required for the course of the experiment. Furthermore, it
should easily undergo a non-reversible conversion when
bleached, should not affect the dynamics of the protein to
which it is tagged and importantly should not dimerize. Early
variants of GFP were susceptible to dimerization, which alters
recovery kinetics [28, 29], thus skewing the results. For most
purposes, EGFP, Venus, or Emerald are the most suitable for
FRAP (for more information on properties of specific fluorescent proteins, see [30]). However, when using EGFP or any
other GFP derivatives it is important to consider that although
infrequently (less than 15% of these fluorophores) they undergo
spontaneous photoswitching—reversible photobleaching with
molecules regaining fluorescence after a period of time
[31]. Therefore, when using these fluorescent proteins for
FRAP it may be a necessary to apply a correction to minimize
the contribution of photoswitching in the signal recovery
[32]. Finally, tagging proteins with EGFP can have an effect
on its behaviour [33, 34]. This can be mitigated if one is careful
in the selection of the site of tagging, in particular avoiding
locations which might affect protein functions or interactions.
However, whenever possible we would recommend confirming
that the tagged protein retains normal behaviour and is able to
substitute for the endogenous protein. Additionally, it is crucial
to use the same tagged proteins in control and experimental
animals or cells.
2. Drosophila embryos are bean-shaped with convex side
corresponding to embryo ventral side and concave to its dorsal
side. The anterior of the embryos is marked by the presence of
micropyle. Therefore, to orient embryos correctly for imaging
using an upright microscope they should be aligned with dorsal
side down on the segment of apple juice agar. We also recommend aligning all embryos in the same anterior-posterior orientation as this simplifies both the transfer to the slide and
imaging.
3. When transferring embryos to a slide, it is important to bear in
mind that the embryos are comparatively fragile. Therefore,
when pressing the surface of the adhesive strip microscope slide
to the embryos positioned on the apple juice segments it is
recommended that a delicate amount of pressure is applied and
only for a brief moment. If possible, use a dark bench to better
visualise the embryos on the apple juice segments which appear
similar to small rice grains to the unaided eye.
4. Circular areas of a Gaussian intensity profile provide the most
straightforward system for the following analysis, although an
approach for calculating diffusion coefficients when using arbitrary bleach area geometries has been proposed [35]. The size
154
Joshua Greig and Natalia A. Bulgakova
should easily undergo a non-reversible conversion when
bleached, should not affect the dynamics of the protein to
which it is tagged and importantly should not dimerize. Early
variants of GFP were susceptible to dimerization, which alters
recovery kinetics [28, 29], thus skewing the results. For most
purposes, EGFP, Venus, or Emerald are the most suitable for
FRAP (for more information on properties of specific fluorescent proteins, see [30]). However, when using EGFP or any
other GFP derivatives it is important to consider that although
infrequently (less than 15% of these fluorophores) they undergo
spontaneous photoswitching—reversible photobleaching with
molecules regaining fluorescence after a period of time
[31]. Therefore, when using these fluorescent proteins for
FRAP it may be a necessary to apply a correction to minimize
the contribution of photoswitching in the signal recovery
[32]. Finally, tagging proteins with EGFP can have an effect
on its behaviour [33, 34]. This can be mitigated if one is careful
in the selection of the site of tagging, in particular avoiding
locations which might affect protein functions or interactions.
However, whenever possible we would recommend confirming
that the tagged protein retains normal behaviour and is able to
substitute for the endogenous protein. Additionally, it is crucial
to use the same tagged proteins in control and experimental
animals or cells.
2. Drosophila embryos are bean-shaped with convex side
corresponding to embryo ventral side and concave to its dorsal
side. The anterior of the embryos is marked by the presence of
micropyle. Therefore, to orient embryos correctly for imaging
using an upright microscope they should be aligned with dorsal
side down on the segment of apple juice agar. We also recommend aligning all embryos in the same anterior-posterior orientation as this simplifies both the transfer to the slide and
imaging.
3. When transferring embryos to a slide, it is important to bear in
mind that the embryos are comparatively fragile. Therefore,
when pressing the surface of the adhesive strip microscope slide
to the embryos positioned on the apple juice segments it is
recommended that a delicate amount of pressure is applied and
only for a brief moment. If possible, use a dark bench to better
visualise the embryos on the apple juice segments which appear
similar to small rice grains to the unaided eye.
4. Circular areas of a Gaussian intensity profile provide the most
straightforward system for the following analysis, although an
approach for calculating diffusion coefficients when using arbitrary bleach area geometries has been proposed [35]. The size
154
Joshua Greig and Natalia A. Bulgakova