4 Notes
1. To make medium for embryo collection, mix water and agar in
a glass beaker. Combine the rest of the reagents in a different
beaker. Heat water and agar in a microwave for 5 min, pausing
periodically (approximately every 30 s) to stir gently. The rest
of the reagents should be microwaved for 2.5 min. The two
mixtures can then be mixed together, and poured into Petri
dishes while still hot.
2. To make heptane glue, place double-sided tape into a glass vial
(use a glass pipette to push tape into the vial), and fill the vial
with heptane. Leave the vial on a nutator for 12–24 h to
dissolve the glue from the tape into the heptane. Use a glass
pipette to transfer the heptane glue into a new vial. The longer
double-sided tape is left in heptane, the thicker the glue and the
more auto-fluorescent. Therefore, it is best to transfer out the
glue or remove the double-sided tape within 24 h of mixing.
3. As microinjections are not feasible using the membrane
method, it is possible to use the less viscous and cheaper
halocarbon oil 27 for mounting, without mixing with halocarbon oil 700. However, for wound healing studies, it is recommended to mix halocarbon oil 27 and 700 to create a more
viscous medium that minimizes embryo leakage upon
wounding.
4. To make a slide to break needles, adhere an 18 Â 18 mm cover
slip to a 25 Â 75 mm microscope slide using a drop of water to
create a seal between the two surfaces.
5. For super-resolution microscopy using SRRF, an acquisition of
>50 FPS for live samples is recommended to minimize artifacts
due to sample movement. Slower acquisition rates can be used
for fixed samples. High magnification (60–100Â) and a small
pixel size (100–150 nm) should also be used.
6. NanoJ-SRRF is written in Java using Aparapi (http://aparapi.
com/), a library that enables running Java code on GPUs (with
support for AMD, Intel, and NVIDIA GPUs). A workstation
equipped with an Intel Core i7-9700K, 64 GB of RAM, and an
NVIDIA Titan Xp can reconstruct one SRRF image from
100 512 Â 512 images within 4 s. Without the GPU, the
total processing time for the same image is 146 s. For comparison, an iMac 2011 with a 2.5 GHz Intel Core i5-2400S processor, 32 GB of RAM and an AMD Radeon HD 6750M
graphics card with 512 MB of dedicated memory requires
200 s to reconstruct the same image, and 520 s if the graphics
card is disabled.
Live Imaging in Drosophila Embryos
213
1. To make medium for embryo collection, mix water and agar in
a glass beaker. Combine the rest of the reagents in a different
beaker. Heat water and agar in a microwave for 5 min, pausing
periodically (approximately every 30 s) to stir gently. The rest
of the reagents should be microwaved for 2.5 min. The two
mixtures can then be mixed together, and poured into Petri
dishes while still hot.
2. To make heptane glue, place double-sided tape into a glass vial
(use a glass pipette to push tape into the vial), and fill the vial
with heptane. Leave the vial on a nutator for 12–24 h to
dissolve the glue from the tape into the heptane. Use a glass
pipette to transfer the heptane glue into a new vial. The longer
double-sided tape is left in heptane, the thicker the glue and the
more auto-fluorescent. Therefore, it is best to transfer out the
glue or remove the double-sided tape within 24 h of mixing.
3. As microinjections are not feasible using the membrane
method, it is possible to use the less viscous and cheaper
halocarbon oil 27 for mounting, without mixing with halocarbon oil 700. However, for wound healing studies, it is recommended to mix halocarbon oil 27 and 700 to create a more
viscous medium that minimizes embryo leakage upon
wounding.
4. To make a slide to break needles, adhere an 18 Â 18 mm cover
slip to a 25 Â 75 mm microscope slide using a drop of water to
create a seal between the two surfaces.
5. For super-resolution microscopy using SRRF, an acquisition of
>50 FPS for live samples is recommended to minimize artifacts
due to sample movement. Slower acquisition rates can be used
for fixed samples. High magnification (60–100Â) and a small
pixel size (100–150 nm) should also be used.
6. NanoJ-SRRF is written in Java using Aparapi (http://aparapi.
com/), a library that enables running Java code on GPUs (with
support for AMD, Intel, and NVIDIA GPUs). A workstation
equipped with an Intel Core i7-9700K, 64 GB of RAM, and an
NVIDIA Titan Xp can reconstruct one SRRF image from
100 512 Â 512 images within 4 s. Without the GPU, the
total processing time for the same image is 146 s. For comparison, an iMac 2011 with a 2.5 GHz Intel Core i5-2400S processor, 32 GB of RAM and an AMD Radeon HD 6750M
graphics card with 512 MB of dedicated memory requires
200 s to reconstruct the same image, and 520 s if the graphics
card is disabled.
Live Imaging in Drosophila Embryos
213