In our new lab here in Regensburg, we are currently re-establishing the method of confocal microscopy. To start with, we used the fly lines which show a defect in the temporal structure of their spontaneous behavior (a project of one of our graduate students). this fly line uses two transgenic constructs (c105 and c232) to drive expression in specified neurons in the fly brain, i.e., the merged expression patterns of c105 and c232. In this case, the transgenes drive expression of green fluorescent protein (GFP). Around 7pm on the evening right after the dissections and the confocal imaging, I reconstructed the image stacks as a 3D video of the expression pattern in the brain and uploaded it to my YouTube channel:
Only an hour or so after I posted the video, Douglas Armstrong sent me an email:
And sure enough, upon closer inspection and comparison with the data displayed in the two links Douglas sent along, it seemed as if only the c105 expression pattern is visible. So as we are establishing the technique here, we need to pay extra attention to threshold effects, preparation and the imaging settings at the two different confocal microscopes we have here. In addition, we need to image flies with the individual drivers, to make sure nothing is wrong with the fly strain that should contain both drivers.
All of these things are important and we probably would have not paid special attention to them as we thought the double driver line was simply a good line to start optimizing the method.
In other words, if I had never published the data online right after we got them, I probably might have spent quite some time not being aware of potential problems with this fly strain.
Thanks so much, Douglas!