Robert Bagnell Jr

1. INTRODUCTION

Advances in our understanding of disease mechanisms have resulted in the need for single-cell analysis. Analytical technologies have become available to accommodate such interrogations. Typically, molecular diagnostic assays begin with a nucleic acid extraction procedure during which tissue architecture and cellular morphology is lost. Laser capture microdissection (LCM) is a technology that enables scientists to examine the processes of individual cells. Whether one is investigating a cell's internal messages or its proteins, isolating that particular cell(s) from a mixed cellular environment is the function of LCM (Fig. 1). This chapter briefly describes the LCM technique by reviewing the current instrumentation and answers some of the most frequently asked questions about LCM.

There is now a vast literature on LCM, which this chapter will not attempt to review. A well-organized listing of the primary papers as well as contemporary work can be found at the Arcturus website http://www.arctur.com. Conn is editor of perhaps the best compilation to date on LCM (1). There is a new methods book on LCM, edited by Murray and Curran (2). LCM was introduced by the National Institutes of Health investigators Liotta, Bonner, and Emmert-Buck in 1996 (3) and 1997 (4). The first commercial instrument was produced by Arcturus Engineering, Inc. (Mountain View, CA) as a result of a Cooperative Research and Development Agreement with NIH. To date, there are four companies that produce LCM equipment.

2. LCM METHODS AND INSTRUMENTATION

Three methods exist that use lasers to collect tiny samples from heterogeneous biological specimens. The first method melts cell-sized spots of a thermoplastic film onto the specimen using an infrared (IR) laser. The plastic cools adhering to the specimen. When the film is lifted, the adhering specimen is also removed. This is the original LCM method created at NIH and first reported by Liotta et al. (3,4). This method is marketed by Arcturus (http://www.arctur.com). The second method uses a special supporting membrane under the specimen. A pulsed ultraviolet (UV) laser cuts the membrane around the desired specimen that then either drops into a collection cap by gravity (Leica; http://www.leica-microsystems.com) or is catapulted into a collection cap by a defocused laser beam (P.A.L.M., http://www.palm-mikrolaser.com). The third method uses an IR laser and a special IR-absorbing plastic film on which the sample is placed. The laser severs the film by heat, thus isolating islands of specimen which remain when the bulk of the film is removed (Bio-Rad; http://www.bio-rad.com).

2.1. ARCTURUS The Arcturus Pix-Cell lie is shown in Fig. 2. An automated version called the AutoPix is also available. The Arcturus LCM process is illustrated in Fig. 3. The specimen is placed on a glass slide with no cover slip. Critical to this process is the LCM cap. The cap, which is sized to fit into a 0.5-mL Eppendorf tube, is made of an optical-grade plastic with a thermoplastic film on the narrow end. The film is placed on the specimen, which is visualized using an inverted microscope equipped with a color charge-coupled device (CCD) camera and video monitor. The specimen is maneuvered utilizing the joy-stick stage positioner to place an area of interest under a target beam that appears on the monitor. The IR laser is activated by pressing a button. The laser passes through the cap from above and causes the thermoplastic film to soften and expand down into the tissue at the position of the target beam. The film adheres to the tissue. Each press of the button is called a "shot." One cap can contain approx 6000 shots. After all areas to be microdissected have been shot, the cap with adherent specimen is lifted away from the slide. The cap is then placed into a 0.5-mL Eppendorf tube for processing of the specimen.

2.2. LEICA AS LMD Figure 4 illustrates the Leica AS LMD. This system is based on the ability of a pulsed UV laser to cut a PEN (polyethylene-naphthalate) membrane covering a glass slide with the specimen on top. The slide is placed on the computer-controlled stage of the upright microscope, specimen side down. Fig. 5. illustrates the stage area. Areas of interest are visualized utilizing the computer-controlled stage and focus system and a color CCD camera. Specimen regions to be microdissected are outlined on the video image using the mouse cursor. Once this is done, the computer system automatically moves the stage to each location. The system guides the UV laser cutting by deflecting the laser beam through the

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ

Fig. 1. Micrometastatic cancer cells in a lymph node (x 40 objective). Only the cells that are forming glandlike structures, indicated by the arrows, are of interest in analyzing the tumor cell genes or proteins.
Fig. 2. Arcturus PixCell IIe LCM system.

objective lens around the previously marked areas. Once cut, the PEN membrane with the specimen attached drops by gravity into the cap of a 0.5-mL Eppendorf tube held in place below the specimen by a special carrier. After microdissection, the cap and tube are removed from the carrier and are ready for further processing.

