6. Make sure that the dipping medium does not completely evaporate by filling the dish to the rim before leaving for the night.
Position an open bottle of water near the stage and within the
microscope heat chamber if one is installed, to provide ambient
humidity.
7. Naming files. A logic and standardized naming system will save
a lot of time and effort. For files to appear in chronological
order, use first the date year_month_day (2020_01_10), followed by the name of the person in charge of the experiment,
and indication of the type of sample (e.g., WT for wild type). In
parallel, an Excel database with all recorded movies is kept
including information of the experiment, identity of each of
the embryos in the file, and imaging parameters. Always maintain the name as you process the images as it will be easier to
trace back.
3.3 Image
Processing: Drift
Correction
During imaging, samples often move or drift within the field of
view. To obtain accurate tracking data, it is essential that these
movements are corrected. This will ensure that any movement
recorded is due to cell displacement and not to changes in the
overall position of the sample (Supplementary Movie 1 shows an
example of drift correction). The idea is to use in focus anatomical
landmarks of the bright field image to generate a transformation
matrix of deformation and drift correction, to then apply this
matrix to each fluorescent channel plane.
1. Open the imaged file including all channels in Fiji (see Note 6).
2. Display it as a hyperstack including all channels, time points,
and z planes. Trim any frame at the beginning and/or end of
the movie that is not required.
3. Separate all channels by clicking on “Image > Colour > Split
channels.” Save the fluorescent channels in separate folders as
individual frames by selecting “Save As > Image Sequence”;
use TIFF format.
4. Open the bright field stack and select one z plane in which
anatomical landmarks are clear (e.g., otic vesicle or somite
borders). Make a stack with one z plane for each time point
in which the chosen landmarks are in focus. “Save As > Image
Sequence” use TIFF format and create separate folder to
save them.
To correct the drift using Fiji, follow steps 5–11.
5. Click on “Plugins > Registration > Register Virtual Stack
Slices.”
6. For the “Source Directory,” select the folder where the in focus
bright field stack was saved.
7. For the “Output Directory,” create a new folder.
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Position an open bottle of water near the stage and within the
microscope heat chamber if one is installed, to provide ambient
humidity.
7. Naming files. A logic and standardized naming system will save
a lot of time and effort. For files to appear in chronological
order, use first the date year_month_day (2020_01_10), followed by the name of the person in charge of the experiment,
and indication of the type of sample (e.g., WT for wild type). In
parallel, an Excel database with all recorded movies is kept
including information of the experiment, identity of each of
the embryos in the file, and imaging parameters. Always maintain the name as you process the images as it will be easier to
trace back.
3.3 Image
Processing: Drift
Correction
During imaging, samples often move or drift within the field of
view. To obtain accurate tracking data, it is essential that these
movements are corrected. This will ensure that any movement
recorded is due to cell displacement and not to changes in the
overall position of the sample (Supplementary Movie 1 shows an
example of drift correction). The idea is to use in focus anatomical
landmarks of the bright field image to generate a transformation
matrix of deformation and drift correction, to then apply this
matrix to each fluorescent channel plane.
1. Open the imaged file including all channels in Fiji (see Note 6).
2. Display it as a hyperstack including all channels, time points,
and z planes. Trim any frame at the beginning and/or end of
the movie that is not required.
3. Separate all channels by clicking on “Image > Colour > Split
channels.” Save the fluorescent channels in separate folders as
individual frames by selecting “Save As > Image Sequence”;
use TIFF format.
4. Open the bright field stack and select one z plane in which
anatomical landmarks are clear (e.g., otic vesicle or somite
borders). Make a stack with one z plane for each time point
in which the chosen landmarks are in focus. “Save As > Image
Sequence” use TIFF format and create separate folder to
save them.
To correct the drift using Fiji, follow steps 5–11.
5. Click on “Plugins > Registration > Register Virtual Stack
Slices.”
6. For the “Source Directory,” select the folder where the in focus
bright field stack was saved.
7. For the “Output Directory,” create a new folder.
92
Zain Alhashem et al.