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Laser capture microdissection


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#1 microbugger

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Posted 03 August 2004 - 09:18 AM

Hello all,

Does anyone have any experiences with Laser Capture Microdissection they'd care to share? Before I go that route, I'm interested in learning from anybody's previous adventures with this technology.

Thanks!

#2 ihcworld

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Posted 06 August 2004 - 09:19 AM

Here is the LCM information link. Hope this helps.

IHC World Laser Capture Microdissection Page

#3 microbugger

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Posted 06 August 2004 - 09:33 AM

Thanks for the link!

I'm also interested in hearing about people's "Hands-on" experience with this technology i.e. ease of use, real world sample stringencies etc.

#4 CellExpert

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Posted 08 February 2012 - 01:42 AM

There are 4 big players in the laser microdissection market:

Molecular Machines and Industries have the CellCut Plus and SmartCut Plus.
They have an adhesive silicone cap that attaches to a membrane. The sample is on the other site of the membrane and will never get into contact with the cap, only with the membrane. On the other site of the sample is the glass slide. So, the sample is sandwiched between them. The membrane and sample is cut with a low power UV laser that has a high repetition rate and short pulse duration. Neither the silicone nor the PET membrane is sticky. They are adhesive (only Van der Waals forces). Therefore, the cap takes out only the cut out regions.

Pro:
  • Absolute contamination free before and during LMD, because sample is sandwiched.
  • Low power laser does not damage cells. Therefore, also single cells can be cut without loss of DNA, RNA, and Protein
  • "PTP" cell arrangement: They collect the cells in a way that the samples are placed in a matrix-order on the cap. highly helpful together with the auto-documentation.
  • Positive sample inspection. You can see every cell on the cap due to PTP. You know exactly how many tissue you work with in downstream.
  • Customizability. If you have any wishes regarding customization you need to go to MMI. They also build their systems on your Zeiss microscope if you want to.
  • Modularity. They have three instruments they can combine into one: a LMD, a capillary based Micromanipulator and an optical tweezer.They can cut single cells and big areas of cells. They keep their morphology and are good in quality.

Cons:
  • They are in general more expensive than Leica and Zeiss.
  • The low power laser is not good for thick or hard tissue. You would have to upgrade to the high power laser (extra cost).
  • The company is not as big as their competitors and not well known.

MMI CapLift process

Arcturus (part of Applied Biosystems --> Life Technologies) has the ArcturusXT
Arcturus was the first LCM company on the market and was very popular. Since ABI bought the company, Carl Zeiss and Leica are the new leader in market share. Arcturus uses the CapSure Isolation method. The cap is molten with an IR laser. The molten polymer touches the sample. An optional UV laser prevents a bad tearing line when the cap is moved up. If UV is used, the morphology of the samples is kept. Except the molten points which hold the sample, the rest of the sample is not damaged.

Pro:
  • Morphology is kept
  • Positive inspection possible
  • Low possibility of contamination (during LCM the sample is sandwiched between cap and membrane)
  • High power laser good for hard to cut samples optional.
  • Fully customizable system: integration of optical tweezer, LMD, Capillary Isolator on every microscope.
Cons:
  • Expensive in comparison to Leica and Zeiss (two laser necessary for same job).
  • Single cells are molten into the cap with an IR laser (loss of quality)
  • Sample must be absolute dry. If wet, the molten polymer does not stick on the cell.
LCM process 1

Leica is a microscopy manufacturer and has the LMD7000 and LMD6500
The isolation process is based on gravity. They move the laser instead of the sample to produce a cutting line. This looks more intuitive on the screen but produces some side effects like an oval focus instead of a circle. After cutting a sample the sample should fall down into a cap that may be filled with lyses buffer. The sample is placed on a membrane slide and is not covered from the other side. Neither with a glass slide nor with a cap. The process is called contact free because nothing touches the sample unless it falls into the cap.
The system is the less expensive you can get and is very popular in Asia therefore. Regarding modularity you can get two systems with different parameters. Because Leica is an optics manufacturer you can only get Leica Microscopes with the LMD.

Pro:
  • Low budget system
  • Two different systems available
  • High power laser good for hard to cut areas.
  • In contrast to Carl Zeiss, large areas can be cut out easily.
  • No heat damage as with Arcturus
Cons:
  • Same Problem as with Carl Zeiss. Sample makes its way down through the air and may get lost. Because there is no initial impulse, electrostatic is an even bigger problem with Leica. The sample may just flap over to the backside of the membrane slide after cutting.
  • No positive inspection of captured samples possible.
  • Single Cell isolation very difficult
  • Dust particles from the air are in the way between sample and cap. Furthermore the cap has a buffer that catches all the dust particles. Because of the heat of the microscope lamp, the buffer evaporates quickly.
gravity collection process

Carl Zeiss has integrated PALM and now sells the MicroBeam
Carl Zeiss is a very popular optics manufacturer and made the proper decision to integrate PALM to sell more of its microscopes into the LMD market. Like MMI, Carl Zeiss offers an optical tweezer combi system. They use a high energy laser to catapult the cut out area into the buffer of the cap. They are similar to Leica except the sample receives an impulse on start. This allows overcoming the electrostatic problem with small samples. As with Leica there is an open buffer in the cap and the way between sample and cap may be blocked with dust particles.

Pro:
  • Single cells and small areas can be isolated
  • Combi system available
  • Isolation of sample from glass slide possible
  • Low consumable cost and low instrument cost
Cons:
  • Cell damage due high energy catapulting
  • Big areas need many shots (= time + cell damage)
  • Similar problems as Leica with contamination, positive inspection, buffer dry out and collection success rate.

laser catapulting process

Recommendation:

Small areas to cut: All except Leica
Big areas to cut: All except Carl Zeiss
Thick and wet tissue: Leica
Low budget: Carl Zeiss or Leica
Living Single Cells: MMI
Living Cells in larger areas: All except Carl Zeiss
Absolute Contamination free: MMI (but I don’t expect contamination to be a major problem with any of the instruments).
Positive Sample inspection: Arcturus or MMI
Extra service in customization and upgrades: MMI only

I worked with several instruments and my favorit clearly was the MMI CellCut Plus.

Edited by CellExpert, 08 February 2012 - 01:44 AM.





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