Introduction
Introduction
Completion of the human genome sequencing initiative revealed the genetic diversity inherent in individuals1. This inherent genetic diversity translates to functional genomic and proteomic diversity, thus creating the need for assays capable of monitoring the genome, transcriptome and proteome in a highly parallel fashion. The most visible example is in clinical oncology. Such assays, collectively termed molecular profiling, are yielding the genomic and proteomic information necessary for designing individual treatment regimens. Historically, morphological analysis of cells and tissue (for example, breast ductal carcinoma in situ) was the basis for defining disease diagnosis and prognosis2, 3. Clinical experience has shown that some tumors have different prognoses based on histology, whereas in other cases similar-looking tumors may have different clinical outcomes. Moreover, when the same standard therapies are administered to a population of cancer patients, only a minority will respond, while others may suffer toxicity without disease benefit. Consequently, the goal of molecular profiling is to provide a rational molecular basis for assessing prognosis and therapy4.
Tumor microenvironment
A common problem encountered by genomic and proteomic researchers in the analysis of tissue arise from the heterogeneous nature of the tissue. Molecular profiling of a pure cell population, which is reflective of the cell population's in vivo genomic and proteomic state, is essential for correlating molecular signatures in diseased and disease-free cells5, 6, 7. Direct microscopic visualization of the cells permits the selection of normal, premalignant and malignant cells, or disease and disease-free cells, as distinct cell populations from the heterogeneous tissue. Heterogeneous tissue may confound molecular analysis because it is currently impossible to discern which cells contribute which cellular constituents to a given tissue lysate. Imagine a tissue section comprised of 80% tumor, 10% stroma and 10% infiltrating lymphocytes. In this example the 10% lymphocyte population may contribute more than 10% of the overall signal. A twofold signal difference is considered significant for gene arrays. There may be greater than twofold differences in cell numbers between different tissue sections from the same tumor, making comparisons of molecular profiles invalid for heterogeneous, non-microdissected samples8, 9. Tumor-stromal interactions are now regarded as important communications for tumor development6, 10, 11, 12, 13, embryonic development14, 15 and wound healing16. LCM enables researchers to isolate specific cells of interest, without contamination from surrounding cells17, 18, 19, 20, 21, 22.
Laser microdissection systems
LCM is a technique for isolating highly pure cell populations from a heterogeneous tissue section, cytological preparation, or live cell culture via direct visualization of the cells21, 22.
There are two general classes of laser-capture microdissection: infrared (IR) capture systems21, 22 and ultraviolet (UV) cutting systems23, 24, 25, 26, 27. The principle components of laser microdissection technology are (i) visualization of the cells of interest via microscopy, (ii) transfer of laser energy to a thermolabile polymer with formation of a polymer-cell composite (IR system) or photo volatilization of cells surrounding a selected area (UV system), and (iii) removal of the cells of interest from the heterogeneous tissue section. LCM is compatible with a variety of tissue types, cellular staining methods and tissue preservation protocols that allow microdissection of fresh or archival specimens. Selective procurement of cells can be obtained with precision of 3–5 
m21, 22.
Although a variety of laser microdissection instruments exist, the term 'laser-capture microdissection' is the industry standard terminology regardless of laser method or type.
The original version of LCM incorporates an inverted light microscope and a near-IR laser to facilitate the procurement of desired cells (this version of LCM was commercialized by Arcturus Molecular Devices (www.moleculardevices.com)) (Fig. 1). After direct visualization of the cells of interest, the operator uses laser pulses to activate a thermoplastic polymer film that expands and surrounds the cells of interest. This polymer-cell composite can be lifted from the slide, effectively microdissecting the cells of interest from the heterogeneous tissue section (Fig. 2). The exact cellular morphology, as well as the DNA, RNA and proteins of the procured cells, remain intact and bound to the polymer. Using LCM, both frozen and fixed tissues can be successfully dissected and recovered cells can be used for DNA, RNA and protein analysis.
Figure 1: PixCell IIe Laser Capture Microdissection instrument.
(a) Infrared laser and inverted light microscope. (b) Laser control tower. (c) PC monitor with live video display. (d) Video monitor. (e) Video camera. (f) Computer.
Full size image (63 KB)Figure 2: Principles of laser-capture microdissection.
(a) A thermolabile polymer is placed on a tissue section on a glass slide. An infrared laser melts the polymer in the vicinity of the laser pulse. The resulting polymer-cell composite is removed from the tissue. (b) Properly melted polymer spots have a dark outer ring and a clear center, indicating that the polymer has melted and is in direct contact with the slide. Only cells lying within the diameter of the black melted polymer will be targeted for microdissection with each laser pulse. Poor spots have a fuzzy appearance, lacking a distinct black ring. (c) Physical forces involved in LCM include an upward adhesive force between the substratum and the tissue, lateral forces between the cells, and a downward adhesive force between the polymer and the cells. (d) A single cell is bound to the thermolabile polymer following microdissection with the infrared laser-capture technique. Photo courtesy of Arcturus Biosciences.
Full size image (38 KB)LCM technology
Laser-capture microdissection instruments (IR and IR/UV systems), as developed at the US National Institutes of Health, exist in manual and automated (robotic) platforms (Arcturus Molecular Devices, Inc.)21, 22. The manual system, PixCell, and the automated system, Veritas, use identical capture principles for microdissection, and the Veritas system has an additional UV laser cutting feature (Fig. 3).
Figure 3: Schematic of UV laser microdissection.
(a) Tissue is mounted on a polyethylene napthalate (PEN) or polyethylene tetraphthalate (PET) membrane. A UV laser can be used to cut away cells of interest or to ablate unwanted tissue, leaving cells of interest intact on the substratum. (b) Veritas UV cutting tools allow defined circular cutting areas or freeform polygon areas to be microdissected.
