Lab update 3-1-08

Mostly, I have been working on immunofluorescence experiments and taking pictures of same. I’ve actually gotten some pretty good images of my experiments and that makes me very happy. I now have good images for:

Figure 1A: Panels 2 and 3
Figure 2B: Panels 1, 2 and 3

There’s still a lot left for me to do, though. Unfortunately, I’m still having problems with the deconvolution software. Fortunately (?) I’m not the only one having problems. While this is definitely inconvenient, at least it means that it’s not just something I’m doing wrong or that my images are too sucky to deconvolve (actually, they look much better before I try to deconvolve them). Importantly, the other person who is having problems with the software said that it was working fine several weeks ago, but is now causing problems. Hopefully, it can be fixed because it’s pretty expensive software.

Anyhow, for now, I’m not doing the deconvolution and the images look fine so I’m going to set up the figures with the images that I have.

Lab update 2-15-08

In Lab: 

This week has been frustrating in several ways with regard to labwork.  First of all, I am attempting to optimize a particular procedure.  “Optimize” is code for “doing lots of experiments that are ultimately failures.”  While, in truth, you are actually moving forward because the optimization needs to be done and each parameter you tweak gets you closer to your goal of the (almost) perfect experiment (there is no such thing as the absolutely perfect experiment), it actually feels as though you are getting nowhere fast and have nothing to show for the 60 hours you just spent in lab.

The other reason this week was frustrating was because I ran smack into my own ignorance.  We have this image processing software that we use for the images that we take with the camera on the microscope.  I had been just merrily using the software, following the steps written up by someone else, when I ran into a problem.  When you run into a problem, that’s when you find out how well you know the system.  The answer for me was, “Not very well at all.”

The process I wanted to do is called deconvolution.  Before this week, I knew that deconvolution “made your image look better.”  How this happened, I really didn’t know (except that it involved math).  This ignorance (while not ideal) was not a problem for me in the past because everything worked the way it should.  But this week I ran into an instance where I had a really crappy looking image and deconvolution made it look even worse.  Which was counter to my thinking of what deconvolution was all about.

I now know that deconvolution is a mathematical way of assigning light back to its original source.  Think of a street light.  Normally, when you are looking at a street light, you see a halo of light surrounding it (particularly if it is foggy out).  The same is true for the fluorescent molecules that I am looking at in my sample.  However, I need to see exactly where the object is.  So now, think of two street lights really close together.  If they are close enough, their halos of light may become combined to look like one halo.  Now, if it was so foggy that you couldn’t actually see the street lights themselves, just the halo, you might think there is only one street light there when in fact there are two.  In my images, I really need to be able to tell if I am looking at one object or two objects really close together.  Deconvolution uses mathematical algorithms to get rid of the halo and only let you see the light that’s actually on the object.  It’s like getting rid of the halo on the street light such that you only see the light that is within the street light itself.

Did that make any sense at all?

Anyhoo, deconvolution is important in astronomy, too, for images coming from telescopes.  Because of this, my husband and I had several very long conversations discussing my image processing woes and how I might be able to resolve them (resolve–hahaha!).  Some couples may watch romantic movies on Valentine’s Day, we discuss image processing.

I was really hoping to be able to cross off some of the images on my list (to indicate completion) on the Paper Progress page, but alas it was not to be for this week.  Therefore, the highlight of my week was when a small rose plant arrived in lab for me for Valentine’s Day from my husband.

The Seminar Sock:

This week, the sock and I went to two thesis defenses (for people who are two years behind me in grad school years;  let’s not talk about how I feel about that).  The first one was from a student whose lab studies the mechanism of recombination during meiosis in yeast.  He had some really fantastic electron micrographs of DNA coated with his favorite protein.  He also had worked on an anti-cancer agent and had some interesting data about that.

The second thesis defense was from a student whose lab studies splicesome assembly and disassembly in yeast.  She had a huge amount of data.  Every time she came to a summary, I thought for sure that must be the last thing because it was so much work and then she went on to test another part of her model.  She’s going to have four papers when she leaves.  Amazing.  Her data was not as cool to look at as the first defense because it mostly consisted of a ton of RNA gels (which, all look about the same; I’m all about pretty pictures in science, this is probably why I’m in the lab that I’m in).  But, her method of systematically building her model was very elegant, as was the model itself.

