7. Mount the de-chorionated embryos on the air-permeable
membrane with the designated dorsal-ventral orientation.
The orientation should be adjusted before the embryos touch
the glue (see Note 14). Finish embryo mounting in 8 min at
40% humidity and 25
C. 20–30 embryos are expected to be
mounted.
8. Add 50 μL of halocarbon 27 on the embryos and cover them
with an 18 Â 18 mm cover glass (No. 1.5). Avoid any shearing.
The embryos should keep their dorsal-ventral orientation.
3.4 Imaging of
Embryos
Here we provide protocols and microscope setups for the Leica SP5
point-scanning confocal system. The protocols are also suitable for
other multicolor confocal microscopes (e.g., Zeiss, Nikon). Image
examples are shown in Fig. 2. Three laser lines are used: a diode
laser for 405 nm, an argon laser for 488 nm, and a HeNe for
591 nm. The powers are set at 0.4 μW, 1.1 μW, and 0.5 μW,
respectively. Light intensities at the sample are estimated to be
13, 36, and 17 μW/cm
2 , respectively.
1. Start imaging when the embryo develops to the desired stage.
Find the embryos with bright-field illumination using a 20Â
objective and save their XY locations.
2. Acquire images through a high-magnification (63Â), high-NA
(1.44) oil-immersion objective. Use HyD photon counters to
provide high sensitivity and a broad dynamic range. Set voxel
size to 107 Â 107 Â 330 nm. For cellular blastoderm, image a
z-stack of 8 μm that covers the entire nuclei at the embryo
periphery.
3. Scan at 700 Hz and accumulate three scans per line. Two
sequential scans are used, alternating with each line, 405 nm
and 591 nm scan simultaneously, followed by the 488 nm scan.
4. Once the acquisition is done, take a full embryo image at the
midsagittal plane using a 20Â objective in order to register
anterior-posterior positions.
5. Take a flat-field image using a Chroma autofluorescent plastic
slide. Reduce power to avoid saturation. The flat-field image is
used to control uneven illumination in the image field of view.
3.5 Correction of
Errors Associated with
Fluorescent Spot
Distance
Measurements
1. Measure three-color TetraSpec beads at different laser powers
to obtain images with intensities matching the dynamic range
of the acquired MS2-, PP7-, or parS-system-tagged spots.
2. Measure a control fly line in which blue, green, and red spots
are physically co-localized. We use a reporter line with alternating MS2 and PP7 stem-loops. Cross males from this line with
females carrying NLS-MCP-3xtagBFP2, MCP-eGFP, and
PCP-mCherry (or PCP-mKate2). Image the embryos with
the same microscope settings.
380
Hongtao Chen and Thomas Gregor
membrane with the designated dorsal-ventral orientation.
The orientation should be adjusted before the embryos touch
the glue (see Note 14). Finish embryo mounting in 8 min at
40% humidity and 25
C. 20–30 embryos are expected to be
mounted.
8. Add 50 μL of halocarbon 27 on the embryos and cover them
with an 18 Â 18 mm cover glass (No. 1.5). Avoid any shearing.
The embryos should keep their dorsal-ventral orientation.
3.4 Imaging of
Embryos
Here we provide protocols and microscope setups for the Leica SP5
point-scanning confocal system. The protocols are also suitable for
other multicolor confocal microscopes (e.g., Zeiss, Nikon). Image
examples are shown in Fig. 2. Three laser lines are used: a diode
laser for 405 nm, an argon laser for 488 nm, and a HeNe for
591 nm. The powers are set at 0.4 μW, 1.1 μW, and 0.5 μW,
respectively. Light intensities at the sample are estimated to be
13, 36, and 17 μW/cm
2 , respectively.
1. Start imaging when the embryo develops to the desired stage.
Find the embryos with bright-field illumination using a 20Â
objective and save their XY locations.
2. Acquire images through a high-magnification (63Â), high-NA
(1.44) oil-immersion objective. Use HyD photon counters to
provide high sensitivity and a broad dynamic range. Set voxel
size to 107 Â 107 Â 330 nm. For cellular blastoderm, image a
z-stack of 8 μm that covers the entire nuclei at the embryo
periphery.
3. Scan at 700 Hz and accumulate three scans per line. Two
sequential scans are used, alternating with each line, 405 nm
and 591 nm scan simultaneously, followed by the 488 nm scan.
4. Once the acquisition is done, take a full embryo image at the
midsagittal plane using a 20Â objective in order to register
anterior-posterior positions.
5. Take a flat-field image using a Chroma autofluorescent plastic
slide. Reduce power to avoid saturation. The flat-field image is
used to control uneven illumination in the image field of view.
3.5 Correction of
Errors Associated with
Fluorescent Spot
Distance
Measurements
1. Measure three-color TetraSpec beads at different laser powers
to obtain images with intensities matching the dynamic range
of the acquired MS2-, PP7-, or parS-system-tagged spots.
2. Measure a control fly line in which blue, green, and red spots
are physically co-localized. We use a reporter line with alternating MS2 and PP7 stem-loops. Cross males from this line with
females carrying NLS-MCP-3xtagBFP2, MCP-eGFP, and
PCP-mCherry (or PCP-mKate2). Image the embryos with
the same microscope settings.
380
Hongtao Chen and Thomas Gregor