Full size image (46 KB)A stationary near-IR laser mounted in the optical axis of the microscope stage is used for melting, or wetting, a thermolabile polymer film. The polymer film is manufactured on the bottom surface of an optical-quality plastic support cap. The cap acts as an optic for focusing the laser in the same plane as the tissue section. The polymer melts only in the vicinity of the laser pulse, forming a polymer-cell composite. A dye incorporated into the polymer serves two purposes: (i) it absorbs laser energy, preventing damage to the cellular constituents, and (ii) it aids in visualizing areas of melted polymer. The short laser pulse durations used, the low laser power levels required (the near-IR laser diode has a maximum laser output of 100 mW), the absorption of the laser pulse by the dye-impregnated polymer and the long elapsed time (0.2 s) between laser pulses combine to prevent any significant amount of heat deposition at the tissue surface that might affect later laboratory analysis. Removal of the polymer from the tissue surface shears the embedded cells of interest away from the heterogeneous tissue section. Extraction buffer applied to the polymer film solubilizes the cells, liberating the molecules of interest.
Infrared LCM platforms are available as manual systems (Arcturus PixCell (www.moleculardevices.com) or Bio-Rad Clonis for live-cell microdissection (http://microscopy.bio-rad.com/principles.htm)), as an automated system (Arcturus AutoPix), as a combined IR/UV system (Arcturus Veritas) or as UV-only systems (Zeiss P.A.L.M. Microbeam, Leica LMD6000 and Molecular Machines & Industries mmi CellCut). Variations of UV cutting systems include UV laser microdissection and catapulting (P.A.L.M. Microlaser Technologies GmbH, http://www.zeiss.com)25, 26, 27, UV laser cutting (Leica LMD6000, Leica Microsystems, http://www.leica-microsystems.com)23 and the mmi Cellcut (MMI AG, www.molecular-machines.com)28, 29. Rather than simply cutting out the cells of interest, any of the UV laser systems may be used to ablate unwanted tissue, leaving the cells of interest intact. The mmi Cellcut system employs a 'touchless' microdissection system in which a UV laser is used to cut tissue from a polyethylene tetraphthalate (PET) membrane slide that is protected from contact with the environment during microdissection. The cut area is captured by placing an adhesive lid (of a microcentrifuge tube) onto the cut area, removing the selected cells from the tissue. The UV cutting systems (Veritas, PALM, Leica and MMI) are particularly useful for microdissection of tissue sections up to 200 m thick, such as plant tissue sections30. A potential limitation of the UV laser systems is the presence of cells with UV-induced damage in the final cell population obtained. These UV-damaged cells are derived from the cells lying directly under the UV laser cutting path. These cells may contribute significantly to the final molecular signal if the number of cells in the perimeter of the cut area is high (>10%) compared to the overall microdissected area.
Expected yield of DNA, RNA and protein
Factors affecting the recovery of DNA, RNA and proteins from microdissected cells include quality of sample, time to preservation before microdissection, type of preservation, fixation method and efficiency of microdissection (Fig. 4). Fixation is the most critical step to ensure high-quality yield of DNA, RNA and protein. Quality of fixation is dependent on the length of time for fixative penetration in the tissue, temperature of fixation and tissue size. The longer the fixative takes to penetrate the tissue, the greater the chance of RNA or protein degradation due to ubiquitous RNases and proteases. Formalin is a fixative of choice in histology labs because of its low cost and rapid, complete penetration of tissue31. The caveat with regard to formalin fixation for molecular analysis is the formation of extensive cross-links between formalin and proteins. Disruption and denaturation of the cross-links produces peptides and protein fragments rather than intact proteins. Although new technologies are being developed to reverse the cross-linking for extraction of sufficient quantity of nucleic acids32 and proteins, high-quality yield of RNA and proteins is routinely best achieved with frozen or ethanol-fixed tissue. The concept and practice of using reversible cross-linkers has been suggested as an alternative to formalin fixation31. Reversible cross-linkers, such as dithiobis(succinimidyl) propionate (DSP), have been applied successfully to LCM with downstream RNA amplification and cDNA synthesis31.
Figure 4: Tissue stability and processing.
Pre-analytical variables such as tissue acquisition delay times and processing delay times greatly influence the quality and yield of cellular molecules, independently of the microdissection process.
Full size image (43 KB)In general, one set of microdissected cells is used for downstream analysis of only one type of molecule. This is due to the need for different solubilization schemes, extraction buffers and denaturing temperatures for each type of molecule. For example, a population of 10,000 microdissected cells could be solubilized in a denaturing buffer at 70 °C for downstream protein analysis, while a second set of 100 cells could be treated with proteinase K at 65 °C for downstream DNA analysis. Examples of cellular yield required for DNA, RNA and protein analyses are listed in Table 1. A typical cell contains approximately 0.01 ng RNA per cell, and therefore if microdissection, extraction and isolation of RNA were 100% efficient, one would need to microdissect approximately 2,000 cells to obtain 20 ng poly(A) RNA. As can be seen from Table 1, it is often necessary to microdissect many more cells than one would calculate to be necessary based solely on the DNA, RNA or protein content of a cell.
Diversity of applications
LCM is applicable to molecular profiling of tissue, permitting correlation of cellular molecular signatures with specific cell populations, as well as comparison of cellular elements within the tissue microenvironment. Applications of laser microdissection include evaluation of tumor microenvironment interactions5, 6, 33, 34, real-time polymerase chain reaction (RT-PCR)35, proteomic and genomic molecular profiling17, 19, 22, 36, 37, 38, 39, 40, 41, forensic analysis of mixed cell samples and hair follicles39, 42, 43, and studies in developmental biology and embryology15, 44, animal models and xenografts11, infectious disease biology45, 46 and plant and cell biology30, 47. Laser catapulting microdissection has been used recently for genetic imprinting and DNA methylation studies of spermatogenesis48. DNA, RNA or protein analyses may be performed with the microdissected tissue by any method with adequate sensitivity. For example, DNA extracted from cells procured via this technique can be used for LOH analysis49, 50, 51, 52, 53, DNA methylation assays48, production of cDNA libraries54, 55, gene expression assays15, 56, 57, real-time RT-PCR and quantitative PCR (QPCR)41, 58, 59, while protein extracted from microdissected cells can be applied to reverse-phase protein microarrays11, 17, 19, 20, 37, 38, 60, 61, 2D gel electrophoresis5, 62, 63, western blotting62, 63, 64 and mass spectrometry65, 66, 67 (Fig. 5).
