2 Materials
This protocol describes a simplified version of the RootChip technology [68, 69]. RootChip is a high-performance microfluidic
device optimized for observation of plant roots. In this version of
the protocol, solutions are changed manually instead of the electronic valve-controlling system described previously [69] for a facile
and economical imaging setup.
2.1 Perfusion
Chamber System
2.1.1 Perfusion Chamber
for a Simplified Version of
the RootChip
1. Fabrication of molds for Polydimethylsiloxane (PDMS):
Design the microfluidic channel patterns using CAD software
and fabricate the mold (Fig. 3a; see Note 1).
2. Preparation of PDMS: Mix PDMS monomer and the curing
agent at a 10:1 ratio (v/v) (Sylgard184; Dow Corning). Degas
the mixture in the desiccator connected to a vacuum pump for
60 min.
3. Pouring PDMS: Once PDMS has been degassed, pour the
PDMS pre-polymer onto the mold. Place the mold in the
desiccator to degas the PDMS for 60 min. Keep the mold
overnight in a 60 ˚C oven to cure the PDMS. Cool down the
PDMS to room temperature and peel it off the mold.
4. Post-processing of the PDMS device: Cut the PDMS in order
to fit a glass coverslip. Three holes are punched on each line:
two are the perfusion delivery channels and one is as an entry
hole of a plastic cone containing a seedling.
5. PDMS devise assembly: The cleaned cover glass (48 Â 60 mm;
Brain Research Laboratories, MA, USA) is bonded to the
PDMS device using a plasma cleaner (PDC-32G; Harrick
Plasma, NY, USA). The bonded PDMS is cured on a hot
plate for 1 h at 60 ˚C (Fig. 3b; see Note 2).
2.1.2 Perfusion Chamber
Holder
1. Fabricate a perfusion chamber holder that is suitable for your
microscope stage (Fig. 3c; see Note 3).
2.1.3 Perfusion System
1. Syringe pump (e.g., YSP-202; YMC, Kyoto, Japan).
2. 50-mL Disposable syringe.
3. Silicone tube (Φ1 mm) (S-1x2; AS ONE, Osaka, Japan).
4. Plastic Luer taper (VRF108; Isis, Osaka, Japan).
5. 15-mL Conical tube.
6. Sealing film for 96-well plate (e.g., SureSeal Thermal; BMBio,
Tokyo, Japan).
312
Akira Yoshinari et al.
This protocol describes a simplified version of the RootChip technology [68, 69]. RootChip is a high-performance microfluidic
device optimized for observation of plant roots. In this version of
the protocol, solutions are changed manually instead of the electronic valve-controlling system described previously [69] for a facile
and economical imaging setup.
2.1 Perfusion
Chamber System
2.1.1 Perfusion Chamber
for a Simplified Version of
the RootChip
1. Fabrication of molds for Polydimethylsiloxane (PDMS):
Design the microfluidic channel patterns using CAD software
and fabricate the mold (Fig. 3a; see Note 1).
2. Preparation of PDMS: Mix PDMS monomer and the curing
agent at a 10:1 ratio (v/v) (Sylgard184; Dow Corning). Degas
the mixture in the desiccator connected to a vacuum pump for
60 min.
3. Pouring PDMS: Once PDMS has been degassed, pour the
PDMS pre-polymer onto the mold. Place the mold in the
desiccator to degas the PDMS for 60 min. Keep the mold
overnight in a 60 ˚C oven to cure the PDMS. Cool down the
PDMS to room temperature and peel it off the mold.
4. Post-processing of the PDMS device: Cut the PDMS in order
to fit a glass coverslip. Three holes are punched on each line:
two are the perfusion delivery channels and one is as an entry
hole of a plastic cone containing a seedling.
5. PDMS devise assembly: The cleaned cover glass (48 Â 60 mm;
Brain Research Laboratories, MA, USA) is bonded to the
PDMS device using a plasma cleaner (PDC-32G; Harrick
Plasma, NY, USA). The bonded PDMS is cured on a hot
plate for 1 h at 60 ˚C (Fig. 3b; see Note 2).
2.1.2 Perfusion Chamber
Holder
1. Fabricate a perfusion chamber holder that is suitable for your
microscope stage (Fig. 3c; see Note 3).
2.1.3 Perfusion System
1. Syringe pump (e.g., YSP-202; YMC, Kyoto, Japan).
2. 50-mL Disposable syringe.
3. Silicone tube (Φ1 mm) (S-1x2; AS ONE, Osaka, Japan).
4. Plastic Luer taper (VRF108; Isis, Osaka, Japan).
5. 15-mL Conical tube.
6. Sealing film for 96-well plate (e.g., SureSeal Thermal; BMBio,
Tokyo, Japan).
312
Akira Yoshinari et al.