Principles Of Laser Capture Microdissection

Recently, a variety of laser-assisted microdissection devices have been developed and are available on the market. Two different technical principles can be discerned. One of them, initially termed laser microbeam microdissection (LMM), uses a pulsed UV laser with a small beam focus to cut out areas or cells of interest by photoablation of adjacent tissue.[1] The second technique is laser capture microdissection (LCM), which is described in more detail below.[2-4]

Laser capture microdissection is based on the selective adherence of visually targeted cells and tissue fragments to a thermoplastic membrane activated by a low-energy infrared laser pulse (Fig. 1). The basic system (marketed by Arcturus, Mountain View, CA) consists of an inverted microscope, a solid-state near-infrared laser diode, a joystick-controlled microscope stage, and hardware and software for laser control and image archiving. A new system for automated microdissection with expanded technical capabilities has recently been introduced by the same company. The thermoplastic membrane used for the transfer of selected cells is mounted on an optically clear cap which fits on standard 0.5-mL microcentrifuge tubes for further processing.

After visual selection of the targeted cells, laser activation leads to focal melting of the ethylene vinyl acetate (EVA) membrane, which has its absorption maximum near the wavelength of the laser. The melted polymer expands into the section, resolidifies within milliseconds, and forms a composite with the tissue. This allows selective removal of the cells attached to the activated membrane. Laser ''shots'' can be repeated multiple times across the whole cap surface to collect large numbers of cells.[2] Depending on the chosen laser spot size, the architectural features of the tissue, and the desired precision of the microdissection, even thousands of cells can be collected within a few minutes. After dissection, the cap is transferred to a tube containing the buffer solutions required for isolation of the molecules of interest. As most of the energy is absorbed by the

Laser housing membrane

-cap tissue section

■ captured cells slide stage objective —

Fig. 1 Schematic representation of laser capture microdissection. A) Activation of the laser leads to focal melting of the thermoplastic membrane attached to the lower portion of the optically transparent cap. B) Lifting of the cap leads top selective detachment of cells adherent to the molten (activated) parts of the membrane. (From Ref. [3].) (View this art in color at www.dekker.com.)

membrane, the maximum temperature reached by the tissue upon laser activation is in the range of 90°C for several milliseconds, thus leaving intact biological macromolecules of interest. The precision of LCM mainly depends on the ability to visually identify the desired cell population on the noncoverslipped slide, and to a lesser degree on the size and configuration of target cells. The current smallest spot size achievable is approximately 7.5 mm. Special caps hovering slightly above the section surface rather than being in direct physical contact with it have been designed for single-cell applications.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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