Figure 5: Example applications of LCM technology.
Microdissected cell populations have been applied to transcript profiling, protein profiling and protein discovery.
Full size image (41 KB)Immuno-LCM
In some cases tumor vascular cells, infiltrating hematopoietic cells and surrounding normal cells have the potential to contaminate microdissected cellular lysates, making interpretation of results difficult29, 56. Immuno-LCM, which involves immunohistochemical staining of tissue before LCM, enhances the ability to identify cells of interest in complex tissues by combining detection strategies based on morphology and immunophenotype criteria29, 56, 68, 69, 70. Immuno-LCM enables the selection of immunophenotypically different yet morphologically similar cells, such as B and T cells, from tissue lacking easily identifiable architectural details. For example, antibodies specific for secretory proteins or proliferation receptors are used for immunohistochemical staining of a functional class of cells. Selection of cells based on functional phenotype enhances the ability to study the significance of genetic alterations in specific function based cell populations68. Prelabeling of cells in vivo in an animal model, by injection of a fluoro-gold label, has been used to label cells while avoiding RNA degradation due to immunohistochemical staining59. A limitation of immuno-LCM is the inability to study protein expression in cells procured with this method due to the binding of the antibody to the protein of interest. Lectin probes are an alternative to immunolabeling. Lectins show promise as markers for brain microvessels in lectin-guided LCM with downstream RNA analysis71, 72. Haqqani et al. used lectin probes for guided LCM of brain microvessels, with downstream isotope-coded affinity tagging (ICAT)-nanoLC-MS/MS65. A limitation of lectin probes is their low affinity and nonspecific binding. Lectins recognize general classes of carbohydrates rather than specific molecules, resulting in potential cross-reaction with a variety of cell types71. Optimal immuno-LCM procedures, in which RNA is not degraded during the staining process, capitalize on the use of high-affinity antibodies, at high concentrations, with short overall staining times56, 68, 70.
Limitations
The major limitation of LCM is the need to identify the cells of interest based on morphological characteristics. Cell identification is usually performed in conjunction with a pathologist, cytologist or technologist trained in cell identification. Further limitations or sources of error are a result of the perishability of tissue molecules after surgical procurement, the choice of tissue staining protocols and the compatibility of tissue fixation techniques with downstream analysis (Fig. 4). Selection of tissue staining protocols should be based on compatibility with alcohol dehydration gradients and with the downstream analysis to be performed with the microdissected tissue. Extensive cross-linking of nucleic acids and proteins in formalin-fixed tissue limits the use of archived formalin-fixed, paraffin-embedded tissue sections for RNA and protein analyses, although some newer RNA and protein extraction chemistries are showing promise for use with formalin-fixed tissue32, 73.
Using the polymer-capture LCM technology21, effective microdissection is a balance between three adhesive forces: (i) maximizing downward adhesive forces between the polymer and the tissue, (ii) minimizing lateral adhesive forces between the cells and (iii) minimizing upward adhesive forces between the slide and the tissue. Minimization of the upward adhesive force is achieved through adequate dehydration of the tissue section during the staining protocol.
Optimal laser-capture microdissection (IR systems) is achieved with tissue sections cut at a thickness of 2–15 m. Tissue sections thinner than 5 m may not provide a full cell thickness, necessitating microdissection of more cells for a given assay. Tissue sections thicker than 15 m may not microdissect completely, leaving integral cellular components adhering to the slide.
Microdissection is performed without coverslips or immersion oils. This results in a tissue section that lacks refractive index–matched image qualities such as color. Depending on the tissue architecture, it may be difficult to distinguish the cells of interest without comparison to index-matched images of the same tissue. The Veritas system addresses this limitation by permitting annotation of the cells of interest from the live video image.
The inverted light microscope in the PixCell platform uses a vacuum to immobilize the slide on the microscope stage. The size of the objective opening limits the usable area on the microscope slide to the middle third of the slide, and therefore tissue sections must be placed in this area of the slide (Fig. 6). Tissue placed at the extreme edges of the slide will not be within the usable area. The Veritas is designed for use with both IR and UV lasers, and thus the usable area of the slide is limited by the area of the membrane-style slides, and is approximately 15 
49 mm.
Figure 6: Tissue placement on slide for effective microdissection.
(a) Optimal placement of tissue is within the middle third of the slide. (b) Incorrect placement of the tissue section near the edges or the end of the slide is a commonly encountered problem.
Full size image (36 KB)Alternative methods
Flow cytometry and fluorescence-activated cell sorting (FACS) is another cell separation method in which cell populations are sorted based on immunolabeling. Flow cytometry is well suited for suspension cells such as circulating hematopoietic cells, but it does not lend itself to analysis of intact tissue cells. Disruption of aggregated living cells profoundly influences gene and protein expression patterns, confounding interpretation of the results12. Thus LCM is the only present technology that can isolate subpopulations of cells from tissues or cell-population monolayers without perturbing the molecular state of the cells.
Future advances
Further technological advances such as touch-screen cell annotation, automated cell microdissection and cell recognition software are leading to the next generation of microdissection capabilities. A variety of procurement methods for pure cell populations are currently available, such as laser cutting (MMI, Leica), laser catapulting (PALM), and scanning laser microdissection (XMD)74. We can envision a future in which healthcare is provided via telemedicine and in which a pathologist reviews and annotates a tissue image, indicating the cells of interest for microdissection. Microdissection could be performed remotely in another location, resulting in seamless integration of cell identification and microdissection.
Artificial intelligence programs for cell recognition could operate in parallel with LCM providing automated cell recognition features. The advent of improved tissue stability and preservation methods will enhance our ability to microdissect and analyze molecular profiles from previously unusable tissue samples.
