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Drosophila melanogaster fruitflies perching on the brim of a bowl of sour dough and waiting for their chance. Image Courtesy: Ursula Gönner, Dolgesheim (Germany).
Drosophila melanogaster fruitflies perching on the brim of a bowl of sour dough and waiting for their chance. Image Courtesy: Ursula Gönner, Dolgesheim (Germany).

Visualize developmental patterns in Drosophila

The marvelous complexity of life is perhaps the greatest source of fascination within the biological sciences. Among the most complex subjects within biology is the development of multicellular life. Differentiating from a single cell, multicellular organisms generate remarkably intricate and complex patterns and forms. It seems, therefore, almost counterintuitive that the long history of studying genetic regulation in ontogenesis is so closely and intrinsically entangled with research on an otherwise unremarkable organism; an organism more commonly recognized as a nuisance (see Fig 1), the fruit fly Drosophila melanogaster [1].

Drosophila melanogaster fruitflies perching on the brim of a bowl of sour dough and waiting for their chance. Image Courtesy: Ursula Gönner, Dolgesheim (Germany).

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The Genetic Cascade

A nuanced genetic machine exists that serves to guide the growth of a single zygote into a complex multicellular organism. Packaged neatly within each cell is a predetermined program, a set of subtle signals and cues that will influence the fate not only of that cell, but of its successors, and its neighbors. The result of this developmental cascade serves to pattern the embryo and the organism. It is a pattern of form and function that is carried into adulthood and passed onto progeny.  

From the moment of fertilization, a series of temporal and spatial cues serve to activate the appropriate genes, the products from these genes activate or suppress other downstream components. This cascade progresses throughout development, marching forward in a in carefully regulated, meticulously directed, and stunningly predictable sequence [2].

The study of developmental biology seeks to elucidate these signals, to characterize them, and to better understand at a granular level how a single cell becomes a complex multicellular organism. To do so, researchers frequently utilize a variety of fluorescent markers that label and identify the product of genetic components within the developmental cascade. Linking the fields of biology and optics, these fluorescent markers are often best viewed using a confocal microscope [3].

Spectral Separation

Properly labeling and tagging multiple gene products within a developing Drosophila embryo via either fluorescent protein expression or fluorescently tagged immuno-labels is already a challenging endeavor, but it represents only half of the imaging challenge. To generate a complete map of a developing embryo a researcher needs an instrument capable of sufficiently spectrally separating different probes, and it must have sufficient optical resolution to localize these labels to the appropriate structure.

Dr. Dianne Duncan manages the Biology Imaging Facility at Washington University in St. Louis.  Among her interests are the genetic control of development and patterning in Drosophila models. Until recently, optical and technical constraints prohibited proper imaging of all four channels in in a 4-plex labeled fluorescent embryo.  Spectral overlap prohibited precise separation fluorophores and scanning speed limitations made high-resolution deep Z-stacks cumbersome. Additionally, seamless tile-scanning on other systems was nontrivial due to challenges with field-uniformity and technical constraints.

The inherent spectral nature of the SP8 platform, coupled with the ability to do a rapid line-sequential scan, allowed for a brilliant, high-resolution, high-signal-to-noise-ratio tile-scan of a complete Drosophila embryo with full spectral separation for all four colors. Post-acquisition image processing using LIGHTNING and LAS X Navigator helped to produce a particularly stunning multi-channel image across a large synthetic field of view:

The Leica SP-detector allows flexible, nanometer-precision tuning of pass bands for optimal efficiency and spectral separation. The continuous multichannel beamsplitter (AOBS) serves for perfect excitation regimes both in sequential and in simultaneous imaging. A white light laser (WLL) completes the concept of the “white confocal”; it allows the user to simultaneously excite using any wavelength or combination of up to 8 wavelengths between 470 and 670 nm. The low-dark noise of Leica’s Hybrid Detectors(HyDs) yield a “virtually noise free image”, while their exceptional sensitivity allows excitation with lower laser intensities to help preserve sample stability over long term imaging [4].

The User´s Voice

“Leica’s spectral detectors allowed us to simply and quickly acquire a 4-channel line-sequential without complications from cross-talk. The Leica Hybrid Detector was instrumental for generation of a clean image with good signal-to-noise ratio at depth.”

Dr. Dianne Duncan, Imaging Facility Director, Washington University in St. Louis, USA.