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BACKGROUND

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

Laser capture microdissection (LCM) is used to isolate specific cells from microscopic regions of tissue, cells, or organisms. LCM is also known as microdissection, laser-assisted microdissection, or laser microdissection. Various tissue microdissection techniques have been used to isolate pure cell populations. LCM was developed to address the problem of tissue heterogeneity, being a mixture of different cell types. Liotta and coworkers (1996) developed an LCM system to overcome the disadvantages of previous tissue microdissection techniques, such as manual microdissection and gross dissection of frozen tissue blocks to enrich for specific cell populations. They developed this system primarily for molecular analysis of solid tumors. The system was rapidly applied for commercial production by Arcturus Engineering (Mountain View, CA).

Three different classes of biomolecules, DNA, RNA, and proteins, can be investigated using LCM specimens (Domazet et al., 2008). LCM technology can fulfill the needs of researchers for routinely performing tissue microdissection. This method has proven invaluable to molecular pathology research and is used in many laboratories worldwide.

BASIC PRINCIPLES OF LCM

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

The basic principle of LCM is the capture of cells or an individual cell onto a thermoplastic membrane from sections of stained tissue (frozen or fixed and wax-embedded sections) or cytological preparations. Cellular components, including RNA, DNA, and protein, can be extracted using the appropriate methods and used for molecular analysis. The LCM system is based on an inverted light microscope, which is fitted with a laser device for visualizing and procuring cells. The two main classes of LCM are infrared (IR) capture systems and ultraviolet (UV) cutting systems (Espina et al., 2006). LCM instruments (IR and IR/UV systems) are available in automated (robotic) and manual platforms.

For IR capture systems, a near-IR laser is attached to a microscope stage. This laser is applied for melting a thermolabile polymer film. A focused laser beam of varying diameter (7.5, 15, and 30 µm) is applied for localized melting of the film over the cells that have been selected. Laser impulses, which are usually 0.5–5 msec long, are able to be repeated, enabling rapid isolation of a large number of cells. The film is produced at the bottom of plastic cap of optical quality. This cap allows the laser to be focused in the same plane as the tissue section. The polymer film melts in the area where the laser impulse is present, resulting in a polymer–cell composite. Only those cells that are within the diameter of the melted polymer are targeted for microdissection with each laser pulse. When the polymer is removed from the surface of the tissue, the embedded targeted cells are sheared away from the tissue section. Cells are solubilized by adding extraction buffer, and the desired molecules are released. In LCM, there are physical forces involved, including an upward binding force between the tissue and substratum, lateral force between cells, and a downward binding force between the cells and polymer. On one slide, a researcher can fire between 1,000 and 3,000 laser shots to capture at least 6,000 cells in approximately 20 min (Kunz and Chan, 2004). A representative setup of an LCM system is shown in Fig. 1.

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Figure 1. Leica LMD 7000 laser microdissection system.

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For UV cutting systems, a UV laser system is used. UV laser systems are useful to get rid of undesired tissue in addition to removing the cells of interest, leaving these cells intact. UV laser systems have the limitation that cells that are damaged by UV might be present in the final cell population acquired. This damage occurs among cells that are in the cutting path.

ADVANTAGES AND DISADVANTAGES OF LCM

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

Advantages of LCM

The components of the LCM system are low cost (Kunz and Chan, 2004). Operating LCM is relatively simple and does not require any moving parts. LCM does not involve manual microdissection or manipulation, which means that it is much faster, and allows one-step transfers. When tissue is transferred to the film, the tissue still has its original morphology. The sterile disposable transfer films minimize contamination. The presence of contaminating cells can also be evaluated. The number and types of cells and structures are easily examined and counted by using a digital microscope to examine the film. Staining the tissue for histology, which is usually required for LCM to enable morphological visualization, does not alter most cellular constituents.

A major advantage of using LCM to obtain specific cells for molecular analysis is that the procedure is performed by directly viewing the cells under light microscopy (Curran and Murray, 2005). LCM is currently the only method that is able to separate cell subpopulations from tissue or cell-population monolayers without disturbing a cell's molecular state. Figures 3 and 4 show representative photographs of cells before and after capture by a laser.