Development of designer ligands for specific molecules may one day overcome the limitations encountered with antibody or lectin probes. These designer ligands could be aptamers, synthetic ligands, single-chain antibodies or antibodies from different species.
Experimental design
The protocols described below illustrate (i) frozen section sample preparation, (ii) hematoxylin and eosin tissue staining, (iii) manual laser-capture microdissection and (iv) automated laser-capture microdissection. Alternative methods of tissue preparation, such as ethanol or formalin fixation with paraffin embedding, are acceptable for RNA and DNA analysis respectively75.
In our experience, it is best to store the block of tissue rather than storing the cut tissue sections. We have had successful protein recovery from frozen rhabdomyosarcoma blocks up to 12 years old when stored at - 80 °C or in the vapor phase of liquid nitrogen. As a general guide, processed and cut formalin-fixed, paraffin-embedded tissue sections for DNA analysis can be stored at room temperature indefinitely; ethanol-fixed sections for RNA or protein analysis can be stored at room temperature for 3 months; frozen sections for RNA can be stored at - 80 °C for 1 month; and frozen sections for protein analysis can be stored at - 80 °C for up to 3 months.
Examples of LCM-compatible stains are hematoxylin and eosin, methylene blue, Wright-Giemsa and toluidine blue. Eosin staining is not necessary for visualization of cells during microdissection. Minimal staining times, in whichever staining protocol is used, limit potential protein alterations due to contact with the staining reagents. Selection of tissue staining protocols should be based on compatibility with the downstream analysis to be performed with the microdissected tissue. Fluorescent stains are compatible with fluorescence-equipped systems and may be used for immuno-LCM. PixCell instruments equipped with fluorescent modules incorporate mercury vapor lamps with blue (455–495 nm excitation, 510 nm emission), green (503–547 nm excitation, 565 nm emission) and red filter cubes (590–650 nm excitation, 667 nm emission). Additional filter cube positions are available for end-user modifications.
Cover slips and mounting media are not compatible with microdissection. In addition, a lack of immersion fluids on any of the optics prevents refraction of light from the tissue image. Thus, the color and detail of a given tissue stain is lost as the stained slide dries (Fig. 3b). Manual LCM methods capitalize on the index of refraction of a wet tissue slide for visualizing and reviewing an index-matched image of the tissue76, 77. An index-matched image or images may be digitally saved and used as a guide, or map, to locate the cells of interest.
The Veritas system combines robotics and optical scanning software for automated microdissection of selected cells. It also incorporates imaging software enabling annotation of cells for microdissection directly from the live video, permitting more accurate identification of cellular morphology during cell selection. Additionally, a series of individual stitched images may be acquired. The image resolution is constant, but the area of the images changes with magnification, permitting precise areas of tissue to be annotated for microdissection.
Annotation software coupled with the refractive index–matched image permits single-point dissection, line dissection or polygon dissection. Algorithm-based cell image recognition software is based on texture, morphology, size, color and contrast of the tissue, permitting automated cell selection in addition to automated microdissection78.
The Veritas visualization system does not include oculars, due to the enclosed system configuration. Instead a PAL-format color camera permits visualization of the slide as a 'roadmap image' for determining the target area of microdissection. The enhancements of the automated system include multiple slide capacity (three slides), area quantitation of microdissected tissue, wetted polymer spot measurement and cell recognition software.
Materials
Reagents
- Specimens for protein analysis: cytospin preps, or ethanol-fixed or frozen tissue sections cut at 2–15 m.
- Specimens for DNA analysis: cytospin preps, frozen tissue sections, ethanol-fixed sections or formalin-fixed, paraffin-embedded tissue sections cut at 2–15 m.
- Specimens for RNA analysis: ethanol-fixed or frozen tissue sections cut at 2–15 m
Caution Observe universal precautions for all human and animal blood or tissue samples. Use appropriate personal protective attire (latex gloves, lab coat, safety glasses, etc.). Handle all biological material as potential biohazard and dispose all biohazardous materials in appropriate container. - Cryopreservation solution (OCT; Sakura Finetek Corp., cat. no. 4583).
- Cryomolds (Sakura Finetek Corp., cat. no. 4728).
- Dry ice
Caution Avoid direct skin contact with dry ice. Vapors can cause asphyxiation. Use with appropriate ventilation. - Mayer's hematoxylin solution (Sigma, cat. no. MHS128)
Caution Hematoxylin is a contact hazard. Wear gloves when handling. - Eosin Y solution, alcoholic (Sigma, cat. no. HT110116)
Caution Eosin Y is flammable and a contact hazard; wear gloves when handling.
Critical Dilute eosin Y 1:1 with 100% ethanol to prevent over-staining of cytoplasmic proteins with eosin. - Scott's Tap Water Substitute Blueing Solution (magnesium sulfate buffered with sodium bicarbonate, ready to use, do not dilute; Fisher, cat. no. CS410-4D)
Critical Mayer's hematoxylin is a mordant dye containing several cationic species that impart a reddish color at low pH. With mild alkaline treatment such as that provided by Scott's Tap Water Substitute, these complexes produce a blue color. Lack of blueing solution may impart unusual staining characteristics, making morphological examination of the cells difficult. - 100% ethanol (ethyl alcohol, absolute, 200 proof for molecular biology; Sigma, cat. no. E7023).
- 70% ethanol (vol/vol) and 95% (vol/vol) in Milli-Q-filter (Millipore)-purified H2O
Critical Prepare fresh ethanol solutions weekly, or more frequently if staining more than 20 slides or if the ambient humidity is >40%. Store solutions at 4 °C up to 1 week when not in use.
Caution Ethanol is flammable and a contact hazard; wear gloves when handling. Do not ingest. - Milli-Q-purified water (Type I reagent-grade water).
