Making a Slide

So. Here's how I make a slide.


1. Line up embryos with a probe (pointy thing).

2. Put them on a slide (see the little white smudge in the middle?).

We put them in halocarbon oil so they can still breathe!

3. Put the slide in the microscope.

4. Turn on the laser. (488nm = blue)

5. Viola! (10x lens)

This is basically what I do for my experiment. Today, I did a time lapse series. I took pictures every hour to see how GFP expression varies over time. Hopefully, I'll be able to fit a line or a curve to my data points tomorrow.

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!

Lord of the Flies

Hi! My name is Caitlin Burk. I'm going to be a senior at Durham Academy next year, and I'm working with Drosophila (fruit fly) genetics in Dr. Dan Kiehart's lab in the French Family Science Center. My lab is interested in (among other things) myosin, a motor protein, and dorsal closure--when the fly embryo closes up on the dorsal side (time lapse video here, it's the one on the left).

The first few days in the lab have been pretty fun. Ruth Montague, one of the other people who works in the lab, has been showing me the ropes. I have learned to change fly cages (to collect embryos), quarantine flies (to prevent mites), to collect virgins (females that have already mated can store sperm from multiple males, and this can result in undesired offspring), and make slides for the imaging microscope. When you make a slide, the embryos, which look like clearish-white hot dogs, have to all be oriented dorsal side up in nice little lines. This can be hard because a) it's hard for me to tell which side is the dorsal one and b) the wet embryos are slippery, on agar, very small (2 lines of 6 embryos is less than 1/4 square cm), and I'm using a probe with an unsteady hand. I'm getting better though. One thing I think is interesting is that on the slides, the embryos are in halocarbon oil. They can still breathe in the oil, and are alive when you look at them under (over?) the confocal microscope. On the scope, you can view your slides in two ways: 1) normally. 2) you can turn on the laser attatched to the microscope and look at the embryos on a computer linked to a camera on the scope. Some embryos have transgenes, or genes that have been inserted by a scientist, that tag myosin with GFP (green fluorescent protein). The laser excites these proteins, and the camera has a lens that can show the fluorescence (if you looked through the microscope yourself, the laser would zap your eyes). One other thing I learned: When scientists discover a fly gene, they can name it whatever they want. There are genes called armadillo, spaghetti squash, hedgehog, etc.

Today I went to my first lab meeting, where Vinay, a grad student, presented the work he's been doing on the formation of fly hair. It was a little over my head, but I was able to actually understand most of what he was saying for about 45 minutes and then asked questions later. I also finally got to meet Dr. Kiehart, who got stuck coming back from Montreal yesterday. We planned out my project, which will deal with the brightness of the fluorescence of different GFP-flies. Fluorescence differs between different flies because of the position of the transgene. In one line of flies, the gene may have inserted itself in a very thick and crowded part of the DNA. Transcription enzymes will have a hard time getting to this section, and the gene won't be expressed very much (dim fluorescence). If in another line of flies, the gene inserts itself in a more spread out and accessible part of the genome, it will be more expressed (bright fluorescence) because the RNA polymerase can more readily transcribe the gene.

Anyway, I have to go read a paper to get ready for my project. More information my project--and why it's helpful for my lab--later (and hopefully pictures too!).