3.3 Transfer of
Seedlings onto
Perfusion Chamber
1. Immediately before transferring the seedling, fill the perfusion
chamber with sterilized control solution by using a syringe.
2. Seven to ten days after germination (see Note 9), plug the
plastic cone into the middle hole of a microfluidic channel.
Choose plastic cones containing seedlings whose roots are
about to exit from the tip.
3. Incubate the perfusion chamber in the control solution overnight in a plant growth chamber.
4. Fill the groove of the chamber holder with water to prevent
plants from drying out.
5. Cover the top of the perfusion chamber with a sealing film
(e.g., SureSeal Thermal; BMBio, Tokyo, Japan).
6. Grow the plants for 1–3 more days for the root tip to enter the
perfusion channel.
3.4 Mount the
Perfusion Chamber
onto the Microscope
3.4.1 Preparation of
Perfusion Chamber
1. Fill two 1 mm diameter-silicone tubes with liquid medium
using a syringe.
2. Connect the silicone tubes to the inlet and outlet ports of the
perfusion chamber.
3. Plug a 50-mL syringe into the silicon tube connecting to the
outlet.
4. Place the end of the silicone tube connected to the inlet into a
conical tube filled with the control solution.
5. Draw the control solution into the perfusion chamber by
gently pulling the syringe connected to the outlet port of
perfusion chamber. Be sure to remove any air in the chamber
by displacing it with liquid.
3.4.2 Assemble the
Perfusion Chamber and
Chamber Holder
1. Wipe the surface of the cover glass gently with paper towel.
2. Attach the cover glass of the perfusion chamber onto the
chamber holder with vinyl tape (Yamato, Tokyo, Japan).
3. Mount the chamber assembly onto the inverted microscope
stage.
3.4.3 Create a Constant
Flow of the Liquid Medium
1. Set the syringe pump to 200 μL/min lane. In this chapter, a
single lane is used for the experiment.
2. Test whether the solution leaks or is successfully collected in
the syringe. Seal if needed to prevent damage to microscope
(Fig. 4).
3.5 Microscopy
1. Set excitation and detection wavelengths for the fluorophores
being used, here GO-Matryoshka (cpsfGFP and LSSmOrange)
(see Note 10).
2. Exposure time is 100 ms (see Note 11).
The Use of Genetically Encoded Fluorescent Biosensors
315
Seedlings onto
Perfusion Chamber
1. Immediately before transferring the seedling, fill the perfusion
chamber with sterilized control solution by using a syringe.
2. Seven to ten days after germination (see Note 9), plug the
plastic cone into the middle hole of a microfluidic channel.
Choose plastic cones containing seedlings whose roots are
about to exit from the tip.
3. Incubate the perfusion chamber in the control solution overnight in a plant growth chamber.
4. Fill the groove of the chamber holder with water to prevent
plants from drying out.
5. Cover the top of the perfusion chamber with a sealing film
(e.g., SureSeal Thermal; BMBio, Tokyo, Japan).
6. Grow the plants for 1–3 more days for the root tip to enter the
perfusion channel.
3.4 Mount the
Perfusion Chamber
onto the Microscope
3.4.1 Preparation of
Perfusion Chamber
1. Fill two 1 mm diameter-silicone tubes with liquid medium
using a syringe.
2. Connect the silicone tubes to the inlet and outlet ports of the
perfusion chamber.
3. Plug a 50-mL syringe into the silicon tube connecting to the
outlet.
4. Place the end of the silicone tube connected to the inlet into a
conical tube filled with the control solution.
5. Draw the control solution into the perfusion chamber by
gently pulling the syringe connected to the outlet port of
perfusion chamber. Be sure to remove any air in the chamber
by displacing it with liquid.
3.4.2 Assemble the
Perfusion Chamber and
Chamber Holder
1. Wipe the surface of the cover glass gently with paper towel.
2. Attach the cover glass of the perfusion chamber onto the
chamber holder with vinyl tape (Yamato, Tokyo, Japan).
3. Mount the chamber assembly onto the inverted microscope
stage.
3.4.3 Create a Constant
Flow of the Liquid Medium
1. Set the syringe pump to 200 μL/min lane. In this chapter, a
single lane is used for the experiment.
2. Test whether the solution leaks or is successfully collected in
the syringe. Seal if needed to prevent damage to microscope
(Fig. 4).
3.5 Microscopy
1. Set excitation and detection wavelengths for the fluorophores
being used, here GO-Matryoshka (cpsfGFP and LSSmOrange)
(see Note 10).
2. Exposure time is 100 ms (see Note 11).
The Use of Genetically Encoded Fluorescent Biosensors
315