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Electron Microscopy Sciences

Technical Data Sheets

Sorting Genotypes: Drosophila Larvae and Arabdopsis Seeds

EMS Catalog #SFA Nightsea

Mouse Brain with EGFP-tagged lentovirus vector
Figure 1. Non-mutant Drosophila melanogaster expressing GFP. © NIGHTSEA/Charles Mazel.

GFP-labeled dorsal striatum
Figure 2. Larval sorting under ambient lighting.

Mouse Brain with EGFP-tagged lentovirus vector
Figure 3. Students sort larvae using NIGHTSEA's SFA in Royal Blue. Dr. Reed now has shifts of two to four undergrads sorting in parallel.

Drosophila Larvae: Using routine fluorescence to sort

The Problem:

Dr. Laura Reed (Dept. of Biological Sciences, University of Alabama, Tuscaloosa) heads 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 requires large numbers of larvae of each genotype. Her program involves testing 84 different genotypes and, for each genotype, 200 or more larvae. A special strain of fruit flies has been genetically engineered to express Green Fluorescent Protein (GFP) driven by an actin promoter (Figure 1). Only the flies without the mutations fluoresce. The clear difference between fluorescent and non-fluorescent larvae makes them easy to sort.

For best results, the larvae need to be collected, sorted, and frozen when at their largest, but before they pupate. However, they are at this stage for only about six hours. With 84 genotypes to test and 200+ larvae per genotype, sorting is a major challenge. While Dr. Reed has 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.

The SFA comes in three different excitation/emission combinations:

Excitation/ Filter application Excitation Emission
"Royal Blue" for GFP, eGFP, fluorescein 440-460nm 500nm
"Cyan" for YFP and similar fluorophores 490-515nm 550nm
"Green" for DsRed, dTomato 510-540nm 600nm

For Dr. Reed, "Royal Blue" provided excellent results (Figure 2, 3).

SFA Advantages

  • Drosophila larva, GFP © Charles Mazel
  • Drosophila larva, GFP © Charles Mazel
  • Drosophila larva, GFP actin © Charles Mazel
  • Drosophila larva, YFP eyes © Charles Mazel
  • Adult Drosophila, UAS-Tomato Tubulin GAL4 © Charles Mazel
  • Drosophila larva, UAS-Tomato Tubulin GAL4 © Charles Mazel
  • Drosophila adult, Venus in muscle © Charles Mazel
  • Drosophila pupa, Venus in muscle © Charles Mazel
  • Drosophila larva, Venus in muscle © Charles Mazel

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 currently works only with GFP (blue excitation / green fluorescence), she only needed to purchase one version of SFA. However, as the needs of her lab grow, additional sets can readily be added.

Finally, as demonstrated by Figure 2, 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 does not need to be in complete darkness.

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

Arabidopsis Seeds

  • White light illumination. Seeds: upper left – bright green fluorescent, lower left – weak green fluorescent, center – control, upper right – bright red fluorescent, lower right – weak red fluorescent.
  • Royal blue excitation with yellow barrier filter. Seeds: upper left – bright green fluorescent, lower left – weak green fluorescent, center – control, upper right – bright red fluorescent, lower right – weak red fluorescent.
  • Green excitation with red barrier filter. Seeds: upper left – bright green fluorescent, lower left – weak green fluorescent, center – control, upper right – bright red fluorescent, lower right – weak red fluorescent.
  • White light illumination. One of each of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.
  • Royal Blue excitation with yellow barrier filter. One of each of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.
  • Green excitation with red barrier filter. One of each of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.
  • White light illumination. Mixture of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.
  • Royal Blue excitation with yellow barrier filter. Mixture of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.
  • Green excitation with red barrier filter. Mixture of the 5 fluorescent seed types – bright green, weak green, control, bright red, weak red.

Arabidopsis thaliana is a small flowering plant that is widely used as a model organism for a variety of genetic studies. Dr. Scott Poethig and colleagues at the University of Pennsylvania have developed a novel transgenic strain of A. thaliana that has chromosomal segments with eGFP on one end and dsRed at the other. The segments can be followed in genetic crosses and manipulated via recombination. The transgenic strains will enable a variety of experiments, including phenotypic analyses of mutations with weak or environmentally sensitive phenotypes. They are intended for use in both research and education.

Dr. Poethig was looking for a cost-effective way to sort the genetically modified seeds in a teaching setting. He learned about the new NIGHTSEA Stereo Microscope Fluorescence Adapter and sent a set of seeds for testing. There were five varieties - strong and weak green fluorescence, strong and weak red fluorescence, and non-fluorescent control. All of the variations were easy to see, even with the room lights on.

In each row the image on the left was taken with white light illumination, the image in the center with the Royal Blue excitation/emission combination, and the image on the right with the Green excitation/ emission combination. Equipment - NIGHTSEA Stereo Microscope Fluorescence Adapter, Motic SMZ168 trinocular stereo microscope, Canon EOS Rebel T2i camera.

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