I also have been working on the sock while taking images at the microscope.  I set up the scope to take a series of images.  I can’t leave the room while it’s doing so because then a ton of light would shine on the scope and I need it to be dark.  The computers are not hooked up to the internet, and it’s too dark to read well.  So, I knit.

Plans for next week:

I’m going to finish optimizing this technique if it kills me!  Then, I’m hopefully going to be able to take some images for my paper and cross off some of the figures on my list.

I’m also hoping to post on this blog a series of entries that I’ve been working on that explain immunofluorescence in layperson terms.  I’ve got to do a bit of tweaking on the posts over the weekend, but I should be able to put the first one up on Monday.  I’m also hoping to follow that up (during the next week) with some pictures of an immunofluorescence experiment in progress, culminating in pictures of the slides that I take on the scope.

Stay tuned!

Lab Update for Feb. 8

This week I tried planning each day in advance; writing a list of things to do and not going home until I got them all done. As usual when I try this type of thing, I grossly overbooked myself and am now exhausted.  Somehow, I thought it would be a good idea to spend 12 hours a day in lab for 4 out of 5 days.  I was wrong.  Well, it was good in terms of data, but bad in terms of my sanity!

I have added a new page to the blog called Paper Progress which has a very basic outline of the structure of my paper so far.  The first three figures are well-planned in terms of structure, but the layout of last three has not yet been planned, so I didn’t elaborate on them.  As I get the data for each figure, I’ll cross it off my list (I’ve been looking for a widget to do this so I can put it in my sidebar, but so far I have not found anything to my liking).  The titles of the figures in the list are not the titles of the figures in real life.  I have not yet determined how much information about my data I can put on the blog without potentially jeopardizing publication of the final paper.  Some journals would consider my blogging about the data publishing it and therefore refuse to publish my paper on the grounds that it has already been published.  I would prefer to publish in an open access journal that would not give me trouble about such things, but where I publish is not a decision that’s entirely my hands.  So, for now, the descriptions are rather vague.

Mostly, I have been trying to get good images of immunofluorescence data  for Figure 1A.  Because the fluorescence fades after time, if I do not capture good images fairly soon after the experiment is complete, I will have to repeat the experiment.   This week, I did several experiments and then today tried to take pictures of these experiments.  The images are not of the quality that I would like and this cannot be changed by the imaging process, so I need to tweak the experiment.  I’m going to leave that until Monday.

This experiment is a huge pain in the @ss because it requires 12 hours of lab time in one day.  There are very long incubation times and this allows me to do other things during those times.  This included evaluating some of the yeast strains that I created for Figure 2A, prepping DNA to create more yeast strains for Figure 2A, creating the yeast strain for Figure 2B, and optimizing the immunofluorescence procedure for Figure 1B.

I realize this probably sounds like gibberish to most people who are not cell biologists.  Therefore, I’m working on a series of posts about basic concepts and techniques.  The first will be about immunofluorescence, hopefully followed by molecular cloning, and transforming bacteria and yeast.  Stay tuned!

It’s still Friday for me

Therefore, I can still call this a Friday lab update!

Having been sick all last week, and still feeling a little under the weather this week, I didn’t accomplish all that much in the lab in terms of experiments.  I started back in lab on Tuesday and all I can really say for my first three days back in lab is that I sent some DNA to be sequenced.  However, I have big plans for the weekend.  Today, I started an immunofluorescence experiment.  It’s a long, involved, and complicated experiment which fortunately can be broken up into several days.  Today’s part took 13 hours to complete.  Tomorrow, I have about 5 hours of work for that experiment at the end of which, I’ll have some slides with yeast cells that will glow green and red and blue under certain wavelengths of light.  The next step will be to look at the slides under the microscope and take pictures.  Unfortunately, the fancy light bulb for our scope is burned out.  And the scope for the neighboring lab is all booked up for Saturday.  So, Sunday, I’ll be spending several hours of quality time on the microscope–looking at my yeast cells and taking pictures if they look good.

