Tuesday, April 01, 2008

I CAN HAS LAZERS!!!!

I CAN HAS LAZERS! I GOTS A NEW LAZER!!!!!

"Wait, what?" you might be saying, dear readers. "Youre a biologist. Um, why are you excited about lasers? Isnt that a physics thing? Another April Fools?"

NOOOOOOOOO!! I CAN HAS A NEW LAZER! Life doesnt suck so hard anymore!*

You know how Im doing this red vs green thing?

I hit an impasse.

Our flow cytometer cant see my red cells.

I can look through a microscope and see loooots of lovely red cells, but the friggen flow cytometer cant see them. Its like the red guys are invisible. Why?

Well, a flow cytometer works by shining a beam of light, a *Dr. Evil hand quotes* 'lazer', on a stream of cells. If your cell contains a green fluorescent protein, the GFPs will be excited by the light and fluoresce... green. The flow cytometer software says "w00t! That little guy is glowing!" and pops him over to a different part of your readout chart. A cell that does not contain any fluorescent proteins will slide on by. The flow cytometer software says "Boo. That guys a dud, but Ill count him anyway." Non-fluorescent cells stay clumped in a background region of your readout.

The catch to all of this is that you have to have the right wavelength of light to excite your fluorescent proteins properly for the computer to register a hit. You cant just shine any old light onto your cells and expect to see green and red and blue, etc.

I was using a beam of light at 488 nm to excite both of my proteins-- the red and the green. But look at this chart (from Clonetech, click for a better version):

Im exciting both proteins at 488. Light green peak for green is at 488. The red peak for red is at 551. At 488 nm, DsRED2 is only at ~30% of brightness, so my dim red cells, reduced another 70%... the poor computer cant see them. Red cells are being counted as non-glowing background cells.

BUT!

I CAN HAS LAAAAZER!!!! We just got a new laser for the flow cytometer... one at 551 nm!!!!!!!!! So I ran a sample I fully expected to fail. Sure I could see red cells underneath the microscope, but with my old protocol nothing would have registered. The cells were way too dim.

But with the new laser? It saw 50 red cells. More than I could see on my own! WHOOOOOO!!! WHOOOOOOO!!!

I can finally move forward with my main experiments!! Biggest roadblocks are gone! WHOOO! WHOOOOO!! LAZERS! WHOOOOOOO!!!





* Arnie has repeatedly offered to let me borrow his lazers, unfortunately, the lazer I need is green, and his lazers are blue. Puppy tries.

35 comments:

Seabhag said...

Ah the joys of excitation and emission spectra. Welcome to my slice of the world. I'm messing around with UV/vis fluoresce for my research project.
So are you running with 2 excitation beams? Or are you just running one at a time and doing two passes?

Aaron Golas said...

Oh yeah? Well MY lab has ROBOTS! Beat THAT!

Bob O'Hara said...

Another demonstration of excitation by lasers.

So, ERV, are you glowing red or green?

ShavenYak said...

C'mon... we all know what you and PZ are really planning to do with that laser. You're going to use it to pop a giant ball of popcorn in Michael Behe's house, aren't you?

Bayesian Bouffant, FCD said...

That graph must be wrong. It shows your excitation levels peaking, then coming back down. Clearly, you're still on the way up.

Tyler DiPietro said...

Hopefully system preparations A-G are all failures so that you'll have to go through with PREPARATION H.

Couldn't resist another Austin Powers reference...

Goblin Inventor said...

Lazers... I <3 lazers. I don't get any lazers... ahh the joys of bioinformatics, pen, paper, and trusty rusty computer =), but most assuredly no lazers. High hopes with your research!

Don Smith said...

So, ERV, are you glowing red or green?

What a whimsical comment from such a boringly named man. Bob. Bob? BOB?!?! That's the best they could come up with!!?!

Did you develop this skill-set as a wordsmith to compensate for that name?

Don Smith said...

Couldn't resist another Austin Powers reference...

Yes, we can tell.
In the future though, please try harder to resist.

At least "Bob" had some bite to his comment.

If you start in with Wayne's World references I'm going to slap the nerd, dork and geek right out of you.

Don Smith said...

Tyler DiPietro,

Just saw your picture. WOOF!
Not too easy on the eyes are you?

Bayesian Bouffant, FCD said...

aaron golas: My lab has laZers, rObOts, a bay-O-wolf cluster, magic 3D specs, and x-raY vision. All your blase are belong to us.

Don Smith said...

All your blase are belong to us.

Guys come on. Don't go OUT of your way to show how extra nerdy you are.

Rob said...

DsRED is also inherently 10X less bright than EGFP, even when excited at the peak. So your red cells were actually maybe 3% as bright as your green cells at 488. Take a look at 'Guide to choosing fluorescent proteins' by Roger Tsien (Nature Protocols I think).

Tyler DiPietro said...

I thought I smelled dried semen on the ground, Ben R must be making a comeback.

Bayesian Bouffant, FCD said...

Guys come on. Don't go OUT of your way to show how extra nerdy you are.

If I were extra nerdy, I would have figured out how to use a font with an accent mark over the e.

Rev. BigDumbChimp said...

don are you always this boring?

Perfectly Candide said...

