3 h after on-chip immobilization or after free movement for 3 h on an NGM plate (control). Ten animals were examined and body bends were averaged among each group. Finally, data from five independent experiments were averaged for each group. Error bars represent standard error of the mean of five independent experiments. All data were analyzed using one-way ANOVA at the 0.05 (*) or 0.01 (**) significance level. Please click here to view a larger version of this figure.
Video 1: Examples of imaging observations of muscular activities in C. elegans enclosed in a worm sheet. Calcium-ion wave propagation corresponding to contraction of the body-wall muscle cells during crawling in HBR4 C. elegans individuals enclosed in a worm sheet. The last ~10 s were observed under bright-field illumination. Please click here to view this video. (Right-click to download.)
Supplementary File 1: Example of dedicated sheet of paper (microbeam irradiation version). Draw an arrow to indicate the position of each animal in the channel. The direction of the arrow corresponds to the head. Please click here to download this file.
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 clogging of animals and eggs in the closed microfluidic channels making it difficult to collect the animals, and thus such chips have tended to be disposable, thereby increasing the cost. In contrast, the microfluidic channels in the current worm sheet are open, making it easier to collect the animals. These worm sheets can therefore be used repeatedly, making them more economical.
Recent technological innovations in PDMS microfluidic chips have shown an increasing trend in structural complexity and multifunctionality16,18,19,20,21,22,23. However, we believe that it is important to make the system simple and easy to use. Indeed, in contrast to the use of conventional large microfluidic chips19,20,21,22,23 that require the attachment of a vacuum pump, the small size and simple design of the worm sheets allow procedures to be conducted easily within a limited space.
The water retention performance of the worm sheets enables long-term observations to be carried out. In addition, the thickness of the sheet allows ion particles to pass through the samples, thus enabling targeted irradiation to be applied to live C. elegans individuals with a precise number of ion particles. These advantages of the worm sheets have greatly expanded the possibilities for biological experiments.
We believe that it is important for biologists to develop new equipment and methods in order to improve the efficiency of their experiments and analyses. The worm sheets and microbeam irradiation software have been developed with this aim in mind, and have the potential to contribute to the success of future innovative experiments beyond their original objectives.
To the best of our knowledge, our group is the first to develop this technology worldwide. However, its standardized use in the future will facilitate the application of targeted microbeam irradiation to animals under live conditions, thus helping to identify the roles of specific cells/tissues in internal processes.
The authors have nothing to disclose.
The authors thank Dr. Atsushi Higashitani for kind advice regarding treatment of C. elegans and Drs. Yuya Hattori, Yuichiro Yokota, and Yasuhiko Kobayashi for valuable discussions. The authors thank the Caenorhabditis Genetic Center for providing strains of C. elegans and E. coli. We thank the crew of the cyclotron of TIARA at QST-Takasaki for their kind assistance with the irradiation experiments. We thank Dr. Susan Furness for editing a draft of this manuscript. This study was supported in part by KAKENHI (Grant Numbers JP15K11921 and JP18K18839) from JSPS to M.S.
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. |