LCM instruments exist in a form of manual and automated robotic platforms [ 3 ]. Regardless of the system used, the fundamental features of the laser microdissection process are visualization of the cells via microscopy, transfer of laser energy to the area of interest, and removal of the cells of interest from the heterogeneous tissue section. The transfer of laser energy may be to a thermolabile polymer thus forming a polymer cell composite as in IR systems, or photovolatilization of cells surrounding a selected area that is specific to UV systems.
UV systems often employ cutting methods that combine UV laser microdissection and catapulting systems [ 7 , 17 ]. The basic principle of LCM is the capture of groups or individual cells onto a thermoplastic membrane from histological sections of stained tissue frozen or formalin-fixed paraffin-embedded Table 1 [ 17 , 18 ]. The system consists of an inverted microscope that is connected to a personal computer for additional laser control and image archiving, a solid state near infrared laser diode, a laser control unit, a joy stick controlled microscope stage with a vacuum chuck for slide immobilization, a CCD camera, and a color monitor [ 19 — 21 ] Figure 1 for an example.
The cap fits on the standard 0. A dye absorbs laser energy, preventing damage to the cellular constituents. It also aids visualizing areas of melted polymer and it aids visualizing areas of melted polymer [ 2 , 3 ]. The cap, that is suspended on a mechanical transport arm and placed on the desired area of the dehydrated tissue section, acts as an optic.
It focuses the laser and brings it to the same plane as the tissue section, and then is lowered in exact apposition to the area of interest [ 3 , 17 , 19 , 20 ]. The polymer melts only in the vicinity of the laser pulse, expands into the section and fills the extremely small hollow spaces present in the tissue. Cells then selectively adhere to the thermoplastic membrane activated by a low energy infrared laser pulse [ 20 ]. Under standard working conditions, the area of polymer melting corresponds quite exactly to the laser spot size [ 19 — 22 ].iptlodz.org/blog/wp-includes/escort-gir-blois.php
Laser capture microdissection and its applications in genomics and proteomics.
As the temperature decreases, it solidifies again within milliseconds and forms a polymer-cell composite that embeds the cells into the plastic membrane [ 2 , 19 , 20 ]. The selected tissue fragments are harvested by simple removal of the polymer from the tissue surface that serves to shear the embedded cells of interest away from the tissue section [ 2 , 3 , 19 , 20 ]. The cap with the dissected cells is then placed in a 0. The long chain polymers within the EVA film then dissolve in the lysis buffer and the cells are released into the solution [ 7 ].
Laser impulses, usually between 0. Up to — cells can be isolated onto a single cap in this fashion [ 7 , 17 ] Figure 2. In the Arcturus Pix Cell II instrument, a current model of the laser micro capture system, the laser beam has 3 settings of diameter [ 5 , 12 ] Figure 1. Varying laser spot size within a narrow range ensures the specificity of dissection,[ 4 , 7 , 10 , 17 — 21 ] and usage of a laser spot at its narrowest diameter 7. The low energy of the infrared laser also avoids potentially damaging photochemical effects [ 19 , 20 ]. The joystick is used for movement of the laser cap around the tissue in order to select multiple areas on the same cap [ 6 , 8 ].
A caveat of the membrane slides is that they are not cover slipped which makes visualization fuzzy [ 8 , 17 ]. However, newer versions of LCM systems have a built in optical system that makes it possible to confirm the histology of the area to be microdissected [ 8 , 17 ]. When using glass slides, they need to be non-charged and non-coated since either feature can interfere with the transfer of tissue from the slide onto the cap.
The cell samples that are obtained can be used for any molecular analytical methods. About 50— cells are adequate for PCR analysis from microdissected material [ 7 ] and can yield nanogram quantities of nucleic acids [ 8 , 24 ]. Of note, histological staining that is required for visualization during microdissection will not affect the quality of the biomolecules In the sample, nor does the process of acquiring cells onto the thermoplastic membrane alter or damage the integrity of DNA, RNA and protein.
Protocols used for molecular analysis from LCM — captured cells are standardized and are available for use by the public at large on the NIH web site http: Newer version for single cell extraction is called cylinder LCM.
The narrow-beam UV laser is used to draw around the cell or cells of interest leaving the desired cell population intact while simultaneously ablating away unwanted tissue [ 7 , 17 ]. The cells of interest are then isolated by catapulting them under pressure onto an overhanging cap. There are two major advantages of this method. First, it avoids any intricate operator dependent step, and second, by ablating the adjacent rim of unwanted tissue, non-specific adherence of tissue to the cap is avoided.
Thus, the heat generated during microdissection is minimal, which reduces the risk of damaging extracted reagents [ 7 ]. The PALM system offers the advantage that there is no physical contact between the cells and plastic because the laser is used to catapult the microdissected cells into a collecting tube under the influence of gravity [ 19 ].
