Researchers have the opportunity to play surgeon — slicing and dissecting out specific sections of tissue or even cell populations — with laser-capture microdissection (LCM). This level of analysis might seem to be difficult when applied to formalin-fixed and paraffin-embedded (FFPE) tissue samples, but many companies are now offering easy and quick LCM solutions.

Leica Microsystems of Wetzlar, Germany, offers the LMD6000 LCM system, which uses an upright microscope for dissection and capture. Christoph Horlemann, the company's product manager for the LMD6000, says this is possible because the transport mechanism for capture is based on gravity, unlike other LCM systems on the market.

'Transport mechanism' refers to the method for delivering a dissected tissue sample from a slide to a collection vessel. Other approaches to this process include that of PALM Microlaser Technologies in Bernried, Germany, which uses a 'pressure catapult' to send sections into tubes from the LCM instrument, and the CapSure system from Molecular Devices in Sunnyvale, California, which uses the laser to extend a polymer onto the tissue sample for capture.

Leica Microsystems' transport mechanism uses slides with a foil covering. The tissue is attached to the foil covering but not the slide, so researchers cut the tissue and the foil together and the sample simply falls by gravity into the collection tube below, explains Horlemann. But even this foil coating might not be needed in the future.

Both Leica and PALM are working with Expression Pathology of Gaithersburg, Maryland, on what may be the next generation of LCM slides. Called Director, these glass slides are based on laser-induced forward transfer (LIFT), a non-contact microdissection technique that uses a thin energy-transfer coating that replaces plastic films or adhesives. The technology was co-developed by researchers at the US Naval Research Laboratory in Washington DC and scientists from Expression Pathology.

The Arcturus XT from Molecular Devices can use either ultraviolet or infrared laser light for tissue dissection. Credit: MOLECULAR DEVICES

Laser energy is transferred to the transfer layer and the layer is vaporized. The laser energy is then converted into kinetic energy, and the selected feature is shot instantly into a collection tube.

LIFT works equally well whether the tissue is collected up (PALM) or down (Leica) as the energy is sufficient to propel the tissue into the collection tube either way. As the transfer layer completely absorbs the laser energy, the biomolecules in the sample are not affected. The use of glass with this coating has other advantages as well.

“Glass slides are quite useful as you can also perform fluorescence and contrast applications without any interfering foil,” says Horlemann.

Molecular Devices, a division of MDS Analytical Technologies, acquired Arcturus in 2006 and is now providing both the Veritas and the Arcturus XT LCM systems. Whereas most LCM systems use an ultraviolet laser for cutting samples, the systems offered by Molecular Devices can have either ultraviolet or the standard infrared laser options.

“The infrared laser is ideal for small areas where a user is looking to pick up only a cell or two,” says Steven Blakely, product manager for the Arcturus LCM systems. “Infrared allows for a gentle collection of cells.”

The use of infrared laser light is critical to the CapSure system because the dye found in the CapSure polymer is activated and becomes adherent with infrared light. Blakely says the CapSure system is particularly useful for FFPE samples that are already mounted on glass slides because the transport mechanism involves only the polymer adhering to the tissue and so can work with any glass slide.

Cutting tissue samples with ultraviolet light offers advantages too, such as dissecting thicker samples. Leica has worked to optimize objectives for its LCM systems with high-energy ultraviolet-light transmission, allowing more power to come in direct contact with the tissue sample. Horlemann says this provides faster cutting of the thicker tissue samples while using less power.

The future of LCM might just lie in automation and increases in throughput. “Now, researchers want to do faster, automated microdissection for proteomics,” says Horlemann. But before anything else, some hardware and software issues needed to be resolved.

Any automated software package has to control all microdissection steps including focusing the microscope, recognizing cells of interest, focusing the laser and defining the area to cut — definitely not a simple task, but one that companies have worked on and made tremendous strides in recent years. Horlemann says that new advances are making automation easier every day. He points to the Director slides, which can allow for contrasting methods and fluorescence, making it easier when attempting to automatically define cells of interest as a step in the right direction.

Although simpler now, further advances will be required before LCM becomes as easy as pushing a button and walking away.

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