- Xylene (Mallinckrodt Baker)
Caution Xylene vapor is harmful or fatal; use with appropriate ventilation and discard in appropriate hazardous waste container. Xylene is flammable and a contact hazard; wear gloves when handling.
Critical The use of xylene substitutes may reduce the efficiency of microdissection for some tissue types. - Protease inhibitors: Complete Protease Inhibitor Cocktail Tablets (Roche, cat. no. 11697498001)
Critical Add protease inhibitors to the 70% ethanol, water, hematoxylin and Scott's Tap Water Substitute staining solutions to limit protein degradation. Complete protease inhibitor tablets are soluble in aqueous solutions. Dissolve the tablets in Milli-Q-purified water. Use this solution to prepare the 70% ethanol. - Protein extraction buffer: 1.0 ml T-PER Tissue Protein extraction reagent (Pierce, cat. no. 78510), 950 l Tris-glycine 2 SDS loading buffer (Invitrogen, cat. no. LC2676 Novex) and 50 l 2-mercaptoethanol (Sigma, cat. no. M3148) (the final extraction buffer is a 2.5% solution of 2-mercaptoethanol in T-PER/Tris-glycine 2 SDS buffer).
- RNA extraction buffer (PicoPure RNA extraction kit, Arcturus Molecular Devices, cat. no. KIT0204).
- DNA extraction kit (PicoPure DNA extraction kit, Arcturus Molecular Devices, cat. no. KIT0103).
- RNase inhibitors (2 units l-
1 Protector RNase inhibitor; Roche, cat. no. 03335399001).
- 3-Amino-9-ethylcarbazole (AEC) chromagen (Dako, cat. no. K3464).
- Acetone (Fisher, cat. no. A16P-4), 20 °C.
- Primary antibody of choice.
- Biotinylated secondary antibody of choice (e.g., anti-rabbit, Vector, cat. no. BA-1000, or anti-mouse, Vector, cat. no. BA-9200).
- Streptavidin-biotin amplification (LSAB kit; Dako, cat. no. K1016).
- Glycerol, 99+% purity (Sigma, cat. no. G-5516).
Equipment
- Laser-capture microdissection apparatus: PixCell II, PixCell IIe, AutoPix or Veritas Laser Capture Microdissection system (Arcturus Molecular Devices)
- Cryostat and/or microtome
- - 80 °C freezer
- Uncoated, precleaned glass microscope slides, 25 75 mm (Fisher, cat. no. 12-550-14) or polyethylene naphthalate (PEN) membrane glass slides (Arcturus Molecular Devices, cat. no. LCM0522)
Critical Use glass slides for capture (IR laser) and membrane slide for cutting (UV laser) - CapSure Macro LCM Caps (Arcturus Molecular Devices, cat. no. LCM0211)
- CapSure HS LCM Caps for RNA downstream analysis (Arcturus Molecular Devices, cat. no. LCM0214)
Critical CapSure HS caps are often used in microdissection of tissue for RNA analysis. A 12-
m rail on the surface of the polymer prevents the polymer from touching the tissue except in the vicinity of the laser pulse. The HS caps are designed with an extraction device, allowing extraction buffer to contact the polymer within a centrally designated area. These features limit any potential RNA contamination from surrounding cells. CapSure HS caps may be used successfully for DNA, RNA or protein extraction. By contrast, CapSure Macro caps are placed in direct contact with the tissue and are not equipped with an extraction device. Any cellular material on the surface of the polymer of a Macro cap will be available for extraction. - 500-l microcentrifuge tubes: Safe-Lock Eppendorf tubes (Brinkmann Instruments, cat. no. 22 36 361-1) or MicroAmp 500 l Thin-walled PCR Reaction Tubes (Applied Biosystems, cat. no. 9N801-0611)
Critical Use of these microcentrifuge tubes is recommended to prevent buffer leakage during extraction (Fig. 7).
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Procedure
Overview
- Points from here (point 1) up to and including point 5 are related to
Frozen tissue sectioningEmbed tissue directly in a cryopreservative solution (OCT) or liquid nitrogen as soon as the specimen is procured. Place OCT-embedded tissue on dry ice and store at -
80 °C.
Critical step Prompt preservation of the sample limits RNA and protein degradation due to nuclease and protease activity (Fig. 4). - Cut frozen sections at 2–15 m thickness (5–8 m is optimal for IR laser capture) on labeled, plain, uncharged, precleaned glass microscope slides. Alternatively, place sections on PEN membrane slides for UV laser cutting. Lung tissue, or other tissue with a thin, open architecture, may be cut on charged or silanized slides to prevent the tissue from nonspecifically adhering to the polymer during microdissection. In general, coated slides are not used for microdissection due to the increased adhesive forces between the tissue and the slide (Fig. 2c).
Critical step Position the tissue section near the center of the slide, avoiding the top and bottom thirds of the slide (Fig. 6). Do not allow the tissue section to dry on the slide at room temperature.Troubleshooting - Place the slide directly on dry ice or keep the slide in the cryostat at - 20 °C or colder until the slides can be stored at - 80 °C or immediately stained and microdissected. Paraffin-embedded sections should be cut and placed on the slide in the same position as described in Step 2. They can be stored at room temperature before microdissection.Pause Point
- The staining procedure is different depending on whether tissue is frozen or paraffin embedded. Use option (A) (or option (B) if additional modifications are needed) for frozen sections, and option (C) for paraffin-embedded sections.
- H&E staining for frozen tissue sections
- Remove slide from freezer and place on dry ice or directly into the 70% ethanol fixative solution.
- Dip the slide, for the time indicated, in each solution as listed in Box 1. Blot the slide on absorbent paper in between the different solutions, to prevent carryover from the previous solution.
Critical step Complete dehydration of the tissue is necessary for minimizing the upward adhesive forces between the tissue section and the slide. Increasing the incubation time to 2 min for the 100% ethanol and xylene rinses (Steps 10–13) may enhance dehydration, increasing the efficiency of microdissection. Skin tissue, cartilage and samples prepared on charged slides may exhibit reduced microdissection efficiency.Pause Point If absolutely necessary, the slide may be left in xylene for a maximum of 5 min before proceeding with LCM.
