Journal of Genetic Syndromes & Gene Therapy
Open Access

Microdissection Testicular Sperm Extraction

microdissection (LCM), also called microdissection, laser microdissection (LMD), or laser-assisted microdissection (LMD or LAM), is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms (dissection on a microscopic scale with the help of a laser).Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss of heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. The total time required to carry out this protocol is typically 1–1.5 h.

A laser is coupled into a microscope and focuses onto the tissue on the slide. By movement of the laser by optics or the stage the focus follows a trajectory which is predefined by the user. This trajectory, also called element, is then cut out and separated from the adjacent tissue. After the cutting process, an extraction process has to follow if an extraction process is desired. More recent technologies utilize non-contact micro dissection.

There are several ways to extract tissue from a microscope slide with a histopathology sample on it. Press a sticky surface onto the sample and tear out. This extracts the desired region, but can also remove particles or unwanted tissue on the surface, because the surface is not selective. Melt a plastic membrane onto the sample and tear out. The heat is introduced, for example, by a red or infrared (IR) laser onto a membrane stained with an absorbing dye. As this adheres the desired sample onto the membrane, as with any membrane that is put close to the histopathology sample surface, there might be some debris extracted. Another danger is the introduced heat: Some molecules like DNA, RNA, or protein don't allow to be heated too much or at all for the goal of being isolated as purely as possible.

For transport without contact. There are three different approaches. Transport by gravity using an upright microscope (called GAM, gravity-assisted microdissection) or transport by laser pressure catapult; the most recent generation utilizes a technology based on laser induced forward transfer (LIFT). With cut-and-capture, a cap coated with an adhesive is positioned directly on the thinly cut (5-8 μm) tissue section, the section itself resting on a thin membrane (polyethylene naphthalene). An IR laser gently heats the adhesive on the cap fusing it to the underlying tissue and an UV laser cuts through tissue and underlying membrane. The membrane-tissue entity now adheres to the cap and the cells on the cap can be used in downstream applications