Disadvantages of LCM

Optical resolution of LCM is limited because sections become dried out owing to a lack of a coverslip, which is necessary for tissue capture (Fend et al., 2000). Commonly used stains, such as hematoxylin and eosin, are sometimes not effective for precise isolation of homogeneous cell populations. Several research groups have solved this problem by adapting immunohistochemical staining techniques for LCM. Immuno-LCM is the process of immunohistochemical staining of tissue prior to LCM. This process assists researchers in determining molecules of interest in heterogeneous tissues by using detection strategies that are based on morphology and immunophenotype (Murakami et al., 2000; Buckanovich et al., 2006) (Fig. 2). One of the limitations of immuno-LCM is that it is unable to be used for studying expression dynamics of the immunostained protein of interest because of antibody binding to the protein of interest.

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Figure 2. Paraffin section delineating the area to be captured by the laser.

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Figure 3. Photograph of a lung section before capturing a cluster of fluorescent neuroendocrine cells, known as a neuroepithelial body.

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Figure 4. The same lung section shown in Fig. 3 was photographed after capturing this neuroepithelial body. The captured body of cells is gone from the tissue section.

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The main limitation of LCM is the requirement for identifying cells of interest based on morphological characteristics (Espina et al., 2006). Cells are usually identified by a cytologist, pathologist, or technologist who is skillful in identifying cells. Other limitations of LCM include tissue molecules perishing after being surgically obtained, the type of protocol for staining tissue, and the suitability of tissue fixation methods with downstream analysis.

Another disadvantage of LCM is that it can be time consuming. When the tissue being studied comprises different intermingled cell types, dissecting out the cells of interest can take a long time.

APPLICATION OF PROTEOMICS TO LCM

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

The field of proteomics involves the identification and characterization of proteins, as well as determination of post-translational modifications. A major application of proteomics is to determine differentially expressed proteins by comparing the patterns of protein expression between normal and diseased cells (e.g., tumor cells) (Lawrie and Curran, 2011). Various proteomic techniques are used for analyzing cells obtained using LCM. The field of proteomics has rapidly expanded, and attention has focused on using LCM combined with protein-based studies.

Two-Dimensional Polyacrylamide Gel Electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (PAGE) is the main tool for separation in proteomic analysis and is compatible with samples obtained by LCM (Craven and Banks, 2001; Xu, 2010). Proteins that are extracted from cells captured by LCM are separated by isoelectric focusing, followed by separation using gel electrophoresis. Different staining techniques are used to observe the separated proteins. Samples are processed without harmful effects and the LCM procedure does not cause any changes. However, staining can affect samples, and these changes are seen in the two-dimensional gel profile. One of the main problems of using LCM for obtaining samples to analyze using 2-D PAGE is the availability of samples; only 600–1,000 proteins are usually resolved from dissected cells.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Protein profiles can be obtained from cells microdissected by LCM, using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) (Palmer-Toy et al., 2000). Matrix solution is microspotted on the captured cells. After being dehydrated by a solvent, the matrix and proteins co-crystallize. The crystals are examined by MALDI MS. The mass spectra that are obtained reflect the protein profiles detected from the cells that have been captured.

SELDI TOF MS using ProteinChip technology (Ciphergen, Fremont, CA) is a sensitive technique for protein identification (Merchant and Weinberger, 2000). This technique uses chips with chromatographic surfaces for separating a small amount of protein based on chemical properties, such as charge, and then analyzes the protein with TOF MS. This method can yield a protein profile from only 25 cells, although larger amounts are preferable. SELDI uses a much smaller amount of material than two-dimensional PAGE, making it well suited to LCM-obtained material.

Protein Arrays

Multiple types of protein arrays are available for determining the protein profiles of target cells (Xu, 2010). In antibody microarrays, the antibody is immobilized in an array format on a substrate. Protein obtained from tissue samples is examined on the antibody arrays, and immunoassays on the chip quantitatively determine the target analytes. The reverse phase (RP) protein array is another type of protein array, which is commonly used combined with the LCM method. Specific cell populations from samples are obtained using LCM and lysed. More than 1,000 proteins are able to immobilized in an array formation on glass-backed nitrocellulose slides. Target protein expression is assessed using a specific antibody, and this is observed by amplified fluorescent or colorimetric assays. This approach is a high-throughput method for investigating activation of signaling pathways and for validating differential expression of target protein biomarkers in clinical cases.

