A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.
Preparation
Procedimento
We describe a new method of preparing Drosophila embryos for live imaging analysis, which we call the hanging drop protocol. Unfortunately, it is not possible to use the hanging drop protocol if working with an inverted microscope. In this case the sandwiching technique (as described in the abstract above) must be used and compression of the embryos remains a concern.
In experiments where cell shape and size are measured in order to calculate forces associated with morphogenetic movement, the use of an upright microscope and the hanging drop protocol is preferable owing to reduced embryo compression. Also, reduced embryo compression using the hanging drop protocol has the consequence of presenting the round, undistorted surface of the embryo whereas the sandwiching technique presents a flattened surface. When using confocal microscopy it is, therefore, necessary to collect a broader Z-stack (more slices per stack) when using the hanging drop protocol versus the sandwiching method. The increased number of slices per Z-stack, however, increases acquisition time as well as any photo-toxicity or photo-bleaching associated with laser excitation. Clearly, the experimental benefit associated with reduced compression is somewhat offset by an increase in Z-stack acquisition times. Despite this increase in Z-stack acquisition time, however, we have observed excellent viability of embryos using the hanging drop protocol.
The hanging drop protocol is also limited to the observation of embryos by fluorescence microscopy, in which the light path of incident and emitted light does not pass through the live-imaging chamber. Live-imaging embryos using DIC microscopy or other non-fluorescence optics could be achieved by modifying the live-imaging chamber to allow a light path from the condenser through the suspended embryo.
A concern when using the hanging drop technique may be undesirable movement or “drifting” of embryos in the field of view during timelapse acquisition. When floating against the underside of the inverted coverslip, we find that the embryos are remarkably stable and do not shift in position when using either dry or oil immersion objectives. Multipoint timelapse acquisition using a motorized microscope stage also does not disrupt the positions of embryos that are prepared using the hanging drop protocol. Any movement of embryos prepared using the hanging drop technique can be eliminated by reducing the size of the halocarbon oil drop and ensuring that separate oil drops are not fusing or wicking along the edge of the live-imaging chamber’s well.
We gratefully acknowledge support to B.H.R. through a Discovery Grant as well as a Research Tools and Instrument Grant from the Natural Sciences and Engineering research Council of Canada (NSERC). We also acknowledge H. Oda and the Bloomington Drosophila stock Center for providing genetic stocks that were used in the example live imaging sequences.