2.3. P.A.L.M. The P.A.L.M. system is illustrated in Fig. 6. The P.A.L.M. system also utilizes a pulsed UV laser for cutting. However, the specimen can be on a variety of substrates, including plain glass slides, slides with a PEN membrane, or culture dishes with a PEN membrane insert. The specimen is placed on the inverted microscope, specimen side up. A

Arcturus Lcm

Fig. 3. Arcturus LCM process.

4 Extract molecules from target cells

Fig. 3. Arcturus LCM process.

Fig. 5. Leica AS LMD stage.

computer controls the stage and microscope focus. A CCD camera presents an image of the specimen on the computer monitor. Marking areas for microdissection is carried out by outlining or "dotting" them in the video image using the mouse cursor. The system then moves to each location and cutting is done by projecting the laser through the objective lens while the computer moves the precision stage around a previously marked area. The specimen is then catapulted (removed by the force of light pressure in the defocused laser beam) into the cap of an Eppendorf tube held above the specimen in a special carrier. Specimens not on a PEN membrane can be microdissected by multiple catapult shots covering the desired area. The cap is then placed on a standard 0.5-mL Eppendorf tube for further processing.

2.4. BIO-RAD CLONIS The Bio-Rad CLONIS system is shown in Fig. 7. This system is based on the ability of an IR laser to cut a PEN membrane that is specially constructed to absorb the IR light energy, thus producing heat. Specimens must be placed on the special multilayered membrane whether on slides or in culture dishes. The sample is placed on the inverted microscope specimen side up. Areas for microdissection are selected utilizing the computer-controlled stage and CCD camera by marking them on the monitor utilizing the mouse cursor. After marking, the system moves to the marked areas and cuts the film by moving the stage over the fixed laser beam, which is projected through the objective lens. Samples are subsequently processed by either removing the membrane containing the unwanted sample or by removing the part containing the desired sample. Both procedures are done by hand, utilizing sharp forceps.

3. SYSTEM COMPARISONS (TABLE 1)

3.1. SPECIMEN CONDITIONS Of the four systems considered here, the Arcturus and Leica systems require that the samples be dry. Also, relative humidity is important for both of these systems. The Leica system requires at least 35% relative humidity (RH), otherwise static charges interfere with the gravity drop of the microdissected sample into the cap. The Arcturus system, on the other hand, requires a nonhumid atmosphere, otherwise the very dry sample will absorb room moisture and the film will not stick to the sample. The P.A.L.M. and Bio-Rad systems can microdissect both dry and wet specimens, including living specimens. The type of surface on which the sample is placed differs among the systems. Arcturus requires that the sample be on a surface that is less adherent to the specimen than the thermoplastic membrane; usually, plain, uncoated glass slides will work. The P.A.L.M., Leica, and BioRad systems require that the specimen be placed on a special PEN membrane for laser cutting. The P.A.L.M. system can microdissect directly from a glass slide by the laser catapulting method.

Samples from a number of sources are suitable for microdissection. The main criterion is that the sample areas to be

Fig. 6. P.A.L.M. system.
Fig. 7. Bio-Rad CLONIS.

microdissected must be visualized on a microscope. Frozen sections on glass slides, metaphase spreads on cover slips, aldehyde-fixed and paraffin-embedded tissue sections, cells on trans-well membranes, cytospins onto glass slides, and tissue cultures grown on specially coated slides or in dishes with special membrane inserts are some of the types of specimen that have been successfully microdissected utilizing the four systems described here. Several histological and immunological stains have been utilized to help visualize the specimen. Each investigator should do some testing to judge whether or not a particular stain has an affect on recovery of RNA, DNA, or proteins, or interferes with a polymerase chain reaction (PCR). Protocols for several histological stains can be found at the Arcturus website.

3.2. SMALLEST MICRODISSECTED AREA Laser spot size on the specimen sets the lower limit on the size of a microdissected area. Spot size is determined by the objective lens numerical aperture, the wavelength of the laser light, and the type and thickness of material the laser has to travel through to reach the sample. Typical laser spot size for the P.A.L.M. system is about 1 |im; for the Leica AS LMD, it about 2.5 |im, and for the Bio-Rad CLONIS, it about 20 |m. The Arcturus system has three spot sizes: 7.5, 15, and 30 |im. All of the systems can microdissect single cells, but for the Bio-Rad system, it is recommended that the cut area around a cell be about 200 | m away from the cell(s) of interest.

3.3. SPECIMEN VISUALIZATION Bright-field and fluorescence imaging are available on all systems. All but Arcturus system can also utilize phase-contrast and differential interference-contrast techniques. All of the systems offer good visualization of the specimen prior to microdissection. The Arcturus, Bio-Rad, and P.A.L.M. systems can visualize the sample after

Table 1 System Comparisons

Instrument

Arcturus

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