1. Prepare necessary materials
NOTE: These preparations are best done days or weeks ahead of time.
2. Prepare egg collection cages
NOTE: Do this at least a day (preferably 2 or 3 days) before the planned injection(s).
3. On the day of injection, prepare the injection solution and load it into the needle
4. Collecting embryos for injection
NOTE: The timing of collection depends on what stage of embryos the injection needs to be performed at. With the timing scheme below, the embryos at the time of preparation for injection will be 0-90 min old, which corresponds to cleavage stages15.
5. Prepare embryos for microinjection
6. Microinject embryos
NOTE: Ensure the microinjection setup includes an inverted microscope, a micromanipulator to hold and position the injection needle, and a commercial microinjector to deliver controlled volumes.
7. Image embryos
NOTE: This protocol utilizes a laser scanning confocal microscope.
8. Analyze LD flow by first using FIJI to prepare a time series of images, and then python for PIV analysis
Following injection, the dye will be localized only at the site where the needle tip was inserted. The dye will then diffuse away from the injection site depending on its diffusive characteristics. Figure 1 shows injection of BODIPY 493/503, soon after injection (panel A) and 24 min later (panel B). After 24 min, the dye has made it to roughly the midpoint of the embryo's long axis.
Analyzing organelle motility can be achieved through dye injections and time-lapse imaging. In Figure 2, an embryo was co-injected with BODIPY 493/503 (Figure 2A) and LysoTracker Red (Figure 2B) and imaged using laser excitation at 488 and 596 nm, respectively. This embryo was then time-lapse imaged (one frame every 30 s for 30 min, 5 min analyzed). The time series was then run through PIV analysis, the output of which is shown via streamlines in Figure 2A,B. Note that the streamlines do not represent the trajectory of individual particles, but the cytoplasmic flow is inferred from analyzing all the particles in that region of the cytoplasm. Through labeling of two independent cellular structures (LDs and acidic organelles), the PIV analysis finds similar flows, with both labels converging on the central region of the embryo where the cytoplasm is flowing into the embryo's interior6.
Currently available FPs allow the labeling of many other organelles and cellular structures. Figure 3 shows the labeling of the ER via ER tracker Green. ER tracker provides a nice resolution of the nuclear envelope, allowing visualization of major cell cycle stages. ER tracker Green is imaged using 488 nm excitation.
Labeling of the mitochondria is tricky, as most dyes tested seem to be trapped in the first mitochondrion they enter. On the other hand, no sign of dye toxicity was detected, making it possible to follow labeled mitochondria through cellularization and the ectodermal nuclear cycle 15. Figure 4 shows an ectodermal cell several hours post-injection with Mitoview 633 (excitation wavelength 633 nm).
BODIPY, Lysotracker, and LipidSpot are robust and can be used to acquire ~500+ images at 512 x 512, line average 3, frame rates from 1/s to 0.1/s. ER tracker Green, SiR-tubulin, and the mitochondrial dyes mentioned are less robust and yield ~50-200 images under the same conditions.
Starting at blastoderm stages, LDs move bidirectionally along microtubules, powered by the opposite-polarity motors kinesin-1 and cytoplasmic dynein5. This motion can be visualized by co-labeling LDs and microtubules via injecting both BODIPY 493/503 and SiR-Tubulin (Figure 5). As LDs frequently reverse their direction of movement (as they switch between kinesin-1 and cytoplasmic dynein), higher frame rates during acquisition better capture critical details of LD motility.
Live imaging of autofluorescent yolk vesicles is possible without any form of dye injection (Figure 6). However, the autofluorescence is dim, and the excitation laser is phototoxic. Thus, live imaging of autofluorescent yolk has a poor signal-to-noise ratio relative to dye injection.
Figure 1: Diffusion of BODIPY 493/503 through the embryo. The dye was injected along the lateral edge toward the anterior end (top right) and diffuses from this injection site into the embryo, labeling LDs. (A) The dye has diffused through portions of the embryo adjacent to the injection site. (B) Roughly 24 min/2 nuclear cycles later, the dye has diffused past the midpoint of the embryo. Scale bar: 100 µm. A 1024 x 1024 frame (line average 4) was acquired every 30 s. Please click here to view a larger version of this figure.
Figure 2: Particle image velocimetry (PIV) for LDs and acidic organelles. An embryo was injected with both BODIPY 493/503 and LysoTracker Red. (A) BODIPY channel. (B) LysoTracker Red channel. (A',B') Streamline diagrams generated by PIV analysis of the two channels generated from 10 sequential frames, including those shown in A and B. A' corresponds to the flow of LDs, and B' corresponds to the flow of acidic organelles. Note that both A' and B' show a left-of-center confluence where embryonic contents are flowing out of the plane of view, into the center of the embryo. Also, note that BODIPY has diffused more than LysoTracker as different dyes have different diffusive properties. Scale bar: 100 µm. A 1024 x 1024 frame (line average 4) was acquired every 30 s. Please click here to view a larger version of this figure.
