Mmr

Protocols

Yes

Yes

No

Yes

Abbreviations: F = frozen; P = paraffin; M = membrane filter; C = culture; I = inverted; U = upright; Bf = bright field; F1 = fluorescence; Ph = phase; TPF = thermoplastic film; LPC = laser pressure catapulting; G = gravity; MMR = manual membrane removal.

Abbreviations: F = frozen; P = paraffin; M = membrane filter; C = culture; I = inverted; U = upright; Bf = bright field; F1 = fluorescence; Ph = phase; TPF = thermoplastic film; LPC = laser pressure catapulting; G = gravity; MMR = manual membrane removal.

it is microdissected. In the case of the P.A.L.M., systems, this is possible as long as the sample has been cut from a PEN membrane. Samples that are microdissected by only catapulting directly from glass end up as flakes in the cap and show no recognizable morphology. In the case of the Bio-Rad system, visualizing the microdissected sample is possible if the microdissection is of the type that removes the unsampled material. For the Arcturus system, the cap with adherent specimen is placed down on a clean area of the slide and the microdissected samples can be seen quite clearly. All of the systems can visualize the area left after microdissection.

3.4. INSTRUMENT PROS AND CONS The Arcturus system is fast and simple to operate. In the Pix-Cell II series, no computer is required to do the microdissection. The attached computer can acquire images and perform other useful functions such as keeping track of the number of shots. It is easy to visualize the microdissected sample and to remove material not adhering to the membrane. For extremely heterogeneous samples in which only one component is desired, the Arcturus system is a very good system as long as the sample is dry The system should be placed in an air-conditioned facility because high RH can adversely affect adherence of the thermoplastic film to the specimen. The mechanical stage requires good manual dexterity to manipulate. Because the system utilizes a vacuum chuck to hold the sample on the microscope stage, materials for microdissection must be on glass slides and placed near the middle of the slide. The Arcturus system is the only system that cannot utilize the laser to ablate unwanted parts of the sample.

Leica AS LMD compares favorably with the Arcturus system. The computer-controlled system allows the user to choose many different areas for microdissection by marking them on the video monitor prior to any microdissection taking place. After marking, the system takes over and performs the microdissection. This is less tedious than the manual Arcturus method. Several different types of area can be marked and these will be microdissected into separate caps by the substage cap mechanism. The microdissected sample cannot be visualized directly. Samples must be placed on slides that are covered with a special PEN membrane. Because the system depends on gravity to collect the dissected sample into the collection cap, static electricity becomes a problem. Static charges on the cap, the PEN membrane, and other plastic materials can divert the dissected sample away from the cap. Therefore, the RH in the region of the cap must be around 35% or better to minimize static charges.

The P.A.L.M. system is the only system that can automatically microdissect both dry and wet samples directly into a collection cap. This is the most universally applicable system of those covered here. As with the Leica, several different types of area can be marked and these will be microdissected into separate caps by the above stage cap mechanism. Movement of the microdissected specimen into the cap is by a mechanism called laser catapulting—a partly understood mechanism in which light pressure from a pulse of the defo-cused laser is sufficient to move the specimen up into the collection cap (5). Normally, samples should be placed on a PEN membrane either on slides or in culture dishes or on cover glasses if chromosomes are to be microdissected. Samples not on a PEN membrane, such as deparaffinized archival histolog-ical sections, can be microdissected by direct laser catapulting.

In this case, the microdissected sample cannot be visualized because it is highly fragmented.

Bio-Rad CLONIS is intended for microdissection of wet samples where the areas for microdissection are well separated from one another, although it can be used for dry samples as well. It is the only system that provides a mechanism for isolating islands of cells while still in culture by creating trenches in the membrane material that cells cannot cross. It can also easily ablate unwanted areas while the cells or tissue are still in culture. Tissues or cells must be placed on the special multilayer membrane. The thickness of the material that can be microdissected can be as great as 200 |im. Because the system uses an IR laser that must pass through the supporting medium, the spot size is quite large in the specimen plane (20-40 |im). This is not ideal for isolating single cells. The system requires that the cut membrane be removed or collected by hand utilizing fine forceps—a process requiring considerable manual dexterity.

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