Single Cell Science: Chips Meet Single Cells with Laser Capture Microdissection

Contents
Introduction
The Principle of Laser Capture Microdissection
PixCell II LCM System
Where Can You Go with LCM?
LCM Meets Chips
How Low Can You Go?
Not the Only Game in Town
References

Introduction (Back to Top)
The ultra-sensitive techniques available today—RT-PCR, cycle sequencing, to mention but a few—put unparalleled power in the hands of researchers to investigate and catalog the rarest of events. Estimates of the sensitivity of some cDNA arrays in measuring mRNA levels, for example, are in the order of less than a copy per cell. However, to take advantage of this kind of sensitivity, it is important to have preparations that match these systems in purity, as even minute amounts of contamination can confound a PCR or sequencing analysis. Many kinds of samples are mixtures of different cell types, for which cell separation tools are either non-existent or lacking. Researchers at NIH have developed a technology, laser capture microdissection (LCM), to fill this void (ref. 1). This technology, which has been commercialized as the PixCell II LCM System by Arcturus Engineering (Mountain View, CA), is a technique for isolating pure populations of cells from microscopic sections quickly and without causing harm to tissue. Using this device and others like it, researchers will soon be able to look at the genetic read-out of individual cells.

Discover magazine, in nominating the PixCell II for its annual award for technical innovation, described the instrument this way: "Tracking the progress of disease within the body can be like hunting for a face in a crowd. Afflicted tissues contain important diagnostic clues that are hidden within a bewildering mix of cells: healthy cells, mildly damaged ones, and ones in advanced stages of disease. PixCell II uses a laser beam to single out and remove the most important cells for further study. The device is already at work in more than 200 medical research institutes worldwide, where it is aiding in the diagnosis of cancer, multiple sclerosis, Alzheimer's, hepatitis, and arteriosclerosis."

The Principle of Laser Capture Microdissection (Back to Top)
The concept behind LCM is simple. A thin, plastic film (ethylene vinyl acetate, or EVA) is placed over a region of interest on a microscope slide, previously identified by the user and positioned at center stage. A pulsed, low power laser beam produces localized heating of the film, which activates it, causing it to adhere to the tissue below it. The portion of the tissue section that is adhered to the film can then be lifted off the slide, leaving the remainder of the tissue behind and intact.

While this is not the first or only method for the microdissection of tissue, it has certain advantages over other methods. First, it is a quick, one-step procedure requiring only seconds to acquire a sample. Second, it leaves intact not only the tissue left behind on the slide, but also the tissue being captured, so that the user can go back and view his handiwork. Third, the targeting can be quite precise, approaching 5 microns, making it feasible to contemplate isolating individual cells (which will be discussed a bit later). Finally, the technology of LCM can be integrated into the familiar platform of a research-grade inverted microscope, making all of the above in the realm of possibility for many cell biology laboratories.

This technology is surprisingly versatile: it can be used with a variety of preparations and staining regimes. Protocols have been developed for working with freshly isolated or fixed preparations, smears, frozen or paraffin embedded material, stained or not. In addition, the number of downstream applications that can be performed on material collected in this fashion is large, among them RT-PCR, cDNA library construction, mutation detection, mRNA expression studies, and microarray analysis.

PixCell II LCM System (Back to Top)
So what has Arcturus Engineering brought to the table? Not long after researchers at the NIH described LCM, a collaborative research and development arrangement (otherwise known as CRADA) was set up between NIH and Arcturus. Through this arrangement, some aspects of LCM have been simplified and made more reproducible and, most importantly, commercially available. A mechanical arm, for example, positions the film, which is permanently bonded to the underside of a transparent vial cap. After the laser beam activates the film (which in the original PixCell System can be set by the user to a 30 or 60 microns), the targeted cells are transferred to the cap surface and the cap placed directly onto a vial for molecular processing. An Image Archiving Workstation is available for controlling and documenting the process, which allows the user to verify that the correct cells have been captured and also recall his session parameters, among other features. Arcturus' latest version of LCM, the PixCell II, has a laser beam resolution to <7.5µm, making single cell-microdissections possible.

Where Can You Go with LCM? (Back to Top)
LCM has been used for isolating pure populations of cells from brain tissue to rectal tissue and most everything in between. The early papers from the group at the NIH demonstrated the efficacy of this technique in procuring cells from a variety of preparations and tissue types—kidney glomeruli, Alzeihmer's plaques, and various cancerous and precancerous tissues—and their subsequent use in a number of downstream applications—PCR, RT-PCR, SSCP, and even enzymatic assays (ref. 1).

In a significant extension of the technology, Fend et al (ref. 2) described a method for rapid immunofluorescent staining of frozen sections followed by LCM, which expands the range of cell types that can be isolated with this technique beyond those with distinct morphological features. In this work, as few as 500 cells isolated on the basis of the presence of a surface marker were used to measure the level of message of not just abundant, housekeeping genes, but cell-type specific genes as well.

