3. Matlab (Mathworks) using the DIPImage toolbox (TU Delft)
or an alternative image analysis software.
4. Image segmentation software (e.g., SIESTA [28] or
equivalent).
5. Fiji [29] and the NanoJ-SRRF plugin [24].
6. For processing and visualization of super-resolution microscopy images, a computer equipped with sufficient RAM
(at least 32 GB). A Graphics Processing Unit (GPU) is recommended, as it will accelerate image reconstruction through
parallel processing (see Note 6).
3 Methods
3.1 Embryo Staging
and Collection
1. Prepare yeast paste by mixing tap water and yeast in a 50 mL
tube using a spatula (see Note 7).
2. Using a spatula or your finger, transfer a small amount of yeast
paste to the surface of the embryo collection plates and spread
the paste (Fig. 1b).
3. To maximize the number and viability of the embryos used for
live imaging, replace the collection plate, allow flies to lay eggs
for 30–60 min, and discard that plate, replacing it with a new
one. Allow flies to lay eggs on the new plate for the appropriate
time (see Note 8).
3.2 Mounting
Embryos
For live imaging, embryos can be glued to a coverslip, or immobilized between a coverslip and an oxygen-permeable teflon membrane. The glue method facilitates proper embryo orientation and
minimizes embryo compression, while maintaining embryos accessible for additional manipulations such as microinjection. However,
the glue method is technically hard for beginners, and it limits the
number of embryos that can be mounted per coverslip, as embryos
are susceptible to dehydration during the alignment process.
Finally, the glue method reduces the size of the field of cells that
can be imaged, due to the curvature of the embryonic surface. In
contrast, the membrane mounting method is easy to use, provides
lots of embryos ready for live imaging, and a large field of cells for
imaging, as the surface of the embryo is flattened during the
mounting procedure. Unfortunately, membrane mounting makes
it difficult to orient the embryos, microinjections are not feasible
with this method, and embryo compression can cause changes in
gene expression [30], and affect the dynamics of morphogenetic
processes [16], so caution should be applied when choosing the
membrane method.
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Gordana Scepanovic et al.
or an alternative image analysis software.
4. Image segmentation software (e.g., SIESTA [28] or
equivalent).
5. Fiji [29] and the NanoJ-SRRF plugin [24].
6. For processing and visualization of super-resolution microscopy images, a computer equipped with sufficient RAM
(at least 32 GB). A Graphics Processing Unit (GPU) is recommended, as it will accelerate image reconstruction through
parallel processing (see Note 6).
3 Methods
3.1 Embryo Staging
and Collection
1. Prepare yeast paste by mixing tap water and yeast in a 50 mL
tube using a spatula (see Note 7).
2. Using a spatula or your finger, transfer a small amount of yeast
paste to the surface of the embryo collection plates and spread
the paste (Fig. 1b).
3. To maximize the number and viability of the embryos used for
live imaging, replace the collection plate, allow flies to lay eggs
for 30–60 min, and discard that plate, replacing it with a new
one. Allow flies to lay eggs on the new plate for the appropriate
time (see Note 8).
3.2 Mounting
Embryos
For live imaging, embryos can be glued to a coverslip, or immobilized between a coverslip and an oxygen-permeable teflon membrane. The glue method facilitates proper embryo orientation and
minimizes embryo compression, while maintaining embryos accessible for additional manipulations such as microinjection. However,
the glue method is technically hard for beginners, and it limits the
number of embryos that can be mounted per coverslip, as embryos
are susceptible to dehydration during the alignment process.
Finally, the glue method reduces the size of the field of cells that
can be imaged, due to the curvature of the embryonic surface. In
contrast, the membrane mounting method is easy to use, provides
lots of embryos ready for live imaging, and a large field of cells for
imaging, as the surface of the embryo is flattened during the
mounting procedure. Unfortunately, membrane mounting makes
it difficult to orient the embryos, microinjections are not feasible
with this method, and embryo compression can cause changes in
gene expression [30], and affect the dynamics of morphogenetic
processes [16], so caution should be applied when choosing the
membrane method.
206
Gordana Scepanovic et al.