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Years ago, tissue samples mounted on glass slides were consigned to a future of histological or immunohistological examination, and then, to the trash bin. Postmicroscopic polymerase chain reaction (PCR) analyses of interesting cellular colonies were impossible, because there was no way to get those cells off the slides, or to isolate those few cells from the vast majority of uninteresting ones. But thanks to laser microdissection (LMD), that is no longer the case. LMD allows researchers to targ
May 13, 2002|
LMD allows researchers to target a specific cell—or even chromosome—and excise it from the cells that surround it on a slide, or in some cases, on a culture dish.1 Researchers can use some LMD systems to destroy a particular cell in the midst of surrounding tissue of interest, a process referred to as negative selection. These instruments have other applications in microsurgery, microinjection, in vitro fertilization, and cell fusion research.
LMD dates to the 1970s, when researchers used lasers to trap and manipulate cells.1 In the mid-1990s, under the National Institutes of Health at the National Institute of Child Health and Human Development and the National Cancer Institute, researchers developed the first LMD system as a research prototype. The NIH later worked in partnership with Mountain View, Calif.-based Arcturus Engineering, to develop a commercial system.2
The field has matured since that first venture, and modern LMD systems vary in the cell-capture method, system configuration, and applications. Some systems offer the possibility of working with cells straight from a culture dish. Others allow researchers to avoid touching a specimen by catapulting or dropping the untouched, dissected cells straight into a capture tube or lifting the protected specimen directly into the lid of a microcentrifuge tube.
In most cases, the stage on which the tissue sits moves either manually or robotically around a stationary laser to excise the desired cells. The Cell Robotics system, for example, has a computer- controlled robotic stage. It is distinguished from other systems, says product manager Deborah Schaefer, because "the stage is our own design and moves very smoothly. It does not move in steps as many stages do, but makes smooth movements even on a circle or diagonal cut."
In contrast, with Leica's AS LMD the laser moves instead of the stage. "Other microdissection instruments direct a stationary beam down the center of the objective lens and move the stage and sample to describe a cut," says Robert Wick, Leica's vice president of microscopy. "We utilize a module with two counter-rotating prisms to 'steer' the beam. This approach is much faster and more accurate."
Some systems offer the user the option of tracing several paths to cut before starting the excision process. MMI's µCUT, for example, can trace the paths of several cells and then do the cutting. It can even group paths that have been defined to allow for separate collection, so a researcher can excise diseased and normal cells and collect each cell type separately.3
Leica's AS LMD, in contrast, uses the company's DM LA upright microscope. "Since we capture the cut sample by allowing it to fall into a reaction tube," says Wick, "the upright configuration is ideal." Most researchers, particularly pathologists, "are more comfortable with this type of microscope," he adds. Several systems also offer fluorescence capability, including the PixCell II, the Leica AS LMD, and the µCUT.
PixCell II, the granddaddy of LMD systems, uses a proprietary four-step Laser Capture Microdissection process to collect tissue. It employs a disposable CapSure™ cap, which is placed over the target tissue area on a slide. These caps, which fit 500-µl microcentrifuge tubes, are lined with thermoplastic film that forms a protrusion when hit by a laser pulse. The protrusion closes the gap between tissue and film, so that lifting the cap will remove the target tissue, leaving it attached to the cap. Because the laser does not physically cut the tissue section, this approach enables the user to positively confirm the excised cells' identity before downstream studies commence.
According to Arcturus, this fusion technique can be used with any common tissue preparation and staining method. The company further claims that the process occurs at temperatures below that of the heating steps in PCR reactions and alters neither DNA nor RNA in "any measurable fashion."
Other companies offer different tissue-capture techniques. Cell Robotics uses a technique that involves "Pick-Up Sticks." Several sticks with film on the ends are placed above the tissue and automatically oriented to the tissue to be collected. Electrostatic forces propel the tissue fragment toward the film, and the film is pushed inside a microcentrifuge tube for collection. Cell Robotics product manager Schaefer says that because the fragment is held inside the tube, rather than inside the cap, "there is no risk of losing the valuable microscopic specimen." She also says that "there is no need for contact between the collecting surface and the original specimen during capture," and that the procedure is motorized for high-throughput collection requirements.
Instead of hitting the desired cells with a laser pulse, MMI's µCUT cuts around the tissue of interest, which is mounted on special membrane-covered slides. This system's strengths, according to Wessel, are that "it is easy to use and the collection process is completely reliable and contamination-free." The process uses an adhesive layer in a collection cap to capture the sample. The µCUT employs no laser fusion or laser irradiation of sample, and the user can watch during cutting and sample removal. The system offers "almost 100% success in [tissue] removal," says Wessel, whereas laser fusion techniques can rip the tissue off a slide and frequently fail.
Bio-Rad's Clonis system also uses film, but in a different way, enabling live-cell applications. Researchers can place the film in a culture dish and culture cells normally. Clonis can then cut around a cell in the culture, and when the film is peeled away, the remaining cells come with it, leaving behind the desired cell. Scientists can also use Clonis to ablate unwanted cells that are then washed away so that the researcher can culture the remaining cells.
