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Hobbes
 
Laser capture microdissection

This is a new technique I've been playing with in the lab that's really cool, and as I had a few screen captures from an experiment I did today I figured someone might be interested. Forgive the horrible quality of the images, as the sections are dry, unfixed tissue with no coverslips--all things that are bad for image quality but required for laser capture.

Basically the idea behind this technique is to use a laser to cut pieces of sectioned sample off a slide and use it for tissue specific gene expression studies. In theory, you can get enough RNA from just a few cells to perform microarray or quantitative PCR, but the more material you collect the better.

In this experiment I've mounted 8 micron thick sections of a day 15 mouse embryo, and I'm cutting parts of the developing kidney to compare gene expression at various stages. I don't have an image of the entire section, but it's very small with the entire kidney about 1-1.5mm in diameter. I'm not going to explain the premise of kidney development here, because this post is too long already but it's not important from a technical point of view. Just take these structures as examples of anything that can be dissected.



In this micrograph you can see a ureteric bud (labeled UB), and the surrounding metanephric mesenchyme (MM).



I tried to take a photo while the laser was cutting the sample, but the software wouldn't allow it, so here's the best I could do. This is the same view, but with the mesenchyme above the UB dissected.



This is a part of the kidney after the removal of the mesenchyme above most of the visible UBs. If you remember that the entire structure is smaller then a pinhead, the areas dissected are very small.



Here's the collected tissue, as it's cut it falls into a test tube below the inverted slide.
This is viewed under a traditional dissecting scope, as the pieces are invisible to the naked eye.

The fluorescent images are labeled with a lectin marker that labels epithelial cells (such as those in the UB or the kidney capsule). More specific labeling is possible with the use of antibodies, but for these test studies this seems good enough.

Last edited by Hobbes; 05-26-2009 at 07:17 PM..
Old 05-26-2009, 07:08 PM Hobbes is offline  
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Stanch
 
cool. I have some interest in comparative embryology but no one I feel comfortable asking simple-minded questions.

What's the advantage of KOH vs. NaOH in dead samples? Is the Southern blot going to rise again?
Old 05-26-2009, 07:57 PM Stanch is offline  
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Wiseass
 
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pretty neat.

were you the guy that looked at the iphone glass under the SEM?

Or was that someone else? If I recall he also changed the color of the case by doing some chemical treatment to it too?

Anyone got that post That'd be great for NerdGasm
Old 05-27-2009, 01:00 PM Wiseass is offline  
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DigitalChaos
 
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pretty neat.

were you the guy that looked at the iphone glass under the SEM?

Or was that someone else? If I recall he also changed the color of the case by doing some chemical treatment to it too?

Anyone got that post That'd be great for NerdGasm

that wasn't him but that guy got raped (by genmayers) for being a retard and completely misusing a really expensive machine.
Old 05-27-2009, 03:20 PM DigitalChaos is offline  
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tmoney1876
 
I should throw one under our 5000x microscope... I wonder if I'll see anything.
Old 05-27-2009, 06:59 PM tmoney1876 is offline  
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Biospider
 
This is incredibly cool. What is the role of the lectin in your markers? Is the fluorophre attached to a lectin that binds to a site on the cells, or is it functionalised with a linker that binds to lectin on the cells? Also, what fluorophore is it? GFP?

When you cut these sections, is the embryo killed in the process? I guess what I'm asking is this: can you check all stages with one embryo, or do you have multiples growing in parallel that are used up at different stages?

How big are the sections you cut? Too small to see with the naked eye, but obviously not too small to still be easily seen under an optical microscope.

I'm guessing the software didn't let you run the camera while running a cutting laser to prevent damage to the hardware.

I should find a way to present some of what I'm doing in my lab - it could be interesting (edit: Let me rephrase It is very interesting! Some people here might think so too )

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Originally Posted by DigitalChaos View Post
that wasn't him but that guy got raped (by genmayers) for being a retard and completely misusing a really expensive machine.

Why? Except time wasted, how expensive is it to run a SEM? I've never used one, but the TEM I have access to only costs me my time + the sample grids.

Please PM/post SEM pictures.
Old 05-27-2009, 09:05 PM Biospider is offline  
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Wiseass
 
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Here's the post on the SEM scan of the iphone and his mod.


http://www.genmay.com/showthread.php?t=775108

can a mod maybe move that into nerdgasm?

seems like it'd be more appreciated here even if the guy did waste funding or whatever to do it...

Last edited by Wiseass; 05-27-2009 at 10:26 PM..
Old 05-27-2009, 10:20 PM Wiseass is offline  
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wwilliam54
 
sounds good
gonna talk to one of my profs about this technique
IN forensics haveing a mixture of several peoples fluids can be a PITA
this might be just the teicket
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Old 05-27-2009, 10:43 PM wwilliam54 is offline  
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Hobbes
 
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Originally Posted by Biospider View Post
This is incredibly cool. What is the role of the lectin in your markers? Is the fluorophre attached to a lectin that binds to a site on the cells, or is it functionalised with a linker that binds to lectin on the cells? Also, what fluorophore is it? GFP?
It's a FITC-labeled lectin that specifically labels some cells.

Quote:
When you cut these sections, is the embryo killed in the process? I guess what I'm asking is this: can you check all stages with one embryo, or do you have multiples growing in parallel that are used up at different stages?
if you saw the entire section you wouldn't ask that--we section the entire mouse. What's cool though is that we can actually get all of the stages we're looking for from one embryo because different parts of the organ are at different stages of differentiation. We're looking at markers that change as the cells become epithelial, so we can compare UB to MM.

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How big are the sections you cut? Too small to see with the naked eye, but obviously not too small to still be easily seen under an optical microscope.
it varies a lot by what we're trying to get. The only real limitation is that the laser doesn't cut a very fine line, so it's tricky to guess how large a space to outline when you're getting something really small.

Quote:
I'm guessing the software didn't let you run the camera while running a cutting laser to prevent damage to the hardware.
nah, that's how you see what the laser's doing it just didn't let me save a screenshot.
using this thing is basically like using a scrollsaw on a very small scale

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I should find a way to present some of what I'm doing in my lab - it could be interesting (edit: Let me rephrase It is very interesting! Some people here might think so too )
please do!
Old 05-28-2009, 04:15 AM Hobbes is offline  
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DigitalChaos
 
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Here's the post on the SEM scan of the iphone and his mod.


http://www.genmay.com/showthread.php?t=775108

can a mod maybe move that into nerdgasm?

seems like it'd be more appreciated here even if the guy did waste funding or whatever to do it...

done
Old 05-28-2009, 08:10 PM DigitalChaos is offline  
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