I’ve also spent some time trying to organize my data into a paper outline.  This was both gratifying and frightening.  I finally have the first half put together in a way that I think (and my advisor agrees) is solid (that is, it tells a nice story).  Unfortunately, the data I already have is not “pretty” enough to include in a publication, so I need to repeat some experiments to try to get nicer pictures.  Even more unfortunately, there are gaping holes in the outline that need to be filled in.  By that I mean the experiments have been planned and we are reasonably sure of what the results will be, but the experiments have not actually been done yet.  That’s a scary place to be right now because there’s always the possibility that the results will be different from what we expect and that will change the scope of the paper.

We will not discuss the second have of the paper.  The second half does not have gaping holes.  This is because there’s nothing there to have a hole in.

Virulent

One thing I was trying to do with this blog was be more consistent about my posting than I am in my other blogs.  Towards this end, I had planned on having a post every Friday talking about what I had done that week in lab.  This week, that post would have looked something like this:

Unfortunately, I seem to have been overtaken by a virus hell-bent on turning my body into walking, talking, coughing snotbag.  I’ve gone through several boxes of kleenexes, and I don’t know how much dayquil/nyquil/sudafed/cough drops/Airborne.  And, I don’t know if I can get anymore dayquil/nyquil/sudafed because the Feds have my driver’s license in their database and the fact that I’ve bought so many products containing pseudophedrine HCl lately might mean that they won’t let me buy anymore.  Or maybe it means that they think I’m running a meth lab and they are planning a raid on my apt.  I can see it now–they burst in only to find me laying on the couch clutching a box of kleenex (”While you’re searching the kitchen, could you bring me a glass of orange juice please, officer?  Thanks.).

Despite the fact that I was obviously sick, I tried to go into lab on Thursday because two people in my lab were giving talks at various events that day.  I thought, hey, a talk, that doesn’t require much effort, I can just sit there.  This only served to confirm that it was a good idea not to do any labwork because obviously, my brain is not functioning at maximum capacity.  Tell me, do you think a person with a hacking cough should go to a seminar?  After spending half of the first talk in the hallway, engaged in a coughing fit, I made my appologies and went home.  Friday, I had to present in lab meeting.  In our lab, lab meeting is a time to discuss lab business (”Is it just me or has the PCR machine been acting funny lately?”  “So, when should we defrost the freezer?”  that sort of thing) and have people talk about their research and get input from the group.  We just recently switched from having one person talk per week to having two people talk per week (but each talks for less time than in the one person per week format) and I was one half of the pair of presenters for the week (since it had been a couple of months since the last time I gave lab meeting, it didn’t matter than I was completely unproductive this week).

I’ve got quite a bit of data these days and I’m trying to organize it into a paper format and find the holes and fill them in.  A paper should tell “a story.”  That is, it should saying something like:

In previous studies, our lab found that Jack and Jill went up the hill and Jack fell down and broke his crown.  Based on those findings, in this paper we wanted to know why Jack and Jill went up the hill and what happened to Jill after Jack fell down.  We will show that the purpose of going up the hill was to fetch a pail of water.  We will also show that after Jack fell down, Jill came tumbling after.  What exactly happened to the pail of water is unknown but based on these findings we speculate that the pail of water also fell down the hill, resulting in the water being spilled.

Right now, I have:

In previous studies, our lab found that Jack and Jill went up the hill and Jack fell down and broke his crown.  Based on those findings, in this paper, we wanted to know why Jack and Jill went up the hill and what happened to Jill after Jack fell down.  We will show that the purpose of going up the hill was to fetch a pail of water.

I’m still trying to figure out what happened to Jill and the pail of water.

Unfortunately, because the lab meeting format has changed, I could only present part of my paper outline–the part that has already been done.  Here, I needed the input from the lab because I need to know these things:

Do I have to show that Jack brought a pail and not a bucket and what the hell is the difference between a pail and a bucket anyway?

I have shown that the substance in the pail is clear and tastes like water.  Do I need to do further tests to show that it really is water?

To which the answers were no and possibly, respectively.  I also got some suggestions for tests to further prove that the pail/bucket contained water.  So, I guess it was productive.  Unfortunately, due to time constraints I didn’t discuss my plans for figuring out what happened to Jill and the pail of water.    Which is okay.  Maybe by the time my next lab meeting rolls around, I’ll have preliminary data on the fate of Jill.