Laser microdissection (LMD)

About this technique

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Laser microdissection (LMD) is a technique used to isolate specimens for biomedical research. A fundamental step in preparation for LMD is to develop a specimen preparation protocol that optimally balances the dissection of the sample and downstream analyses. LMD is often used to collect samples from which RNA is to be isolated. As RNA is particularly sensitive to degradation, the following description emphasises precautions required for this end use.

To maintain and preserve RNA integrity it is best to avoid formalin fixation as it has an adverse effect on nucleic acids and consequently downstream analysis. It is recommended that alternative fixatives for paraffin-embedded tissues are used, e.g. methacarn, DSP or HOPE.

All materials used for RNA work need to be prepared RNAase free by treating with RNase ZAP or RNase AWAY. All solutions (including staining solutions) are prepared in DEPC-treated water. Alternative treatments to remove RNases are autoclaving and UV treatment. For UV irradiation treatment of slides, place them in a UV crosslink chamber and deliver at least 1 joule of energy (maximum power for 30–45 minutes). For autoclave treatment of slides, place slides into a steel basket or slide holder and place the basket into a beaker or jar and cover tightly and autoclave at 121°C for 20 minutes. Note that sterilising slides by autoclaving or UV treatment does not guarantee complete destruction of RNases, therefore it is recommended to purchase RNase-free certified slides.

Fresh frozen tissues are preferred for isolating RNA and proteins. Best quality RNA is achieved from tissues frozen immediately after surgery, which minimises RNA degradation over time by endogenous RNases. Tissue is placed in a disposable cryomould in OCT (optimum cutting temperature) and immersed in pre-cooled isopentane (e.g. Pyrex beaker) in liquid nitrogen. The samples can then be stored at -70°C until ready for cryosectioning.

Before sectioning, the cryostat should be cleaned with 70% ethanol and RNase Zap (including the disposable microtome blade). A sheet of aluminium foil should be placed down in the cryostat to keep an RNase free area.

Sections of between 5 and 20 um in thickness can be used, however 7-10 um achieves the best balance between good quality sections and laser microdissection. In general terms, the thicker the section, the greater the laser 'power' required to dissect cells.

Specially coated slides are required for LMD applications to enable the dissected regions to be readily released from the slide. The tissue sections are mounted on these foiled membrane slides (2–3 sections per slide depending on the size of the sample).

Once the frozen sections have been mounted on the appropriate slides, there are three approaches. They can either be fixed and stained immediately for LMD or stored in sealed slide boxes (seal with masking tape) at -70°C until required. They can also be fixed and then stored at -70°C for LMD at a later date. Prior to LMD, the slides should be thawed and slowly adjusted to room temperature e.g. from -70°C to -20°C freezer for 30 mins. The slides are then taken out to thaw at room temperature in a slide box with silica gel desiccant. This helps to avoid formation of water condensation. This precaution is critical for RNA quality, especially when working with small numbers of cells, since water (moisture) activates RNases. The presence of water also affects the efficiency of the laser. If moisture is still present, place slides in 40°C oven for 5 mins.

Sections can be fixed with ice-cold acetone for 2-3 mins. 70% or 100% ethanol for 30 secs or a mixture of ethanol: acetic acid (19:1) to increase the adhesion of the tissue to the membrane slides. LMD can be performed on stained or unstained cells. Suggested stains are toluidine blue, haematoxylin and eosin or methyl-green.

To perform the dissection, the cell or groups of cells to be isolated are marked out on an image of the sample on a computer screen. The focused laser then cuts along the pre-marked path with very high precision. During excision, the membrane is vaporised which releases the dissected region from the slide. This is then either catapulted into the cap of a microcentrifuge tube for subsequent analyses, or the excised part falls into the collection cap by means of gravity. These methods depend on the instrument being used.

0.2 ml and 0.5 ml microcentrifuge tubes are best for collection and the caps may be filled with lysis buffer to prevent the degradation of nucleic acids. The instruments allow viewing of the caps to confirm the collection of dissectates.