6. Calculate the volume of all other somites and plot.
7. For the somitocoel, use the draw tool to determine the perimeter by hand and then calculate as a sphere.
3.5 Morphology
Analysis
1. Take a single slice from the top of the somite at time zero and
the same slice at a later time.
2. Download imageJ correlator plugin at https://imagej.nih.
gov/ij/plugins/image_correlator.html.
3. Run the image correlator plugin in ImageJ to determine the
correlation factor.
4. Repeat for all samples and plot the mean and S.D. for the top of
all somites.
5. Repeat for mid and bottom sections.
3.6 Real-Time
Accurate Cell-Shape
Extractor (RACE)
Analysis
1. Take the image stack of interest and crop out a region at the lip
and the region opposite the lip of the somite in X, Y, Z. Save
these regions.
2. Download source code from https://bitbucket.org/jstegmaier/
race.
3. Run one set of data and adjust threshold and watershed levels
so that all cells are detected. By eye, determine that no artifacts
are being detected. Log threshold and watershed values and
apply these to all data sets.
4. Run all data and plot results.
4 Notes
1. Time-lapse images were captured using a TriM Scope II (multiphoton) microscope from LaVision Biotec GmbH, Bielefeld,
Germany equipped with a Chameleon Vision II pulsed laser
from Coherent Inc., Daventry, UK. Imaging used a 20Â PlanApochromat objective (0.8 NA) on a Zeiss AxioObserver D1,
and fluorescence (525 Æ 25 nm) captured using a highsensitivity GaAsP detector.
2. Temperature (38
C) was maintained using a Heating Insert P
S1 controlled by TempModule S and AxioVision software (Carl
Zeiss UK, Cambridge).
3. Set chamber to 38
C and the lid to the chamber to 38.5
C to
avoid condensation.
4. We used ImSpectorPro software.
5. Each image file will be 30–100 Gb in size.
6. Use freshly delivered eggs that have been stored for no more
than 1 week at 16–17
C.
4D Live Imaging and Analysis of Chick Embryo Somites
179
7. For the somitocoel, use the draw tool to determine the perimeter by hand and then calculate as a sphere.
3.5 Morphology
Analysis
1. Take a single slice from the top of the somite at time zero and
the same slice at a later time.
2. Download imageJ correlator plugin at https://imagej.nih.
gov/ij/plugins/image_correlator.html.
3. Run the image correlator plugin in ImageJ to determine the
correlation factor.
4. Repeat for all samples and plot the mean and S.D. for the top of
all somites.
5. Repeat for mid and bottom sections.
3.6 Real-Time
Accurate Cell-Shape
Extractor (RACE)
Analysis
1. Take the image stack of interest and crop out a region at the lip
and the region opposite the lip of the somite in X, Y, Z. Save
these regions.
2. Download source code from https://bitbucket.org/jstegmaier/
race.
3. Run one set of data and adjust threshold and watershed levels
so that all cells are detected. By eye, determine that no artifacts
are being detected. Log threshold and watershed values and
apply these to all data sets.
4. Run all data and plot results.
4 Notes
1. Time-lapse images were captured using a TriM Scope II (multiphoton) microscope from LaVision Biotec GmbH, Bielefeld,
Germany equipped with a Chameleon Vision II pulsed laser
from Coherent Inc., Daventry, UK. Imaging used a 20Â PlanApochromat objective (0.8 NA) on a Zeiss AxioObserver D1,
and fluorescence (525 Æ 25 nm) captured using a highsensitivity GaAsP detector.
2. Temperature (38
C) was maintained using a Heating Insert P
S1 controlled by TempModule S and AxioVision software (Carl
Zeiss UK, Cambridge).
3. Set chamber to 38
C and the lid to the chamber to 38.5
C to
avoid condensation.
4. We used ImSpectorPro software.
5. Each image file will be 30–100 Gb in size.
6. Use freshly delivered eggs that have been stored for no more
than 1 week at 16–17
C.
4D Live Imaging and Analysis of Chick Embryo Somites
179