Tuesday, April 3, 2012

Adventures in photobleaching

There are a couple of ways to act when you make a Really Big Mistake in lab.

1) Correct it, tell no one, and pretend it didn't happen. This method is preferable when you do something small and stupid. Like one time some sequencing results didn't look very good. And I realized that I had completely diluted my primers to the wrong concentration. Like, mMols off. This would be an okay mistake not to tell anyone else around you in lab, just as long as you fix it and make sure never to make that mistake again. (And also that no one else will be using your primer stocks.) (This also isn't exactly how it went for me because it took someone else pointing out that I had made a blaring error in my primer dilutions, so everyone knew about it in the end.)

2) Admit you made a mistake, ask for help, correct it, and then pretend it didn't happen (but never make that mistake again.) This method is especially useful when something big is going wrong, and you can't figure it out on your own. This is when you just have to suck it up that you'll look like an idiot for about 5 minutes while someone is explaining what you did wrong (or as I prefer to see it, what you weren't doing right!) but in the end it's all for the best because a) hopefully you learned something, and b) you won't make that mistake ever again. And it's a win-win! Because when you come across another poor soul who's made the same mistake as you, after you help them figure out what they weren't doing right, you can choose whether or not to admit to them that you too made the same mistake.

3) Ask for help, figure out that you were a complete idiot about something, and laugh uproariously about it. This might include just yelling PHOTOBLEACHING every 20 minutes or so for the rest of the evening after you figured your problem out. 


So this may or may not have happened to me today.


I will start off by saying that my lab does not do fluorescent microscopy. No one in my lab has fluorescent microscopy experience, and so we're looking at some fluorescence in our cells (just to check transfection levels) and using a scope in a lab downstairs that we've never really been formally trained on. The one caveat is that I come from a family of fluorescent microscopists--so this what follows is sort of unforgivable.

Here is what happened.

We walked downstairs with our little flasks of cells, and we put them under the microscope. We turned on the light, turned on the laser, made sure we were on the right filter, opened the shutter, and looked at our cells.

"Ooh!" My post doc says, "Pretty glowing cells!"

"Awesome," I say, "Let's put it on the screen!"

(I am clearly paraphrasing here.)

So, with the fluorescence still on, we manually switch the scope from eye to camera, we press "Live", and we wait for the picture to come up on the screen.

(If you're keeping tally of time blasting cells with fluorescence, we're now clocking in at 2 minutes.)

It takes another few seconds for the image to come up, then of course we have to refocus slightly for the image to be in focus for the camera.

Then we take a picture.

(Fluorescence still open, in case you were wondering.)

Then we hmmm and hawww and write down the settings we used so we use the same settings for all the other flasks.

Then we go get another flask.

I will skip the rest of the nitty gritty details, because the short of it is, we kept looking from eye to camera and back again, and since apparently neither my post doc nor I were feeling incredibly competent today, we kept forgetting to manually change the scope from eye to camera, all with the fluorescence on. 


And then we wondered aloud to each other, "Why are all of these flasks getting progressively dimmer?" And of course, being scientists, we say, let's get that first flask out that was really bright!

So we get the first flask out and everything is really dim.

In retrospect:

It didn't even occur to me that this was the problem until later I was talking about what I was doing to someone who actually knows how to do microscopy, and they were just like, You focused with the fluorescence ON?? My post doc still isn't quite convinced that this is the problem, but I think she just doesn't want to admit that it's the problem because it makes us look like complete idiots.

And me?

I am perfectly ok with looking like a complete idiot. Because, you know, how else would you learn? (By actually listening carefully to what people tell you to do so you never make mistakes? Where's the fun in that?)

And for the rest of the evening, yes, I have yelled PHOTOBLEACHING aloud at odd intervals. Because, arrrghhh.....PHOTOBLEACHING!








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