Clonis is not limited to use with culture dishes; it can also be used with fixed tissues. In fact, a key aspect of the Clonis system is its ability to cut extra-thick sections. Typically, according to product manager Morris, there is no lower limit on section thickness, though there is an upper limit: LMD cannot usually handle sections more than 40-µm thick, and often, "much nearer 20." But the ability to obtain thick sections is important, he says, because it "ensures a greater yield of biochemicals and a more representative sample." Additionally, the ability to cut thicker sections is important for the cutting of viable tissue sections, which, Morris notes, typically should be at least 70-µm thick.
In LPC, a "photonic cloud" provides an energy pulse, which launches the excised specimen into a microfuge tube cap. According to Schuster, the cells do not absorb the energy, and this form of capture can be used with live cells without harming them. "PALM technology," concludes Schuster, "makes the dream of 'beaming' come true."
Because no unwanted material contaminates the process, the specimen retains its purity and "downstream analysis yields better results," Schuster says. He adds that because the system is fully automated, "using it does not require highly specialized training, and samples can be processed very quickly." The system requires no unique disposables such as film, foil, or specially lined caps.
Leica's capture technique is less akin to Star Trek than it is to Newtonian physics: It relies on gravity to land the specimen in the collecting cap. Tissue is placed on a special foil on a glass slide that researchers can process normally and then place upside down on a microscope stage. When the laser passes through the glass it melts the foil as it cuts around the tissue's edge, and the foil and sample fall into a collection tube positioned immediately below the stage. According to Leica's Robert Wick, this system is easy to use, has high throughput and accuracy, and offers contamination-free cell collection without irradiation. Leica offers multiple collection tubes in an automated, programmable cassette for its users.
Regardless of the cell-cutting method, researchers will want to know whether the cutting process actually worked. All systems offer an inspection mode so that when cutting is complete, the operator can inspect both the fragment that has been removed and the area from which it was excised. These systems also offer archiving capabilities so that the image of the captured fragment can be stored.
Not all systems can be used with live cells, and not all can be used with archived slides. The Cell Robotics system can excise cells from archived slides. The Clonis, PALM MicroBeam, and Cell Robotics systems, and the Leica AS LMD will work with living cells, but when it comes to fixed or fresh-mounted tissue, the Leica AS LMD and MMI µCUT require special films or foils; researchers will encounter difficulty using slides prepared with other methods.
Regardless of the source, though, once the tissue is harvested, the real work begins. One common application is to examine gene expression profiles. Gary W. Miller, an assistant professor of pharmacology and toxicology at the University of Texas at Austin, uses the PixCell II in his examination of the dopamine neurons in the substantia nigra pars compacta. "Standard dissection of the midbrain area can yield dozens of different cell types, which makes it difficult to know which neurons are responsible for changes in gene expression," says Miller. With the PixCell II, "we are able to isolate an enriched population of dopamine neurons from mice that have been exposed to pesticides or other toxicants thought to be involved in Parkinson's disease. We then isolate RNA from the cells to examine gene expression by real-time PCR and microarrays."
Researchers can also use PCR to amplify nucleic acids harvested from isolated cells and create cDNA or genomic libraries. Genes isolated from harvested cells can be cloned, and nucleic acids or proteins from these cells can be scrutinized on Southern, Northern, or Western blots. With systems that offer optical trapping and live-cell capabilities, scientists can apply in vitro fertilization techniques—from drilling a hole in the zona pellucida to bringing the sperm and egg together—as well as microinjection and cell fusion methods.
IN FOCUS | Emily Willingham
|Courtesy of Brinkmann Instruments|
"A really nice feature of this system is the ability to do single cell dissection," says Joel Lopez, technical marketing manager for cell biology at Brinkmann, Eppendorf's North American distributor. "Using the Micro-Dissector, you can perform single-cell dissection without killing the neighboring cell." On the other hand, Lopez continues, it is possible to kill the neighboring cell when using an LMD system.
Because tissue pretreatment is not necessary, both living and archived tissue are candidates for the MicroDissector. In addition to the MicroChisel, the system comes with the aspiration pipetter and filter micropipette tips. Users can choose between Filtertip MDS for large samples of tissue (0.2-mm diameter) or TransferTip® MDS for aspirating small samples, such as a single cell. To use the system, a micromanipulator—such as Eppendorf's TransferMan® NK2—mounted on an inverted microscope is required. For delicate cell-transfer techniques, Lopez recommends Eppendorf's CellTram™ vario. "It's a manual microinjector that offers great performance when you need to transfer samples manually or when you are working with delicate samples," he says. "It can be used for stem cell transfer, for example."
To cut tissue, the user manually circumscribes the area with the MicroChisel, using the foot pedal to determine the amount of force. Lopez says the quality and performance compares favorably to that of LMD systems. "My first time," he says, "it took a while, but now it takes only a few minutes." To provide a visual for other researchers, the system can be used with a monitor and camera.
As for concerns about how ultrasonic dissection compares to LMD, Lopez says, "With our system, you can do the same work with tissue with potentially less damage in the capture." Lopez adds that the cutting method is completely safe for subsequent analysis of nucleic acids, for example. The MicroDissector list price is approximately $13,600 (US); the TransferMan NK2 micromanipulator price is approximately $14,000.