may be interested in images 30 s apart or maybe 5–10 min for a
global view of the cells in motion.
12. To ease image processing, do not change the size of the imaging region, laser intensity, or detector gain throughout the
experiment.
13. Check the microscope once an hour for the first 3 h, then check
the image every few hours to re-adjust the focus and potentially
XY position to keep the region of interest in focus as the
embryo grows and shifts position.
3.5 Image
Processing
1. Open Fiji software and concatenate all time-lapses that were
saved and restarted through the experiment.
2. Drift, growth, and the heartbeat of the embryo all contribute
to mis-alignment of the images over many hours. After concatenation, align the image using the descriptor-based series registration plugin [3]. If the embryo has moved a lot during
imaging, first try adjusting the parameters in the plugin. If
this fails, partial registration over different blocks of time can
be aligned separately then concatenated back together.
3. If the embryo drifted or grew by a large amount in the
Z direction, sometimes registration may work in the XY direction but not in Z. Imaris (Bitplane Inc) has a registration
algorithm that may suffice, or a manual registration can be
done if the XT module is available.
4. Tracking of cells or other analysis can be performed at this
point to answer your specific scientific questions.
4 Notes
1. Volume of medium. 7 mL of EC culture medium is an estimate.
More culture medium will provide a buffer if the sample begins
to dehydrate on the microscope, but more medium will raise
the sample and may interfere with the working distance on the
microscope.
2. Objectives. A water dipping objective is not recommended for
this method because the embryonic tissue needs access to
oxygen. The area covered by even a small droplet of water
will become necrotic or pull on the rest of the tissue. A long
working distance objective is ideal; however, a 20Â 0.8NA or
10Â 0.5NA are possibilities.
3. Remounting. If the embryo has fallen off the paper or become
folded, it may be corrected by flooding the EC culture dish
with Ringer’s solution and gently reorienting the embryo.
Remove most of the water, then lay down a new paper ring.
Live Imaging of EMT in Chick Neural Crest
113
global view of the cells in motion.
12. To ease image processing, do not change the size of the imaging region, laser intensity, or detector gain throughout the
experiment.
13. Check the microscope once an hour for the first 3 h, then check
the image every few hours to re-adjust the focus and potentially
XY position to keep the region of interest in focus as the
embryo grows and shifts position.
3.5 Image
Processing
1. Open Fiji software and concatenate all time-lapses that were
saved and restarted through the experiment.
2. Drift, growth, and the heartbeat of the embryo all contribute
to mis-alignment of the images over many hours. After concatenation, align the image using the descriptor-based series registration plugin [3]. If the embryo has moved a lot during
imaging, first try adjusting the parameters in the plugin. If
this fails, partial registration over different blocks of time can
be aligned separately then concatenated back together.
3. If the embryo drifted or grew by a large amount in the
Z direction, sometimes registration may work in the XY direction but not in Z. Imaris (Bitplane Inc) has a registration
algorithm that may suffice, or a manual registration can be
done if the XT module is available.
4. Tracking of cells or other analysis can be performed at this
point to answer your specific scientific questions.
4 Notes
1. Volume of medium. 7 mL of EC culture medium is an estimate.
More culture medium will provide a buffer if the sample begins
to dehydrate on the microscope, but more medium will raise
the sample and may interfere with the working distance on the
microscope.
2. Objectives. A water dipping objective is not recommended for
this method because the embryonic tissue needs access to
oxygen. The area covered by even a small droplet of water
will become necrotic or pull on the rest of the tissue. A long
working distance objective is ideal; however, a 20Â 0.8NA or
10Â 0.5NA are possibilities.
3. Remounting. If the embryo has fallen off the paper or become
folded, it may be corrected by flooding the EC culture dish
with Ringer’s solution and gently reorienting the embryo.
Remove most of the water, then lay down a new paper ring.
Live Imaging of EMT in Chick Neural Crest
113