This process avoids the potential risk of modification of molecules of interest due to heating and cooling of the thermoplastic membrane. Of course, the original quality and subsequent handling of the tissue is of fundamental importance [ 25 ]. Laser microbeam microdissection systems use a much finer laser beam diameter 0. Thus, UV LCM systems are ideally suited for the precise microdissection of single cells although this is potentially more time-consuming, especially when a large number of cells are required to be microdissected. This is unlikely to be a significant issue for nucleic acid based molecular analysis, as it can often be performed on very few cells, but the time taken to acquire the large number of cells for proteomic studies may become a significant factor in experimental planning and design.
In this method, an adhesive lid of a microcentrifuge tube is placed onto the cut area and the selected cells are removed from the tissue. It is very often hard to visualize cells for microdissection because non-coverslipped slides are used. This is particularly difficult when isolating cells that are morphologically very similar B and T lymphocytes , or present within a heterogeneous background such as Reed Sternberg cells in Hodgkin lymphoma [ 3 , 7 ]. Immuno-LCM can help to overcome this issue. Immunohistochemistry HICK is performed to mark the cells expressing a type-specific antigen such as CD3 for T-cells or prolactin for pituicytes , and the cells are captured under direct visualization.
It is also possible to simultaneously detect multiple different messages in pathologically altered tissue with the immunohistochemical tagging. One variation of immuno LCM is prelabeling of cells in vivo in an animal model, by injection of a fluorogold label. It has been used to label cells to avoid RNA degradation due to immuohistochemical staining [ 7 ]. The nature of investigation is determining the choice of fixative. RNAse free reagents should be used at all times for RNA based investigations, because protection of sample from degradation is important.
It is also important to note that the specimens need to be dehydrated as well, since the presence of water interferes with the bonding of polymer to the captured cells.
Laser capture microdissection and its applications in genomics and proteomics.
Example of microdissected cancer cells with LCM. Melanoma cells before A and after B, C microdissection. At present, 3 distinct classes of biomolecules can be analyzed in LCM specimens: Therefore, it is possible to perform genomic analyses on samples derived from one single cell, whereas for protein this may not be possible with the current generation of proteomic tests. Recent studies of the identification of prostate specific genes by the analysis of prostate expression sequence tags ESTs have shown the power of LCM in creating tissue specific expression libraries.
For example, it was initially believed that highly expressed T cell receptor found in prostate libraries generated from microdissected tissue stems from the contaminating T cells in the prostatic interstitium, but the subsequent analyses showed that the transcript did in fact originate from prostate epithelial cells. In the future similar revealing results can be expected for other tissues.
Of course, to produce useful information, it is most important to have primary tissues of superior quality [ 1 ]. In most cases, solid tumors progress in a multistep pathway. A continuous accumulation of genetic aberrations within the indigenous cells result in neoplastic transformation [ 17 ]. In carcinomas, this continuum of changes may involve amplification or gain of function mutations in dominant oncogenes, or loss of function by deletion, mutation or methylation in recessive tumor suppressor genes.
In Knudson's two hit hypothesis of tumor suppressor gene function, one parental allele is lost by deletion, while the second is inactivated by mutation or by some other mechanism such as aberrant methylation. This means that in order to examine the integrity of parental alleles at a given polymorphic locus of tumor suppressor genes in non-cancer tissue, two different or heterozygous alleles would be present: In cancer tissue, the normal allele is lost, i.
LOH has been used for a long time in cancer research both for mapping of tumor suppressor genes, as well as for studying the frequency of involvement of known or putative tumor suppressor genes in various cancers using flanking or intragenic polymorphic markers. The use of microdissection in the study of cancer has made a significant difference in the application of LOH analysis [ 31 , 37 — 40 , 42 — 57 ]. Before the widespread availability of microdissection techniques, many valuable samples had to be discarded because the desired purity could not be achieved. The study of genetic losses in preneoplastic lesions was virtually impossible to perform.
Microdissection had revolutionized the approach to LOH research in many ways. First it made this study feasible. Besides LOH analysis, other genome analyses can be performed from microdissected samples, such as analysis of patterns of X-chromosome inactivation to assess clonality, restriction fragment length polymorphism RFLP and single strand conformation polymorphism SSCP analysis for assessment of mutation in critical genes such as Ki-ras and P53 , and most recently, the analysis of promoter hypermethylation. Hypermethylation of promoter sequences in tumor suppressor genes is a frequent and early event in carcinogenesis.