That is nothing. At Westphalia Labs, the most noble Baron of Thunder-ten-tronckh has paid for laboratory phasers for each and every researcher, and student. Heck even the janitors get one...it makes clean up really easy.

Don Smith said...

If I were extra nerdy, I would have figured out how to use a font with an accent mark over the e.

Don't sell yourself short.

Don Smith said...

don are you always this boring?

For some reason non-straight men who are interested in finding 'good' cookbooks, who listen to jazz n' funk, claim to have a general interest in "stuff" and photography find me boring.
So, I wouldn't say I'm "always this boring", just that some very particular segment of the population tends to view me that way.

Torbjörn said...

A "very particular segment of the population" finding you boring being all of us, of course. (Yawn.)

[The nerd, dork and geek in me couldn't refrain from pointing out that the troll, kook, and bore forgot to establish a set intersection.]

Don Smith said...

A "very particular segment of the population" finding you boring being all of us, of course.

Yes, all of you, in that particular segment of the population, that I had mentioned above find me boring.

I think Abbie needs to know that not all of the fans of her blog are dweebs. I enjoy her blog posting only to come to the comments to find all of the nerds trying to out-geek each other.

The nerd, dork and geek in me couldn't refrain

Don't worry. I'm quite handy at helping nerds, dorks and geeks like yourself become more able to 'refrain' when need be.

Tyler DiPietro said...

I find it odd that non-dweebs like don seem to have so little to occupy themselves aside from trolling blogs.

Tyler DiPietro said...

BTW Don,

"If you start in with Wayne's World references I'm going to slap the nerd, dork and geek right out of you."

If you ever happen to be in the Portland, Maine area, I'll be glad to take up on that offer. Work usually puts me in a really shitty mood anyway.

If you ever want to show that you're something more than a pants-shitting little bitch taunting random people behind what is almost guaranteed to be a pseudonym, give me a buzz. I'm in the phone-book.

Don Smith said...

If you ever want to show that you're something more than a pants-shitting little bitch taunting random people behind what is almost guaranteed to be a pseudonym, give me a buzz. I'm in the phone-book.

What? And lost my fists in your sick ass eyebrows? Wind up getting nerd juice all over me?
Nah, I'd rather just make fun of you.
What vintage loser-80s shop you swipe those glasses out of?

Also, that was me doing YOU a favor by saying I'd slap the nerd, geek and dork out of you. Learn to be alittle more thankful.

Tyler DiPietro said...

"Nah, I'd rather just make fun of you."

Because you are, indeed, a tiresome little bitch. Q.E.D.

The offer's always open, though.

Rev. BigDumbChimp said...

"non-straight men"

Is that supposed to be an insult?

I noticed you picked and chose the parts of my profile to attack (including making a wildly assumptive attack).


And yes you are quite boring, and apparently lonely.

Damian said...

Don Smith (aka Bigger than Squirrels), is that you?

No wonder you have to troll blogs looking for some action. Move along now, Don, I can't stomach leaf patterned shirts.

Nice 'tache, by the way.

"If I ever....."

Torbjörn Larsson, OM said...

Yes, all of you, in that particular segment of the population, that I had mentioned above find me boring.

Still opening up for the set universe to find you boring. Probably because you know deep down it's true - or perhaps you are just dumb as a rock, not reacting to the environment.

I'm quite handy at helping nerds, dorks and geeks like yourself

You really don't get subsets.

Perusing your repetitive comments you are more of dork and geek than the common visitor here. You don't reach nerd level though, because that would necessitate some knowledge and/or intellectual capability, which you clearly don't exhibit.

Rrr said...

@ damian: If he is the same squirrel, I wonder if he knows that Moonshadow was written by a converted MOSLEM!! From norway.

"What would you do if I sang out of tune...?"

@ torbjörn: You didn't sufficiently stress the boring repepepetitititititivitititty of this ork's comments.

:-)

William Wallace said...

"If you ever want to show that you're something more than a pants-shitting little bitch taunting random people behind what is almost guaranteed to be a pseudonym, give me a buzz. I'm in the phone-book."

Just checked under "Tyler DiPietro" and "food stamp pimp" on line at yahoo and white pages, and neither are listed. I would love to meet you in person, though.

Tyler DiPietro said...

Tut, tut, William. Can't resist getting the backs of losers who threaten random people over the internet unprovoked? Somehow I'm unsurprised.

Nonetheless, if you're ever near Portland and want to meet up, it's cool. I'll need more than your ridiculous pseudonym to go by, though.

Ian H Spedding FCD said...

ObNitpick...isn't it "laser" - Light Amplification by Stimulated Emission of Radiation.

Blake Stacey said...

Actually, the original acronym was LAYZOR, but the scientific community of the 1960s decided that "Yowsers!" and "Zap!" lacked the appropriate gravitas for such an important piece of technology.

The Inoculated Mind said...

Hello ERV, I've tagged you with a blog meme: Seven Things.
http://www.inoculatedmind.com/2008/04/07/seven-things/
Your mission, should you choose to accept it, is to describe seven random/weird things about yourself.

Anonymous said...

Grr dsRed sucks. Did you put the laser into a Calibur or what?

Also, you probably have everything working now, but really watching the PFA treatment time and concentration helps w/dsRed (I assume you use PFA since you are doing HIV)