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NIGHTSEA Applications


Using routine fluorescence to sort Drosophila larvae


The Challenge


Dr. Laura Reed (Dept. of Biological Sciences, University of Alabama, Tuscaloosa) was heading a research program to investigate whether mutations in specific genes in fruit flies, Drosophila melanogaster, affect triglyceride storage.


To gather sufficient material for analysis, Dr. Reed required large numbers of larvae of each genotype. Her program involved testing 84 different genotypes and, for each genotype, 200 or more larvae. A special strain of fruit flies had been genetically engineered to express Green Fluorescent Protein (GFP) driven by an actin promoter (Figure 1). Only the flies


Fluorescing Zebrafish


Here are comments from a faculty member at Colgate University:


Students in Developmental Biology Lab were examining the effects of


Figure 1. Non-mutant Drosophila melanogaster expressing GFP.


without the mutations fluoresced. The clear difference between fluorescent and non-fluorescent larvae made them easy to sort.


For best results, the larvae needed to be collected, sorted, and frozen when at their largest, but before they pupated. However, they were at this stage for only about six hours. With 84 genotypes to be tested and 200+ larvae per genotype, sorting was a major challenge. While Dr. Reed had a large pool of undergraduates available for sorting, the greater challenge was that she only had access to borrowed time on another lab's research fluorescence stereo microscope.


The Practical Solution


Dr. Reed visited the NIGHTSEA booth at the annual Drosophila Research Conference and tested the Stereo Microscope Fluorescence Adapter (SFA) system.


She immediately realized the potential of putting both her undergraduates and four of her existing lab-grade stereo microscopes to work. The SFA provided a practical, economical solution for her limited equipment.


For Dr. Reed, the Royal Blue excitation/emission set provided excellent results.


SFA Advantages


NIGHTSEA’s Stereo Microscope Fluorescence Adapters offer a number of advantages. First, they require no modification to your existing microscope. They just click into place, making them easy to use and easy to exchange, either on one microscope or between different microscopes in the lab.


Secondly, SFAs are economical and expandable. Since Dr. Reed worked only with GFP (blue excitation/green fluorescence), she only needed to purchase one version of SFA. However, as the needs of her lab grew, additional sets could readily be added.


Finally, as demonstrated by Figure 2, the SFA’s bright illumination and excellent barrier filters allow many fluorescence experiments to be conducted under near-ambient lighting. In this case, the overhead lights were turned off and the blinds closed, but the room did not need to be in complete darkness.


As for Dr. Reed? Using NIGHTSEA’s SFA, she could routinely have shifts of two to four undergrads at a time, sorting Drosophila larvae in parallel. 84 genotypes? 200 larvae per experiment? Problem solved!


Figure 3. Students sort larvae using NIGHTSEA’s SFA in Royal Blue. Dr. Reed had shifts of two to four undergrads sorting in parallel. Zebrafish – GFP fluorescence Figure 2. Larval sorting under ambient lighting.


pharmacological agents on development of zebrafish embryos. In order to better visualize the development of the nervous system and vasculature, we used transgenic fish that expressed GFP either throughout their nervous system or in the developing vasculature. The NIGHTSEA system easily adapted to our dissection scopes and allowed students to observe the development of their fish at several different time-points. They could readily observe the transgene expression, and it helped solidify the phenotypes they were observing and allowed them to determine an optimal time to fix their fish for analysis under the compound microscope.


For quick screens it actually worked perfectly well in a bright room. For more intimate looking (more than


presence/absence calls), we turned out the room lights. Worked better than I’d hoped it would.


These pictures of fluorescing zebrafish embryos and juveniles were taken using the NIGHTSEA Stereo Microscope Fluorescence Adapter.


Zebrafish embryos – histone H2B- Dendra2


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