13. Name your processed files consistently with the original files
but with a different name to prevent overwriting.
14. Always keep the same donor and acceptor channels throughout
all your analyses. You can identify them by plotting the intensity of a region containing fluorophores against the wavelength
(or channel number) (select the multichannel stack then Image>Hyperstacks>Hyperstack to Stack, then select a region and
Image>Stacks>Plot Z-axis profile). The plot should display
two maxima (around 490 and 530 nm), if not, do not analyze
further: the cell has likely not processed the full construct
properly.
15. Before this step, you may filter some noise by local averaging of
pixel levels, at the expense of spatial resolution (for instance,
Process>Filters>Gaussian Blur).
16. For E-cadherin-TSMod, the donor channel often exhibits
intracellular aggregates, probably synthesis or degradation
intermediates in which the acceptor fluorophore does not
emit. Hence, the acceptor channel is preferred.
17. You can explore the different tools available on Image J to
process binary images (Process>Binary>. . .) to improve segmentation from the thresholded image.
18. More information on how to use the ROI Manager tool can be
found here (https://imagej.nih.gov/ij/docs/guide/). Size
and shape selection can be very useful to exclude regions too
small or round to be cell–cell contacts. In the ROI Manager,
you can delete unwanted regions (>Delete) or add missing
regions (draw with any selection tool then >Add).
19. This does not need to be done for every experiment but can be
performed from time to time to assess the microscope stability.
20. The Ecad-TSModΔcyto is sensitive to intermolecular FRET
[14]. This normalization thus corrects for possible FRET
changes due to intermolecular FRET among other causes.
Acknowledgements
This work was supported in part by the Centre national de la
recherche scientifique (CNRS), the French National Research
Agency (ANR) grants (ANR-17-CE13-0013, ANR-17-CE090019, ANR-18-CE13-0008), France BioImaging infrastructure
(ANR-10-INBS-04), La Ligue contre le Cancer (Allocation de
recherche doctorale to HC), the European Union’s Horizon
2020 research and innovation programme (Marie SkłodowskaCurie grant agreement No 665850-INSPIRE to PSC). PSC
298
Helena Canever et al.
but with a different name to prevent overwriting.
14. Always keep the same donor and acceptor channels throughout
all your analyses. You can identify them by plotting the intensity of a region containing fluorophores against the wavelength
(or channel number) (select the multichannel stack then Image>Hyperstacks>Hyperstack to Stack, then select a region and
Image>Stacks>Plot Z-axis profile). The plot should display
two maxima (around 490 and 530 nm), if not, do not analyze
further: the cell has likely not processed the full construct
properly.
15. Before this step, you may filter some noise by local averaging of
pixel levels, at the expense of spatial resolution (for instance,
Process>Filters>Gaussian Blur).
16. For E-cadherin-TSMod, the donor channel often exhibits
intracellular aggregates, probably synthesis or degradation
intermediates in which the acceptor fluorophore does not
emit. Hence, the acceptor channel is preferred.
17. You can explore the different tools available on Image J to
process binary images (Process>Binary>. . .) to improve segmentation from the thresholded image.
18. More information on how to use the ROI Manager tool can be
found here (https://imagej.nih.gov/ij/docs/guide/). Size
and shape selection can be very useful to exclude regions too
small or round to be cell–cell contacts. In the ROI Manager,
you can delete unwanted regions (>Delete) or add missing
regions (draw with any selection tool then >Add).
19. This does not need to be done for every experiment but can be
performed from time to time to assess the microscope stability.
20. The Ecad-TSModΔcyto is sensitive to intermolecular FRET
[14]. This normalization thus corrects for possible FRET
changes due to intermolecular FRET among other causes.
Acknowledgements
This work was supported in part by the Centre national de la
recherche scientifique (CNRS), the French National Research
Agency (ANR) grants (ANR-17-CE13-0013, ANR-17-CE090019, ANR-18-CE13-0008), France BioImaging infrastructure
(ANR-10-INBS-04), La Ligue contre le Cancer (Allocation de
recherche doctorale to HC), the European Union’s Horizon
2020 research and innovation programme (Marie SkłodowskaCurie grant agreement No 665850-INSPIRE to PSC). PSC
298
Helena Canever et al.