LCM Meets Chips (Back to Top)
In the first study of its kind, research published in Nature Medicine earlier this year combined LCM technology with DNA microarray analysis, and provided convincing evidence for both the reproducibility and the accuracy of these combined technologies (ref. 3). Two kinds of dorsal root ganglia cells (large and small) were isolated separately and analyzed for gene expression on a microarray containing some 400 plus cDNAs. In looking at multiple sets of cells of each of the two cell types (with 500–1,000 cells per set) analyzed separately, the researchers found a high correlation in the data obtained from each cell type. In addition, the validity of the assignment of expression to one cell type or the other was established by performing in situ hybridizations back to tissue sections with individual cDNA's that showed expression in one or the other cell type in the microarray experiment. The PixCell II was key in making it possible to extract these two cell types from the unwanted surrounding cell.

In work presented last month at American Association of Cancer Researchers (AACR), scientists at the NCI, FDA, and Ciphergen, the Palo Alto-based ProteinChip company, analyzed LCM-collected material from various stages of three different cancers (breast, prostate, and colon) using Ciphergen's ProteinChip. Using this sensitive and rapid method for creating protein fingerprints of cell or tissue types, the researchers found distinct patterns for the different stages of the diseases (ref.4).

How Low Can You Go? (Back to Top)
Much of the work to date has used collections of cells (500–1,000 seems to be the norm, requiring multiple captures) but according to Arcturus, there is a great deal of interest in isolating smaller populations or even individual cells. In the early LCM, the size of the laser beam size was the limiting factor. However the PixCell II, with its small beam size (<7.5µm), makes the procurement of single cells possible.

Two reports have already come out analyzing single cells captured with LCM. Earlier this year, Suarez-Quian et al (ref. 5), using a LCM prototype fashioned at the NIH for capturing single cells (called the cylinder LCM or cLCM, since it replaces the flat transfer surface with a cylindrical-shaped one), demonstrated the capture of individual cells from a variety of preparations and tissues. Also reported are the results of experiments with individual hepatocytes, visualized and captured by virtue of immunostaining with hepatitis B virus (HBV). Groups of from one to six cells were analyzed by PCR for the presence of HBV DNA. With a single, 40-cycle round of PCR, HBV DNA was detectable by ethidium bromide staining in a single cell. A second report, coming from the Mayo Clinic (ref. 6), demonstrated the technology offered by Arcturus' PixCell II LCM System in showing an analysis by RT-PCR of levels of pituitary-specific mRNA species in single rat anterior pituitary cells.

Not the Only Game in Town (Back to Top)
PixCell II is not alone. A German company makes an instrument it calls P.A.L.M. (Positioning and Ablation with Laser MicroBeams, which doubles as the company name as well). In a report published last August in Nature Biotechnology, a group of German scientist using PALM to capture individual cells detected a point mutation in a single colon adenocarcinoma cell, isolated from archival material, to boot (ref. 7). This machine differs from the PixCell II LCM System in several notable respects, though. With PALM, the laser is used as a cutting tool (in PixCell II, it's used as a heating tool to activate the membrane), which destroys the tissue surrounding the ablated area. PALM also uses a different technique for capturing the ablated tissue, using the energy of the laser to literally catapult the selected material off the slide into a collector. The catapulting technique removes the possibility of monitoring the results of the capture, which can be done with the transfer membranes in LCM. However, unlike the PixCell, which is dedicated to laser microdissection and capture, the PALM is more versatile, and can be used as "laser tweezers" in a variety of ablation experiments.

References (Back to Top)

1. Emmet-Buck et al., "Laser capture microdissection," Science, 274: 998–1001, 1996.
2. Fend F et al., "Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis," American Journal of Pathology, 154:61–6, 1999.
3. Luo, L. et al., "Gene expression profiles of laser-captured adjacent neuronal subtypes," Nature Medicine, 5:117–122, 1999.
4. Paweletz, C.P. et al., "A novel, proteomic approach to monitor carcinogenic disease progression using sruface enhanced desorption ionization spectroscopy (SELDI) of laser capture microdissection (LCM)-derived cells from cancer tissue," Proceedings of the American Association for Cancer Research, 40:411, 1999.
5. Suarez-Quian, C.A. et al., "Laser Capture Microdissection of Single Cells from Complex Tissues," Biotechniques, 26: 328–335, 1999.
6. Jin L. et al., "Analysis of anterior pituitary hormone mRNA expression in immunophenotypically characterized single cells after laser capture microdissection," Laboratory Investigations, 79: 511–2, 1999.
7. Schutze K and Lahr, G., "Identification of expressed genes by laser-mediated manipulation of single cells," Nature Biotechnology, 16: 737–742, 1998.

For more information on LCM: Arcturus Engineering Inc., 1220 Terra Bella Avenue, Mountain View, CA, 94043. Tel: 650-962-3020.

By Laura DeFrancesco