effectively with charge-coupled device cameras than by photomultiplier tubes. With greater detection sensitivity, exposure time can
be reduced without affecting the signal to noise ratio. Therefore,
spinning disk confocal microscopy allows for faster imaging for
longer periods of time and with lower levels of photobleaching. A
disadvantage of spinning disk microscopy is the pinhole crosstalk
effect, which can affect the axial resolution of the image. A limited
axial resolution is problematic to image developmental processes
deep within the specimen. To overcome this and other limitations
of spinning disk confocal microscopy, including the potential for
photobleaching or phototoxicity, other techniques such as lightsheet microscopy have been developed [22, 23].
Super-resolution microscopy enables the visualization of small
molecular complexes and single fluorophores, surpassing the diffraction limit. However, the application of super-resolution microscopy often requires special, expensive equipment; and results in
slow image acquisition and/or increased phototoxicity, making
super-resolution microscopy often not amenable for application
in vivo. A novel approach, super-resolution radial fluctuation
microscopy (SRRF) was recently developed [24], providing a balance between resolution, cost and speed of image acquisition.
SRRF localizes individual fluorophores based on the radial symmetry and persistence in time of their emission (in contrast with noise,
which is neither symmetric nor temporally correlated). Using this
approach, SRRF can achieve lateral resolutions of 80–100 nm using
conventional fluorophores, with temporal resolutions of only a few
seconds. Imaging with SRRF does not require special optics or
high-power laser excitation. Therefore, SRRF can be used to conduct super-resolution imaging of dynamic molecular events in living cells [25].
In this chapter, we describe methods to image collective cell
movements with high spatial and temporal resolution in the context
of developing Drosophila embryos. The Drosophila embryo is amenable to genetic, pharmacological and biophysical manipulations,
thus rendering it an excellent system to probe the mechanisms of
collective cell migration. Furthermore, transgenic fluorescent lines
useful to study molecular dynamics during coordinated cell movements are freely available (see Table 1 for examples). In this chapter,
we illustrate how to prepare embryos for time-lapse spinning disk
confocal microscopy. We introduce methods to wound the embryonic epidermis, and we demonstrate how to use quantitative
metrics to measure the efficiency of collective cell movements.
Finally, we demonstrate how to apply SRRF to image protrusive
activity in cells that move in a coordinated manner. Overall, the
methods presented here enable multi-scale, in vivo imaging of
cellular and molecular dynamics during collective cell migration.
Live Imaging in Drosophila Embryos
201
be reduced without affecting the signal to noise ratio. Therefore,
spinning disk confocal microscopy allows for faster imaging for
longer periods of time and with lower levels of photobleaching. A
disadvantage of spinning disk microscopy is the pinhole crosstalk
effect, which can affect the axial resolution of the image. A limited
axial resolution is problematic to image developmental processes
deep within the specimen. To overcome this and other limitations
of spinning disk confocal microscopy, including the potential for
photobleaching or phototoxicity, other techniques such as lightsheet microscopy have been developed [22, 23].
Super-resolution microscopy enables the visualization of small
molecular complexes and single fluorophores, surpassing the diffraction limit. However, the application of super-resolution microscopy often requires special, expensive equipment; and results in
slow image acquisition and/or increased phototoxicity, making
super-resolution microscopy often not amenable for application
in vivo. A novel approach, super-resolution radial fluctuation
microscopy (SRRF) was recently developed [24], providing a balance between resolution, cost and speed of image acquisition.
SRRF localizes individual fluorophores based on the radial symmetry and persistence in time of their emission (in contrast with noise,
which is neither symmetric nor temporally correlated). Using this
approach, SRRF can achieve lateral resolutions of 80–100 nm using
conventional fluorophores, with temporal resolutions of only a few
seconds. Imaging with SRRF does not require special optics or
high-power laser excitation. Therefore, SRRF can be used to conduct super-resolution imaging of dynamic molecular events in living cells [25].
In this chapter, we describe methods to image collective cell
movements with high spatial and temporal resolution in the context
of developing Drosophila embryos. The Drosophila embryo is amenable to genetic, pharmacological and biophysical manipulations,
thus rendering it an excellent system to probe the mechanisms of
collective cell migration. Furthermore, transgenic fluorescent lines
useful to study molecular dynamics during coordinated cell movements are freely available (see Table 1 for examples). In this chapter,
we illustrate how to prepare embryos for time-lapse spinning disk
confocal microscopy. We introduce methods to wound the embryonic epidermis, and we demonstrate how to use quantitative
metrics to measure the efficiency of collective cell movements.
Finally, we demonstrate how to apply SRRF to image protrusive
activity in cells that move in a coordinated manner. Overall, the
methods presented here enable multi-scale, in vivo imaging of
cellular and molecular dynamics during collective cell migration.
Live Imaging in Drosophila Embryos
201