It is a potentially reversible, and thus can be used as a surrogate biomarker in chemoprevention trials [ 7 ]. With the help of combination of microdissection with a primer extension preamplification PEEP and whole genome amplification WGA , it became possible to use smaller and smaller samples of cells, thereby refining the study of preneoplastic lesions, which has opened a whole new frontier in cancer research.
For example, the comparative genomic hybridization CGH , until recently, was possible only with large amounts of DNA extracted from tumor tissues. Now, thanks to the new technology, CGH can be performed on 20— microdissected cells from FFPE tissue and even fewer in material obtained from precipitating fixatives such as methanol. Formalin fixed archival tissue can be used in all of these methods.
The sample size is very small, no more than 50 — dissected cells per PCR and even fewer cells if material is obtained from cryostat sections or methanol- fixed specimens [ 7 ]. A useful parameter to determine how tumors differ from the normal tissues they are derived from is differential gene expression [ 7 ]. It is quite possible that the identification of gene expression patterns related to neoplastic transformation, inflammation or tissue repair will have far reaching consequences in the prognostic and diagnostic field, preventive medicine and for novel treatments tailored for specific genetic alterations [ 19 , 20 ].
Gene expression can be studied by a variety of available methods such as differential display, representation difference analysis RDA , SAGE, ESTs, and differential gene chips [ 4 , 7 ]. Similar issue as in DNA-based studies in the analysis of gene expression is the problem of contamination with inflammatory and stromal cells [ 7 , 20 , 60 ]. Therefore, there has been an increasing need to apply microdissection methods in the expression studies as well, because confirmation by HICK or in situ hybridization is laborious, time-consuming and might not always be possible.
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Proteins perform all the necessary functions of the cell. The existence of a DNA sequence does not guarantee the synthesis of a corresponding protein, nor is it sufficient to describe its function and cellular locations. DNA sequence also does not give information about context dependent posttranslational processes such as glycosylation, phosphorylation or sulphatation, or how proteins link together into networks and functional machines in the cell [ 4 ].
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Proteomics is a complementary approach to study gene expression and provide additional information regarding the effects of post-translational modification. A variety of techniques such as western blotting, high resolution two dimensional polyacrylamide gel electro-phoresis 2-D PAGE , mass spectrometry and peptide sequencing can be used for the analysis [ 7 , 61 , 62 ]. Mass spectrometry, such as surface enhanced laser desorption ionization SELDI mass spectrometry, has facilitated the study of gene expression at the protein level leading to the recent expansion of proteomics-based research [ 4 , 5 , 7 , 61 ].
In the context of protein analysis of LCM procured samples, a number of factors must be considered, such as tissue type, molecule s being studied or the method of the downstream analysis. There are limitations resulting from the amount of samples available for analysis, because there is no amplification step for protein analysis [ 5 ]. Electrophoresis of cell samples in 2-D PAGE first separates individual proteins by charge, and then by size. Proteins have a distinct advantage compared to RNA; they are much more stable and this disparity has important consequences for the measurement of these molecules in biological materials, especially in clinical samples.
All of the current techniques require tissue homogenization and hence do not account for the cell of origin contributing the measured protein content. HICK has been used for a long time to identify cell-specific protein expression. However, there still remains to be resolved issue of artifacts secondary to fixation technique, antigen-antibody affinity, extent of antigen retrieval and absence of a reliable quantification protocol. Recently, LCM has been applied to the study of protein alterations in tumors and their preneoplastic lesions, which is an important step toward formulating treatment and intervention strategies [ 4 , 7 ].
Prostate specific antigen PSA levels in microdissected benign and malignant tissues can be measured with a rapid, sensitive and quantitative chemo-luminescent assay. This quantitative technique can potentially be extended to analyze a large variety of proteins in pure populations of normal and tumor cells. More than proteins or their isoforms from a sample of about 50, microdissected cells can be resolved in 2-D PAGE and the dysregulated products in cancer cells identified.
Novel tumor specific alterations can be identified by sequencing of the altered peptide products in cancer cells [ 7 ]. Based on the chemical characteristics of proteins i. Using SELDI biochip, Paweletz et al have successfully classified protein population into molecular weight classes and shown distinct protein expression patterns of normal, premalignant and malignant cancer cells procured by LCM from human tissues [ 4 ]. Efficient detection of gene mutations is becoming increasingly important in the pathological diagnosis, classification and treatment of tumors.
The current detection method, however, is labor intensive, a major barrier of tumor mutational analysis for routine clinical use. LCM plays a major role in this area because captured tumor tissue is enriched in tumor-associated genetic alterations prior to molecular analysis, which eliminates the time consuming intermediate steps in mutational analysis, and thus allows more rapid and efficient tumor genotyping [ 63 ]. For example, the prostate gland is composed of epithelial and stromal cells. Prostatic carcinomas PCA often grow in an infiltrative pattern, with individual tumor acini infiltrating through stroma and directly adjacent to benign prostatic epithelium.
