8. Placing the oil on the dish where the embryos are rather than
on the objective has several advantages. First it prevents the oil
from running down the objective before being held between
the objective and the cover glass on the bottom of the dish. It
also restricts the oil to the area where the embryos are, so that
the oil does not spread over large area, resulting in too little oil
between the objective and area to be imaged.
9. A 60Â or 63Â oil objective is necessary to get sufficient quality
images of detailed morphology of adherens junctions and contractile myosin.
10. Determine the exact age of the embryo by the depth of the
cellularization, as shown by any proteins localizing around the
cellularization front such as E-cadherin or myosin. DIC can be
helpful to determine the age if no fluorescent proteins in the
embryo associate with cellularization structures.
The pattern of E-cadherin or myosin around cellularization front in the precisely oriented embryo should appear very
symmetric. Since cellularization proceeds faster in the mesoderm, the pattern will appear asymmetric in a slightly tilted
embryo.
Normally the footprint of the ventral side of the embryo on
the glass is a smooth oval. When embryos leak content due to
damage, the footprint will appear abnormally big and show
wrinkles due to loss of internal pressure. The outline of the
footprint can also appear wavy. Additionally, avoid embryos
that have landed on debris. Damaged embryos often also
show defects in cellularization. The cellularization front in a
healthy embryo will appear very even on both the XY plane or
in the z direction, both in terms of cell sizes and cellularization
speeds. Cells in damaged embryos may have shrinking cells
surrounded by stretched cells. If the significantly damaged
cell is outside field of view, cells in the field of view often appear
stretched to a certain direction. Do not image such an embryo.
11. To image embryos from cellularization to gastrulation, a good
starting point of imaging format is the following: physical size
of each pixel: 0.12 μm; digital dimension of each image:
1024 Â 512; Z stack depth: 10–15 μm with 1 μm interval;
temporal interval: 1 min. Scanning goes much faster along
X than Y, to save time, always use more pixels along X rather
than the other way around. For most confocal microscopes,
each stack will take much less than 1 min. Therefore, there is no
need to use bidirectional imaging as less frequent and spreadout exposures to lasers appears to reduce photobleaching compared to concentrating all the laser dosage in a short period of
time. At the end of an imaging session, zoom out to examine if
the imaged area is significantly darker than the rest of the
embryo.
76
Lingkun Gu and Mo Weng
on the objective has several advantages. First it prevents the oil
from running down the objective before being held between
the objective and the cover glass on the bottom of the dish. It
also restricts the oil to the area where the embryos are, so that
the oil does not spread over large area, resulting in too little oil
between the objective and area to be imaged.
9. A 60Â or 63Â oil objective is necessary to get sufficient quality
images of detailed morphology of adherens junctions and contractile myosin.
10. Determine the exact age of the embryo by the depth of the
cellularization, as shown by any proteins localizing around the
cellularization front such as E-cadherin or myosin. DIC can be
helpful to determine the age if no fluorescent proteins in the
embryo associate with cellularization structures.
The pattern of E-cadherin or myosin around cellularization front in the precisely oriented embryo should appear very
symmetric. Since cellularization proceeds faster in the mesoderm, the pattern will appear asymmetric in a slightly tilted
embryo.
Normally the footprint of the ventral side of the embryo on
the glass is a smooth oval. When embryos leak content due to
damage, the footprint will appear abnormally big and show
wrinkles due to loss of internal pressure. The outline of the
footprint can also appear wavy. Additionally, avoid embryos
that have landed on debris. Damaged embryos often also
show defects in cellularization. The cellularization front in a
healthy embryo will appear very even on both the XY plane or
in the z direction, both in terms of cell sizes and cellularization
speeds. Cells in damaged embryos may have shrinking cells
surrounded by stretched cells. If the significantly damaged
cell is outside field of view, cells in the field of view often appear
stretched to a certain direction. Do not image such an embryo.
11. To image embryos from cellularization to gastrulation, a good
starting point of imaging format is the following: physical size
of each pixel: 0.12 μm; digital dimension of each image:
1024 Â 512; Z stack depth: 10–15 μm with 1 μm interval;
temporal interval: 1 min. Scanning goes much faster along
X than Y, to save time, always use more pixels along X rather
than the other way around. For most confocal microscopes,
each stack will take much less than 1 min. Therefore, there is no
need to use bidirectional imaging as less frequent and spreadout exposures to lasers appears to reduce photobleaching compared to concentrating all the laser dosage in a short period of
time. At the end of an imaging session, zoom out to examine if
the imaged area is significantly darker than the rest of the
embryo.
76
Lingkun Gu and Mo Weng