BIOTECHNOLOGY: Nitrogen laser cuts and catapults cells

Scientists studying gene sequences and how genes in a single cell are regulated and expressed need a tool to examine the DNA in single cells. To understand how cells change—whether transforming into an embryo or a tumor—biologists would like to isolate and analyze single cells, one at a time.

While extremely sensitive methods exist for analyzing tiny amounts of material, such as PCR (polymerase chain reaction) based DNA amplification or RT (reverse transcriptase) PCR, these methods are also extremely sensitive to contaminants. Two researchers at the Academic Hospital Munchen-Harlaching (Munich, Germany) have reported an extension of a previously available method that allows exactly this level of dissection and isolation without contamination from other cells. Karin Schutze and Georgia Lahr have demonstrated how their method can be used to isolate a single cancerous cell and analyze the cell to detect point mutations.

According to the researchers, the method avoids mechanical contact to capture clean samples of any shape and size between one and several hundreds of micrometers in diameter. This is small enough to allow clusters of cells, or individual cells, to be isolated. The researchers used a commercially available, semiautomated nitrogen-laser system (Robot-MicroBeam; Palm, Wolfratshausen, Germany) to both cut the study cells from fixed tissue and remove them. The automation is another important benefit of this work, allowing efficient, relatively quick, sample recovery.

The focused nitrogen-laser output at 337 nm is used at two different pulse energies to accomplish two steps: cutting around the sample and then ejecting the sample from its resting place onto a collection surface. Tissue to be sampled is first prepared by normal methods—made into formalin-fixed and paraffin-embedded tissue sections on clear slides. The samples can be stained. With laser microbeam microdissection, the beam enters through the glass slide and ablates the tissue in a zone from 1 to 10 µm wide around the sample. At this wavelength, the cells adjacent to the ablated zone are not affected.

Laser catapulting

Laser pressure catapulting involves removing the circumscribed cells with the same laser. The beam, which enters the sample from below the slide, is focused slightly below the target specimen, and the energy is increased to about twice the level used for microdissection. With only a few—and in some cases, one—pulses from the laser, the samples are ejected from the surface of the slide and catapulted up to 8 mm away onto the collection surface—typically either a small piece of glass or the cap of a common microfuge tube. For small samples, the specimen is usually fragmented, but the DNA survives intact. The wavelength of the laser is far away enough from the absorption maxima of proteins and DNA to avoid damaging them.

An alternative method, which uses polyethylene membrane rather than a glass slide, has been developed for catapulting intact samples as large as 1 mm in diameter. The 1.35-µm-thick membrane does not interfere with PCR detection methods.

Isolation and transfer of a single cell took less than 30 s. Larger samples such as cell clusters required more time, but still less than two minutes per sample.

To test the method, the researchers used a microscope to look for the catapulted samples on the collection surface. About 95% of the larger, clustered and pooled cell samples were detected. Although only 63% of the single-cell samples were detected, this may be more a problem with microscopically locating a single cell than a failure of the method. The researchers note that "from some of the caps with microscopically undetected cells an RT-PCR signal was found," indicating that genetic material was catapulted onto the caps.

The researchers demonstrated the specificity of the single-cell analysis. They used the method to isolate a single tumorous cell from an archival colon adenocarcinoma and then performed mutation studies using RT-PCR analysis. The analysis showed that the point mutations occur at expected spots and demonstrate that a single cell is sufficient to analyze expressed genes.

The method is expected to aid molecular biologists in preparing cell-specific cDNA (complementary DNA) libraries to search for specific genes and to determine cell-to-cell patterns of gene expressions.