Figure 3: ER tracker labels syncytial nuclear divisions. A syncytial blastoderm embryo was microinjected with ER tracker and a portion of its surface was imaged over time. (A) Spindle assembly during a nuclear division. (B) Abscission of the nuclear envelope during the same division. (C) Subsequent interphase. (D) The onset of the next division is indicated by centrosome appearance (occurrence of circular ER-free regions, marked by arrowheads). Note the gradual dye bleaching. Excitation wavelength: 488 nm. Scale bar: 5 µm. A: initial frame, B: 3 min elapsed, C: 10 min elapsed, D: 13 min elapsed. A 1024 x 1024 frame (line average 4) was acquired every 30 s. Please click here to view a larger version of this figure.
Figure 4: Mitoview 633 labeling of mitochondria. An embryo was injected with Mitoview 633 during the syncytial blastoderm stage and imaged 4 h later, after cellularization. The image shows a neuroectodermal cell of an embryo in germ-band extension. Scale bar: 5 µm. A 1024 x 1024 frame (line average 4) was acquired every 30 s. Please click here to view a larger version of this figure.
Figure 5: Co-labeling of LDs and microtubules. A cellularizing embryo was injected with a mixture of BODIPY 493/503 (yellow A,B,C) and SiR Tubulin (magenta A',B',C'). A'', B'', C'' show the merged channels. Panels A, A', and A'' show the initial frame, panels B, B' and B'' show the frame after 5 s, and panels C, C' and C'' show the frame after 10 s. Scale bar: 5 µm. A 512 x 512 frame (line average 3) was acquired every 2.5 s. Please click here to view a larger version of this figure.
Figure 6: Imaging yolk vesicle autofluorescence during syncytial cleavage stages. (A) At the start of the acquisition. (B) After 8 min. (C) After 16 min. Excitation wavelength: 405 nm. Low excitation intensity was used to keep the embryo alive. Scale bar: 100 µm. A 1024 x 1024 frame (line average 4) was acquired every 30 s. Please click here to view a larger version of this figure.
Supplemental data. Please click here to download this File.
AUTODO | abcepta | SM1000a | Stains lipid droplets in violet/blue |
BODIPY 493/503 | ThermoFisher | D3922 | Stains lipid droplets in green |
Desiccant Beads –Desiccant-Anhydrous Indicating Drierite | W.A. Hammond Drierite Company | 21001 | |
Dissecting microscope SteREO DiscoveryV20 with transillumination base | Zeiss | 4350030000000000 | |
Double sided tape-Permanent Double-Sided Tape | Scotch (3M) | – | Sold by many vendors |
ER-Tracker Green (BODIPY FL Glibenclamide) | ThermoFisher | E34251 | Stains the ER/nuclear envelope |
Femptotip II | Eppendorf | H129354N | Ready to use |
Glass capillaries -Glass Thinw w/fil 1.0mm 4in | World Precision Instruments | TW100F-4 | Must be pulled |
Glass coverslip | – | – | Buy the appropriate refractive index for your objective lens |
Glass slides -Double Frosted Microscope Slides precleaned | FisherBrand | 12-550-343 | |
Halocarbon oil 27 | Sigma-Aldrich | H8773-100ML | |
Halocarbon oil 700 | Sigma-Aldrich | H8898-50ML | |
Heptane greener alternative anhydrous, 99% |
Sigma-Aldrich | 246654-1L | Heptane is considered toxic by the USA's OSHA |
Confocal microscope | Leica | Sp5 | Many different types of confocal microscopes will work. |
LipidSpot 610 | Biotium | #70069 | Stains lipid droplets in far red |
LysoTracker Red | ThermoFisher | L7528 | Stains lysosomes in red |
MitoView 633 | Biotium | #70055 | Stains mitochondria |
Needle loading pipette tips – 20uL microloader tips | Eppendorf | # 5242956.003 | |
P-1000, Next Generation Micropipette Puller | Sutter Instruments | P-1000 | The settings used are heat-470, pull-70, velocity-60, delay-90, pressure-200, ramp-479 at 1×1. They refer to the intensity and length of the heating, the timing of pulling force and whether the force is applied linearly. Using these conditions, one capillary generates two usable needles with fine openings at the tip. |
SiR-Tubulin | Cytosketon Inc | CY-SC014 | Made by Spirochrome; Cytoskeleton Inc. is the North American distributor. Stains microtubules in far red |
TransferMan | Eppendorf | 5178 NK | |
ViaFluor 405 | Biotium | #70064 | Stains microtubules in violet/blue |
Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.
Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.
Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.