- Modified H&E staining for skin tissue or other tissues with strong intracellular adhesion
- Remove slide from freezer and place on dry ice or directly into the first solution.
- Carry out a staining procedure much as for option (A) but including any additional slide or tissue treatments that may be required, such as incorporation of glycerol slide coatings or modified staining protocols with glycerol; one such approach for frozen tissue sections is delineated here, as adapted from ref. 79. Dip the slide, for the time indicated, in each solution as listed in Box 2. Blot the slide on absorbent paper in between the different solutions, to prevent carryover from the previous solution.
- H&E staining for formalin-fixed, paraffin-embedded tissue sections
- Dip the slide, for the time indicated, in each solution as listed in Box 3. This procedure is necessary because paraffin-embedded tissue sections must be de-paraffinized before staining and rehydrated to allow staining of the tissue elements.
Critical step Complete deparaffinization is essential for DNA and RNA extraction (Steps 1–2). - Allow the stained slide to air dry. Dust or debris on the slide may be removed by blotting the dried slide with a PrepStrip sample preparation strip (Arcturus Molecular Devices).
Critical step Xylene dissolves the LCM cap polymer. It is imperative that the slide be completely dry before cap placement for microdissection. When dry, the slide will appear as a grayscale (non–refractive index–matched) image.Pause Point If absolutely necessary, the slide may be left in xylene for a maximum of 5 min before proceeding with LCM.
- Dip the slide, for the time indicated, in each solution as listed in Box 3. This procedure is necessary because paraffin-embedded tissue sections must be de-paraffinized before staining and rehydrated to allow staining of the tissue elements.
- H&E staining for frozen tissue sections
- Perform microdissection with either the manual PixCell System (A), the automated Veritas System (B) or the Veritas UV Cutting Laser (C).
- Manual laser-capture microdissection (PixCell System)
- Turn on power for microscope and laser control box. There is no warm-up period required for the PixCell II/IIe (Fig. 1).
- Load the CapSure cap holder assembly with LCM caps: remove the CapSure cap holder assembly from the LCM instrument platform and press in the locking pins on each end to hold the cassette in the load position. Slide a CapSure cartridge onto the cassette until it stops. Two cartridges may be loaded onto the cassette module.
- After the cartridges are loaded, pull the locking pins out to lock the cartridges in place. Slide the cap holder assembly onto the PixCell II/IIe.
- Access the LCM software program by double clicking on the Arcturus software icon.
- Enter your user name or select a name from the list. Click on "Acquire data."
- Enter a study name or select a study name from the list. Click on "Select."
- Enter the Slide # and Cap Lot #. If desired, notes concerning the slide or study may be entered as "Notes."
- Click the checkbox for "Stamp images with name, date & time" if this information is to be imprinted on the images created during LCM. Click "Continue."
- Move the joystick into the vertical position to ensure proper positioning of the cap in relation to the capture zone.
Critical step - Place the microscope slide containing the prepared and stained specimen for microdissection on the stage.
- Locate the cells of interest using either the oculars or the monitor. After the target area for dissection is in the viewing area, with the joystick still in the vertical position, press the "Vacuum" switch on the front of the Controller to activate the vacuum and hold the slide in place during microdissection.
- The Live Video and PC screen displays the current image on the microscope. The images may be saved as you work by selecting the appropriate icon from the toolbar (see Box 4).
- Slide the CapSure cap holder assembly backward or forward so that a cap is sitting at the "Load" position. Swing the placement arm over the cap. Placing one hand over the counterbalance to prevent jarring of the cap and improper seating, lift the placement arm and place the cap onto the slide. This is the transfer position.
- Enable the laser by turning the key switch located on the front of the controller and then pressing the "LASER ENABLE" button. Pressing this button will activate the target beam when the placement arm is in the transfer position.
- Verify that the laser is in focus according to Box 5.
Critical step The laser should not be refocused when changing objectives or spot sizes. It is only necessary to focus the laser, with the small (7.5-m) spot size setting and the 10 objective, for each initial cap placement and anytime the cap is repositioned on the tissue. Microdissection may be performed with any suitable laser spot size and a 4 , 10 or 20 objective after the initial laser focus. - TroubleshootingPress the red pendant button to fire a test laser pulse. Observe the wetted polymer after the laser is fired. Firing the laser pulse causes the polymer to melt in the vicinity of the laser pulse. There should be a distinct clear circle surrounded by a dark ring (Fig. 2b).
Critical step The dark ring produced by pulsing the laser is caused by a combination of migration of the dye and changes in the thickness of the polymer wall at the site of the laser pulse, permitting visualization of the melted polymer. The black ring should be sharp in appearance with a clear center. This pattern indicates proper laser focusing, adequate laser operation and acceptable performance of the polymer. - Adjust the "Power" and "Duration" of the laser pulse with the up and down arrows on the front of the controller to obtain a melted polymer spot with a diameter similar in size to the selected laser spot size. Generally, increasing the Power (maximum 100 mW) will achieve better polymer melting and contact with the slide, while increasing the duration will yield spots with larger diameter. These settings can be adjusted to customize the melted polymer spot to the type and thickness of the tissue to be dissected. The suggested settings for different spot sizes for the PixCell II system with a Macro cap are: small spot (7.5 m): 45 mW, 750 s; medium spot (15 m): 35 mW, 1.5 ms; large spot (30 m): 25 mW, 5.0 ms.
- Single-cell microdissection is possible by adjusting the power and duration settings such that a very narrow area of the polymer is melted with each laser pulse. Select the 7.5-m spot size setting and manually adjust the laser power and duration. Spot sizes less than 7.5 m are achievable because the power and duration, and thus the melted polymer spot diameter, may be adjusted independently of the laser spot adjustment lever. Suggested settings for single-cell microdissection are power 45 mW and duration 650 s using Macro caps. Increase power if using HS caps.
