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Holtzman, S., Kaufman, T. (2013). Large-scale imaging of Drosophila melanogaster mutations. 
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FBrf0220532
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Personal communication to FlyBase
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To date, relatively few imaging projects have been undertaken that show a multitude of mutations in Drosophila melanogaster.  One such project, undertaken by Jennifer Childress in Georg Halder's lab, shows a representative sample of popular mutants and phenotypes (Childress et al., 2005).  For many years, we also wanted to document many of the existing Drosophila mutations by performing a large-scale imaging project.  However, due to the limitations of extended focus software and added processing time in Photoshop (Jennifer Childress, personal communication), this was not economically feasible.  Leica has developed a new imaging system, Live Z Builder, that collects focused pixels in real time, allowing the user to manually capture an image with a dissecting scope, motorized focus and special software.   Preparation time for most imaging is reasonable, with an average of just a few minutes per fly image.  No Photoshop editing was performed other than cropping, adjusting brightness between specimens, sharpening, and labeling mutant phenotypes (in other words, no editing was performed on the image itself for out-of-focus pixels, imaging artifacts, etc.).   This ease of imaging made a large-scale project for Drosophila mutations possible.
The microscope utilized for imaging was a Leica MZ16, with a DFC450 camera on a motorized mount.  The software that was used to collect the stacked image in real time was Live Z Builder, part of the Leica Application Suite.
In most cases, mutations were imaged that were single mutations, or were the only mutation visible in the body part imaged.  In some cases, double mutants were imaged when judged to be easy to determine; for example, some eye shape mutations were imaged that also contained eye color mutations as well.  In a few instances, more complex genotypes were imaged when there was no alternative.  All flies imaged were females except where noted.  Fly stocks were obtained from the Drosophila Stock Center and were cultured at 25°C except for one eye color mutant, drb, which is temperature-sensitive and should be grown at 18°C (Lindsley and Zimm, 1992).
All flies were imaged on a Dutch Boy E26-1 Tournament Blue paint chip for blue background color.  For eye shape, bristle, and eye color imaging, flies were collected within one day of eclosion (and after five to seven days for aged eye color mutants), placed in Eppendorf tubes, and were frozen at -80°C for one hour.  Flies were then thawed at 22°C (room temperature) for 30 minutes before imaging.   Two fiber-optic light boxes were used with each fiber-optic arm set at 90 degrees to encompass the stage.  A cylindrical cross-section of a foam coffee cup was placed around the flies to diffuse light evenly around the specimens (Jennifer Childress, personal communication).
For live imaging of whole flies for body color and wing mutants, a combination of CO2 gas anesthesia and microscope cold stage was used.  The cold stage uses Peltier technology to chill a metal plate, upon which the blue paint chip and microscope slide were placed for the blue background (cold stage constructed by Jim Powers at Indiana University).  After flies had been anesthetized for at least one minute with CO2, they were then placed on the cold plate (set to 8.3°C, which in turn chilled the glass slide on top to 11°C) and allowed to wake up.  The flies were imaged as soon as they stood up. 
References
Childress, J., Behringer, R., and Halder, G. (2005). Learning to Fly: Phenotypic Markers in Drosophila. Genesis 43(1). Cover Illustration.
Lindsley, D.L. and Zimm, G.G. (1992).  The Genome of Drosophila melanogaster. Academic Press, San Diego, CA. 
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    English
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