APPLICATION OF GENOMICS TO LCM

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

Samples obtained by LCM can be subjected to any type of molecular analysis. DNA and RNA are easily extracted and used for polymerase chain reaction (PCR) and analysis of gene expression (Maitra et al., 1999). DNA and RNA analyses require less material than protein analyses. Therefore, genomic analyses can be performed on samples from a single cell, whereas this might not be possible for protein. LCM can be applied to a variety of methods to discover new genes and efficiently analyze their function in specific cells. There are standard protocols for molecular analysis of LCM-captured cells; these are accessible by the public on the NIH website (http://dir.nihcd.nih.gov/lcm.htm).

DNA can be used for analysis of a single or a few genes by PCR. Additionally, DNA acquired from microdissected paraffin-embedded tissue sections can be used for genome-wide screening techniques. DNA analysis also encompasses methods, such as dideoxy fingerprinting, clonal analysis, and direct sequencing. Other applications are designed to identify loss of heterozygosity and single-stranded conformational polymorphisms (Bonner et al., 1998).

RNA molecules are able to be preserved during LCM. Therefore, measurement of gene expression and regulation is feasible in a cell type-specific manner in complex tissues (Fink and Bohle, 2011). However, isolation of nucleic acids involves some steps that may fragment and degrade the RNA. Frozen tissue is optimal for RNA, but this type of tissue does not provide clear histological detail, and is inconvenient for storage and handling. RNA, as well as protein quality, quickly degrades after staining. Therefore, LCM should be performed and completed within 1 hr of staining a tissue section. An important recognition is that an optimal preparation strategy is not currently available. Messenger RNA from microdissected cancer lesions is used for microchip microarrays for differential display, as starting material for producing cDNA libraries, and in other methods for identifying new genes or mutations. The Cancer Genome Anatomy Project (CGAP) is using LCM to catalog genes that are expressed during solid-tumor progression.

EXPRESSION MICRODISSECTION

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

Expression microdissection is a conceptual and practical advance in technology that provides considerable improvement in laser dissection based on operator-independent cell selection by molecular targeting (Hanson et al., 2011). Until recently, expression microdissection was not available to investigators. However, this method has now been adapted to several different commercially available laser sources to make it more accessible to researchers. Unlike other approaches, expression microdissection does not require visualization of cells or use of a microscope (Chimge et al., 2007). A film of clear ethylene vinyl acetate is placed on a histological slide containing immunohistochemically stained cells. A light source irradiates the whole tissue section. The chromogen absorbs the light energy, leading to focal heat activation of the film and bonding it to target cells. The film is removed from the tissue section to obtain the targeted (immunostained) cells for later molecular analysis.

Advantages of expression microdissection that have facilitated dissection-based analyses include subcellular precision, increased dissection rate, elimination of variance caused by individual operators, and removal of targeting difficulties (Tangrea et al., 2004). In particular, expression microdissection is useful for studies lacking molecular amplification methods. A limitation of expression microdissection is that mRNA measurement in frozen tissue sections is affected by the immunostaining process and is difficult to accomplish using any of the immuno-based dissection methods.

FUTURE DIRECTIONS

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED

Since the original invention of LCM in the 1990s, this technology has considerably evolved. This field has various challenges, including the requirement for better precision and the need for a relatively large amount of biomolecules for some downstream analysis platforms. Future directions for LCM include the analysis of many different tumors for new diagnostic biomarkers and therapeutic targets. Further development of laser-based microdissection systems will result in more widespread use of these technologies. In particular, integration of sophisticated image analysis software to enable automatic dissection of pre-defined cells of interest is anticipated. The development of automated laser microdissection systems should considerably increase the accuracy and speed at which cells are able to be microdissected and obtained for molecular analysis, as well as assist higher-volume and larger-scale molecular analysis of various cell types.

