12. Confocal microscope: The described method requires an
upright confocal microscope.
13. Water dipping lenses: Since imaging is performed in water, the
use of dipping lenses is critical. These lenses are available in the
market with different magnifications and Numerical Aperture
(NA) from different suppliers. We found that Â25 0.95NA,
Â40 0.8NA, and Â63 0.9NA lenses are very useful for imaging
different sizes of samples at high resolution.
14. Air permeable tape (e.g., paper surgical tape).
3 Methods
To image the vegetative meristem and leaves of small seedlings, we
use a similar approach to that described in Heisler and Ohno in
2014 for the inflorescence meristems [3]. In summary, seedlings at
3 days after stratification (3 DAS) are transplanted to a new sterile
Petri dish containing solid growth medium. To expose the first pair
of leaves and vegetative meristem, one of the two cotyledons is
carefully removed in water, then the Petri dish is mounted on the
stage of an upright confocal microscope, equipped with water
dipping lenses. Below we describe in detail the above-summarized
procedure.
3.1 Seed
Germination
1. Sterilize seeds by vapor sterilization [7].
2. Sow seeds on Petri dishes containing solid growth media,
typically GM plus Vitamin.
3. Seal dishes with air permeable tape and place them at 4
C for a
minimum period of 3 days to synchronize the germination
4. Transfer dishes to the growth room (at 22
C in continuous
light, 50% humidity) and place them vertically in a tube rack.
This allows the roots to grow parallel to the medium and
facilitates the transfer of seedlings to a new square Petri dish
for imaging without damaging the roots. For this experiment,
we usually use seedlings 2.5–3 DAS.
3.2 Seedling
Preparation
At 2.5–3 DAS the vegetative meristem with the first pair of leaves is
located centrally between the two cotyledons. In order to expose
them, a cotyledon will need to be removed.
1. Transfer the seedlings to a clean dish containing solid growth
medium under a dissection microscope (see Note 1).
2. Use a sterile blade to make a vertical slit in the medium just
next to the seedling before gently inserting the seedling root
into the slit (Fig. 1a–c).
Confocal Microscopy of Arabidopsis Seedlings
297
upright confocal microscope.
13. Water dipping lenses: Since imaging is performed in water, the
use of dipping lenses is critical. These lenses are available in the
market with different magnifications and Numerical Aperture
(NA) from different suppliers. We found that Â25 0.95NA,
Â40 0.8NA, and Â63 0.9NA lenses are very useful for imaging
different sizes of samples at high resolution.
14. Air permeable tape (e.g., paper surgical tape).
3 Methods
To image the vegetative meristem and leaves of small seedlings, we
use a similar approach to that described in Heisler and Ohno in
2014 for the inflorescence meristems [3]. In summary, seedlings at
3 days after stratification (3 DAS) are transplanted to a new sterile
Petri dish containing solid growth medium. To expose the first pair
of leaves and vegetative meristem, one of the two cotyledons is
carefully removed in water, then the Petri dish is mounted on the
stage of an upright confocal microscope, equipped with water
dipping lenses. Below we describe in detail the above-summarized
procedure.
3.1 Seed
Germination
1. Sterilize seeds by vapor sterilization [7].
2. Sow seeds on Petri dishes containing solid growth media,
typically GM plus Vitamin.
3. Seal dishes with air permeable tape and place them at 4
C for a
minimum period of 3 days to synchronize the germination
4. Transfer dishes to the growth room (at 22
C in continuous
light, 50% humidity) and place them vertically in a tube rack.
This allows the roots to grow parallel to the medium and
facilitates the transfer of seedlings to a new square Petri dish
for imaging without damaging the roots. For this experiment,
we usually use seedlings 2.5–3 DAS.
3.2 Seedling
Preparation
At 2.5–3 DAS the vegetative meristem with the first pair of leaves is
located centrally between the two cotyledons. In order to expose
them, a cotyledon will need to be removed.
1. Transfer the seedlings to a clean dish containing solid growth
medium under a dissection microscope (see Note 1).
2. Use a sterile blade to make a vertical slit in the medium just
next to the seedling before gently inserting the seedling root
into the slit (Fig. 1a–c).
Confocal Microscopy of Arabidopsis Seedlings
297