Lutchman et al analyzed dermatin, a cytoskeleton protein encoded by a gene on chromosome 8p21 [ 64 ]. Rubin et al studied loss of heterozygosity at 10q23, a region that has been associated with many tumors including glioblastoma multiforme, melanoma, endometrial carcinoma, and PCA [ 65 , 68 ].
Since pure tumor samples are very difficult to isolate, prostate tumor cell lines were initially used for gene expression assay. Bubendorf et al were one of the first researchers who investigated amplification of androgen receptor gene with FISH on tissue microarrays of PCA. They found that both Myc and androgen receptor genes were over expressed in hormone refractory PCA with respect to clinically localized hormone-sensitive PCA. They also found that syndecan-1 was prognostically significant in PCA [ 69 ]. LCM can also aid in the diagnosis of many dermatological diseases.
Routine diagnosis of cutaneous B- or T-cell lymphomas is challenging. Yazdi et al have introduced a LCM-based clonality assay to overcome these diagnostic dilemmas [ 66 ]. Using this technique, the authors were able to determine whether clonal T-cell receptor TCR gene rearrangement obtained by PCR stems from lymphoma or some inflammatory skin diseases [ 66 ].
The so-called intermediate endpoint biomarker IEB can serve as a reliable surrogate at the target site to monitor success of chemoprevention of cancer. Selection of an IEB requires that there is a consistent association between the biomarker and cancer and that biomarker modulation should be observed after chemotherapeutic interventions.
These biomarkers can be used to select high risk patients for cancer development, and for evaluation of the efficacy of novel chemopreventive agents, which can greatly decrease the cost of clinical trials. In a similar study, Mao et al have observed that years after active smoking cessation genetic changes similar to those in lung cancers can persist in histologically normal or mildly abnormal bronchial epithelium [ 71 ].
These data suggest that there exists a definite time lag between phenotypic and genotypic response in epithelial tissues. LCM can be applied for biomarker discovery in multiple human tissue types and organ systems.
LCM in combination with DNA transcriptome profiling has been successfully used to identify differentially expressed genes between the supragranular and infragraunlar cellular layers of human neocortex [ 72 ]. Demuth et al used LCM in brain biomarker research. In a so-called 3D spheroid in vitro invasion assay, they have evaluated the transcriptome of invasive glioma cells and their stationary equivalents [ 73 ].
LCM and whole genome expression microarrays, coupled with quantitative RT-PCR revealed that the activity of MAPK3, a key activator of p38 in glioma, and p38 were strongly correlated in their study set. With the help of LCM, Buckanovich et al isolated RNA from frozen or formalin-fixed paraffin-embedded breast tissue sections and identified a set of 12 novel ovarian cancer biomarkers termed as tumor vascular markers, which were distinct in comparison with vascular markers from either normal ovary or other tumor types [ 2 , 74 ].
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Other areas where LCM has also been used include evaluation of tumor microenvironment, forensic analysis of fixed cell samples and hair follicles, studies in developmental biology and embryology, animal model xenografting, infectious disease biology, plant cell biology, spermatogenesis [ 1 , 2 ]. The profound impact of molecular profiling on biomedical research and disease management is already taking place.
Biomedical research community at no previous time has been more poised for rapid discovery and application of discoveries toward improved patient care. Consequently, it is of the utmost importance that efforts in molecular profiling be maximized for accuracy and relevance. For decades many questions in biomedical research have been waiting to be answered. The use of microdissection methods, optimal tissue fixation and database integration for molecular profiling will yield many answers to those questions.
Several challenges in these areas must be addressed. Protocols that maximize biomolecule preservation without sacrificing histopathology, and rescue biomolecules from cross-linking must be developed and improved. The benefits of rapid and precise microdissection, such as those provided by LCM, must be actualized not only through research, but also in a diagnostic capacity.
Besides microdissection, methods must be developed that provide high-throughout analysis of tissue without sacrificing information regarding spatial relationships. The next few years hold great promise for the use of molecular information in disease management, including design of optimal lower risk, patient tailored treatment. National Center for Biotechnology Information , U. Int J Clin Exp Pathol. Barbara Domazet , 1 Gregory T. Author information Article notes Copyright and License information Disclaimer. Describe the connection issue. SearchWorks Catalog Stanford Libraries.
Laser capture microscopy and microdissection. Responsibility edited by P. Imprint Amsterdam ; Boston: Physical description xxxv, p. Series Methods in enzymology v. Marine Biology Library Miller. Find it at other libraries via WorldCat Limited preview. Bibliography Includes bibliographical references and indexes. Contents Comparison of Current Equipment. Internal Standards for LMD.