Chapter 9 - Ultraviolet Laser Microbeam for Dissection of Drosophila Embryos

Abstract

This chapter describes the use of ultraviolet (UV) laser microbeam interrogation strategies, combined with confocal microscopy, to investigate the developmental process of dorsal closure. Drosophila embryos that carry GFP-fusion transgenes are mounted to allow high spatial and temporal resolution imaging under conditions that allow development to proceed unimpeded. With the help of QuickTime videos of the time-lapsed image stacks, changes in specimen morphology that result from laser surgical interrogation are therefore described both qualitatively and quantitatively. Drosophila embryos that carry GFP-fusion transgenes are mounted to allow high spatial and temporal resolution imaging under conditions that allow development to proceed unimpeded. For each confocal microscope system, modifications to the optical path of the microscope are necessary to allow simultaneous imaging and laser surgery. The advent of spectral variants of GFP mentioned earlier, the proliferation of other unrelated fluorescent proteins, each with different excitation and emission characteristics. Once the mirrors are aligned properly to guide the unmodified beam through the objective, the polarizer, the individual lenses that comprise the beam expander and the telescope are added, in that order.