The LCM system developed at NIH and Arcturus is basically
an inverted microscope fitted with a low-power near-infrared laser. Tissue sections are mounted on standard glass slides, and a transparent,
100-mm-thick, ethylene¨Cvinyl acetate film is then placed over the dry section. The laser provides enough energy to transiently melt
this thermoplastic film in a precise location, binding it to the targeted cells. The laser diameter can be adjusted from 7.5 to 30¦Ìm
so that individual cells or a cluster of cells can be selected. Because the plastic film absorbs most of the thermal energy and the
pulse lasts for a fraction of a second, little or no detectable damage of biological macromolecules occurs.
After the appropriate cells have been selected, the film and adherent cells are removed, and the unselected tissue remains in contact with the glass slide. These cells can then be subjected to appropriate extraction conditions for ensuing molecular analysis. To improve the convenience of the technique, the transfer film can be mounted on a transparent cap that fits on a 500-¦ÌL microcentrifuge tube.
LCM is compatible
with various common methods for the preparation of tissue sections. Tissues are typically fixed by alcohol-based precipitation techniques.
Aldehyde-based fixation may also be used, but covalent cross-linking of macromolecules can potentially interfere with subsequent analysis
of RNA or protein. Sections of 6-mm thickness can then be prepared from paraffin-embedded or frozen tissue and mounted on glass slides.
These sections may be stained by standard techniques such as hematoxylin and eosin, methylene green nuclear stain, fluorescence in
situ hybridization, or immunohistochemistry for identification of tissue morphology and cell populations of interest. Because the
section thickness is less than that of a cell, up to 20 cells may need to be selected to obtain a complete genome or expression profile.
Up to 3000 transfers can be performed on one film, representing more than 6000 cells, depending on their size. The cells are then
lysed and extracted in an appropriate buffer for the analysis of DNA, RNA, or protein . Remarkably, single cells captured by this
technique have been successfully analyzed by techniques based on nucleic acid amplification.
The major disadvantage of LCM is that it isolates minute amounts of material, which limits analysis to amplification-based techniques or collection of numerous cells, as in protein analysis. Although this technique is faster than previous microdissection methods, isolation of large numbers of cells from many sections can require considerable time. Another disadvantage is that cover slips and mounting solutions are not compatible with LCM and, as with other microdissection techniques, visualization of samples can be difficult. Arcturus has equipped its microscopes with a diffusion filter to improve the viewing of cells. For an experienced histopathologist, this suboptimal visualization should not be a problem. Another limitation of LCM is that it is not compatible with live-cell analysis, but for these applications flow cytometry may be used.
Reference: ED Brignole. Modern Drug Discovery (2002), 3:69-70