- To perform microdissection, visually locate the cells of interest and align the target beam directly over the cells. Using the target beam to guide the dissection, press the pendant switch for single shots. For a rapid fire of pulses, press and hold the pendent switch. The laser pulse frequency interval can be adjusted on the controller by selecting "REPEAT" and then selecting the desired time between laser pulses. Rapid fire of pulses is beneficial if the tissue section contains large areas of relatively homogeneous cells, as may be seen with certain carcinomas (for example, rhabdomyosarcoma or ovarian carcinoma). The operator manipulates the stage and tissue in a continuous motion while the laser is activated, essentially forming a continuous line or area of melted polymer spots. This reduces operator fatigue and increases productivity of microdissection. When using the rapid-fire pulse mode, review the tissue morphology and histology before microdissection to ensure that the areas targeted for microdissection are truly homogeneous and do not contain significant populations of other cell types.
A drawback of the PixCell system (but not the Veritas) is the inability to microdissect directly from an index-matched image of the tissue. Map images may be saved while the slide is wet, providing a guide for microdissection73. Index-matched images may be obtained with either system by rewetting the tissue with a drop of xylene before microdissection. It is imperative that the slide be completely dry before cap placement for microdissection because xylene dissolves the polymer.
Critical step With frozen sections for protein analysis: limit total analysis time for staining and microdissection to less than 1 h. With frozen sections for RNA analysis: limit total analysis time for staining and microdissection to less than 30 min, if possible, to prevent RNA degradation.
Critical step A phenomenon termed 'polymer depletion' occurs when microdissecting large, polygon-shaped areas from the perimeter toward the center. As the laser melts the polymer downward onto the cells, the polymer is depleted on the edges of the laser fire area. As more and more polymer is melted onto the cells in a localized area, this depletion effect becomes more apparent. This can be prevented by microdissecting large, enclosed areas from the center of the area toward the perimeter. - After the desired number of cells have been collected on a cap, remove the cap by lifting it off the slide using the placement arm. Swing the placement arm away from the slide. Assuming an average epithelial cell diameter of 7 m and a laser spot size of 30 m, the operator can expect to collect, on average, 5–6 cells per laser pulse. Using this information, it is possible to estimate the number of cells captured based upon the number of laser pulses counted during microdissection (which is automatically counted on the toolbar on both the PixCell and Veritas systems) and the efficiency of microdissection.
Troubleshooting30- m laser spot sizeNumber of pulses 5 % efficiency = total cells captured15- m laser spot sizeNumber of pulses 3 % efficiency = total cells captured7.5- m laser spot sizeNumber of pulses 1 % efficiency = total cells captured - If desired, the microdissected material may be viewed by placing the cap on an area of the slide without tissue. Turn the vacuum off by depressing the "vacuum" button on the Controller. Position the slide so that the tissue is not in view on the monitor or through the oculars. Swing the placement arm, containing the cap, back onto the slide. Use the joystick to manipulate the cap above the objective. Observe the cap for microdissection of the desired cells and for debris and/or adhesion of nonspecific tissue to the polymer surface.
Critical step Estimate the percent efficiency of microdissection by observing the polymer for cellular material within the diameter of the melted laser spot. Efficiency of microdissection is a critical factor for estimating the number of cells procured by LCM.
Critical step If viewing the cap reveals debris or nonspecific tissue adhesion, this may be removed by blotting the polymer surface with a CapSure Cleanup pad or the tacky side of an adhesive note. Do not use 'super sticky'–style adhesive notes. - TroubleshootingLift and rotate the cap arm until the cap is over the "cap removal site." Lower the arm and then rotate the arm back toward the slide. The cap will remain at the cap removal site. Blot the cap as described in Step (xxi) if necessary.
- Insert the polymer end of the cap into the top of a 500-l microcentrifuge tube. The sample is now ready for extraction of the desired components, or alternatively the cap-tube assembly may be stored for extraction at a later date (Fig. 7).
Figure 7: Scheme for extraction of protein from multiple LCM caps.
Multiple LCM caps can be solubilized in 1 volume of denaturing extraction buffer, thus concentrating the amount of protein per buffer volume. Caps are sequentially placed on tubes, with the resulting lysate transferred to clean tubes for extraction with the next cap. Due to the expansion of the microcentrifuge tube during incubation at 70 °C, it is imperative to transfer the whole-cell lysate to a clean, room-temperature tube before extraction of the cells from the next cap.
Full size image (42 KB)
Critical step RNA should be extracted immediately after microdissection. Condensation in the microcentrifuge tube during storage may be a potential source of RNase contamination. Microdissected cells for DNA analysis may be stored desiccated at room temperature up to one week before extraction. Protein extraction should be performed just before the downstream analysis. - Images saved during microdissection may be saved on the LCM computer hard drive as described in Box 6.
- After all dissections are completed, put the PixCell II/IIe in shut-down mode by first pressing the "Laser Enable" button to disable the laser. Turn off the power to the PixCell II/IIe, the controller and the Video Monitor. An abbreviated procedure for the work bench is described in Box 7.
- Automated laser-capture microdissection (Veritas System)
- Turn power on. The Veritas requires a warm-up period of approximately 1 h if the instrument is turned off78.
- Click on Veritas icon. Enter user name and password.
- Load slide(s) and caps. Click OK on the Materials loading screen.
- Click and drag red camera box on the roadmap image to an area of interest.
- Focus live video image. Adjust light intensity.
- Acquire static images as follows: Click on red rectangle (region of interest) tool in the toolbar; click and drag on the roadmap image to draw a region of interest; then click on region of interest tool to close tool.
- Click and drag red camera box on the roadmap image to an area that will represent the center of the cap.
- Click and drag cap onto the slide after verification that the slide is dry.
- Focus laser. Adjust the target beam until the beam reaches the point of sharpest intensity and most concentrated light, with no rings or coronas. The laser should now be focused for any objective.
