Drosophila Drosophila Drosophila (try saying it 3 times fast)

Hi! Sorry for the long absence, it's been pretty busy. Anyway, in the last few weeks my project has really developed. As I mentioned before, I will be measuring the fluorescence levels of several different GFP fly lines. My lab will use this information to choose bright or dim embryos depending on the needs of their experiment (ex: you have a red fluorescent protein you want to image with the green at the same time. the red is dim, so you want to choose a dim green as well so the faint red is not drowned out).
Because my project compares different pictures taken at different times, we needed to figure out a way to make a "control" in each picture. The pictures are different because the embryos fluoresce under a 488nm laser. That laser can vary in power and make 2 different fluorescing embryos look the same. Different embryos also require different laser powers to create 'optimal' pictures--not too bright or too dim.
The first thing we tried was fluorescent beads. The idea made sense: squirt some of these 6um beads (embryos are about 150 micrometers by 400um) onto each slide. Take pictures. Use the ratios of bead brightness from each picture to compare embryo brightnesses (ex: in one picture, the beads have a pixel value of 1. in the other, 2. to compare, double the brightness of the embryo in picture one).
But in practice, this was not so easy. First of all, the beads needed to be kept in the dark and at around 4C all the time. This meant I had to do all the squirting in the dark in the 'cold room' where my lab works with proteins. Needless to say, it was pretty hard. I would either miss the spot on the coverslip (if the embryos and beads weren't in the same frame for pictures, it wouldn't work), the oil I was injecting into would freeze (you put embryos in halocarbon oil to keep them alive under a coverslip), or if I did get the beads right, they would mess themselves up. Once, the computer measured the 3% beads as WAY brighter than the 30%. So beads weren't so helpful. (one of the grad students had ordered them a long time ago to do what I am now, but got frustrated and decided it wasn't worth the time)
But luckily we found a paper. And it described a method that did not include beads. Our new method allows us to adjust laser power for each embryo and then normalize the values to make them comparable. Basically, you view the results as if you had taken each picture at 1% laser power. First, you raise the laser power to make the embryo you are viewing a certain brightness. You take the picture, take the mean brightness of the embryo (with a histogram on the computer) and divide by the laser power you used (ex: mean brightness=100 laser power=25% normalized brightness=4).
I'm really enjoying the new method we found for two reasons: 1) I get to use the really fancy 510 confocal microscope (I will post pictures) 2) I feel like I got to have creative/intellectual input when it came to how we were going to implement it. No one in the lab has done it before, so we kind of got to start from scratch.
Anyway, now I'm going to put up some pictures from a few weeks ago. Next time: my side projects-- inverse PCR and gene mapping!