LITERATURE CITED

  1. Top of page
  2. BACKGROUND
  3. BASIC PRINCIPLES OF LCM
  4. ADVANTAGES AND DISADVANTAGES OF LCM
  5. APPLICATION OF PROTEOMICS TO LCM
  6. APPLICATION OF GENOMICS TO LCM
  7. EXPRESSION MICRODISSECTION
  8. FUTURE DIRECTIONS
  9. LITERATURE CITED
  • Buckanovich RJ, Sasaroli D, O'brien-Jenkins A, Botbyl J, Conejo-Garcia JR, Benencia F, Liotta LA, Gimotty PA, Coukos G. 2006. Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment. Cancer Biol Ther 5:635642.
  • Charboneau L, Tory H, Chen T, Winters M, Petricoin EF, III, Liotta LA, Paweletz CP. 2002. Utility of reverse phase protein arrays: applications to signaling pathways and human body arrays. Brief Funct Genomic Proteomic 1:305315.
  • Chimge NO, Ruddle F, Bayarsaihan D. 2007. Laser-assisted microdissection (LAM) in developmental biology. J Exp Zool B Mol Dev Evol 308:113118.
  • Craven RA, Banks RE. 2001. Laser capture microdissection and proteomics: possibilities and limitation. Proteomics 1:12001204.
  • Domazet B, Maclennan GT, Lopez-Beltran A, Montironi R, Cheng L. 2008. Laser capture microdissection in the genomic and proteomic era: targeting the genetic basis of cancer. Int J Clin Exp Pathol 1:475488.
  • Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. 1996. Science 274:9981001.
  • Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF, III, Liotta LA. 2006. Nat Protoc 1:586603.
  • Fend F, Kremer M, Quintanilla-Martinez L. 2000. Pathobiology 68:209214.
  • Fink L, Bohle RM. 2011. Laser microdissection and RNA analysis. In: Murray GI, editor. Laser capture microdissection: methods and protocols. New York: Humana Press, Springer. p 167185.
  • Hanson JC, Tangrea MA, Kim S, Armani MD, Pohida TJ, Bonner RF, Rodriguez-Canales J, Emmert-Buck MR. 2011. Expression microdissection adapted to commercial laser dissection instruments. Nat Protoc 6:457467.
  • Kunz GM, Jr., Chan DW. 2004. The use of laser capture microscopy in proteomics research—a review. Dis Markers 20:155160.
  • Lawrie LC, Curran S. 2011. Laser capture microdissection and colorectal cancer proteomics. In: Murray GI, editor. Laser capture microdissection: methods and protocols. New York: Humana Press, Springer. p 245253.
  • Maitra A, Wistuba II, Virmani AK, Sakaguchi M, Park I, Stucky A, Milchgrub S, Gibbons D, Minna JD and Gazdar AF. 1999. Enrichment of epithelial cells for molecular studies. Nat Med 5:459463.
  • Merchant M, Weinberger SR. 2000. Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. Electrophoresis 21:11641177.
  • Murray GI, Curran S. 2005. An introduction to laser-based tissue microdissection techniques. Methods Mol Biol 293:38.
  • Murakami H, Liotta L, Star RA. 2000. I.F.-L.C.M. laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication. Kidney Int 58:13461353.
  • Palmer-Toy DE, Sarracino DA, Sgroi D, LeVangie R, Leopold PE. 2000. Direct acquisition of matrix-assisted laser desorption/ionization time-of-flight mass spectra from laser capture microdissected tissues. Clin Chem 46:15131516.
  • Simone NL, Bonner RF, Gillespie JW, Emmert-Buck MR, Liotta LA. 1998. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet 14:272276.
  • Tangrea MA, Chuaqui RF, Gillespie JW, Ahram M, Gannot G, Wallis BS, Best CJ, Linehan WM, Liotta LA, Pohida TJ, Bonner RF, Emmert-Buck MR. 2004. Expression microdissection: operator-independent retrieval of cells for molecular profiling. Diagn Mol Pathol 13:207212.
  • Xu BJ. 2010. Combining laser capture microdissection and proteomics: methodologies and clinical applications. Proteomics Clin Appl 4:116123.