- Suggested laser settings are power 70 mW and pulse 2,500 s. Fire the laser by double clicking on the live video in any area under the cap lacking tissue. Check polymer wetting. Observe the wetted polymer after the laser is fired for adequate polymer melting (Fig. 2b).
- If the suggested settings above fail to provide an adequate melted polymer spot, these settings can be adjusted anytime after a cap is placed on the tissue section and before microdissection. The size of the wetted polymer may be adjusted by changing the power and duration of the laser. These settings can be adjusted to customize the melted polymer spot to the type and thickness of the tissue to be dissected. In general, increasing the power provides more energy to melt the polymer, pushing the polymer toward the tissue. Increasing the duration generally increases the diameter of the melted polymer.
- Measure the spot size diameter by clicking the mouse on one side of the wetted polymer spot and then clicking on the opposite side of the spot. The measured diameter is displayed in the spot size window.
- Annotate static images or the live video image for the cells of interest. The annotation tools are single point, line or polygon.
- Capture cells by right clicking on the static/live video image and selecting "capture selected cells." The capture process will continue until all annotated areas for that particular image have been captured.
- Click and drag the cap to QC station to view the captured cells embedded in the polymer. The cap may be returned to the original slide for capturing additional cells, to another slide or to the unload tray.
- Click and drag cap to unload tray.
- Click Instrument/Open instrument door.
- Remove cap. If viewing the cap reveals debris or nonspecific tissue adhesion, this material may be removed by gently blotting the polymer surface with the CapSure Cleanup pad or an adhesive note.
Critical step Prompt removal of the cap from the instrument and storage of the cap, or proceeding immediately with RNA extraction, prevents potential molecular degradation. - Insert the polymer end of the cap into the top of a 500-l microcentrifuge tube. The sample is now ready for extraction of the desired components, or alternatively the cap-tube assembly may be stored for extraction at a later date.
Critical step RNA should be extracted immediately after microdissection. Condensation in the microcentrifuge tube during storage may be a potential source of RNase contamination.Pause Point Microdissected cells for DNA analysis may be stored desiccated at room temperature up to 1 week before extraction. Protein extraction should be performed just before the downstream analysis. - Repeat Steps (vii)–(xix) for additional static images. To microdissect a new slide, click Start new session. Unload slides and all caps in the unload tray. Insert a new stained slide for microdissection and repeat Steps (iv)–(xix).
Critical step Remove all caps from the unload tray before starting a new session. The fork arm used to transport caps will crash, and possibly be damaged, if it encounters a cap in the unload tray as it attempts to deposit another cap from a previous session. - To exit and shut down, click File/Exit, and Save images if needed. Unload all caps in the unload tray, remove unused caps and remove slides.
- Do not turn the instrument power off. The instrument power may remain on for daily operation, eliminating the need for an initial warm-up period.
- Veritas UV Cutting Laser microdissection
- The Veritas requires a warm-up period of approximately 1 h if the instrument is turned off.
- Click on Veritas icon. Enter user name and password.
- Load slide(s) and caps. If using membrane slides, click on membrane slide button.
- Click OK on the Materials loading dialog box.
- Double click on the roadmap image to position the red camera box over an area of interest.
- Focus live video image.
- Click Tools/Cutting Laser to view the cutting laser dialog box.
- Adjust the UV laser power by selecting either low, medium or high power. Fine tune the laser with the slider bar in the cutting laser dialog box.
- Click "Pulse" to fire the UV laser.
- Set the spot size for the UV cutting laser. Click "spot" on the cutting laser toolbar and either enter a defined value or else perform a cut, use the ruler tool to measure the width of the cut, click "set spot" and enter the width measured with the ruler. Click "OK" to close the dialog box.
- For membrane slides, set the cut properties for each capture group. Click on a capture group. Click "enable tabs." Enter values for the minimum number of tabs, spacing and size. Click "OK."
- Click on the live video image.
- Select the "cut and capture" tab on the microdissection dialog box. The knife icon allows freeform outlines to be drawn for microdissection. The circle icon allows circular areas to be outlined for microdissection. The ablation tab allows areas to be marked for photo volatilization with the UV laser.
- Annotate the live video image with the cutting tool of choice.
- Right click on the live video and select "cut selected groups."
- A cap will be placed on the tissue after the UV laser cuts the desired area(s). Once the cap is in place, the near IR laser will tack the polymer to the cut tissue.
- Click and drag the cap to the QC station or to the unload tray.
- Click Instrument/Open door.
- Remove the cap. Insert the polymer end of the cap into the top of a 500-l microcentrifuge tube. The sample is now ready for extraction of the desired components, or alternatively the cap-tube assembly may be stored for extraction at a later date.
Critical step RNA should be extracted immediately after microdissection. Condensation in the microcentrifuge tube during storage may be a potential source of RNase contamination.Pause Point Microdissected cells for DNA analysis may be stored desiccated at room temperature up to 1 week before extraction. Protein extraction should be performed just before the downstream analysis. - Repeat Steps (xii)–(xix) for additional areas and caps. To microdissect a new slide, click Start new session. Unload slides and all caps in the unload tray. Insert a new stained slide for microdissection, and repeat Steps (v)–(xix).
Critical step Remove all caps from the unload tray before starting a new session. The fork arm used to transport caps will crash and possibly be damaged if it encounters a cap in the unload tray. - To exit and shut down: Click File/Exit, and save images if needed. Unload all caps in the unload tray, remove unused caps and remove slides. Do not turn the instrument power off. The instrument power may remain on for daily operation, eliminating the need for an initial warm-up period.
- Manual laser-capture microdissection (PixCell System)
Timing
Step 4A or 4B, including Box 1 (frozen section tissue staining): 7–10 min
Step 4C, including Box 2 (paraffin-embedded tissue staining): 17–20 min
Step 5A or 5B, including Box 3 (microdissection with IR capture laser): 3,000 laser shots in 30–45 min
Step 5C (microdissection with UV cutting laser): 
1,000 m2 in 5 min.
Anticipated results
Tissue section thickness, number of laser shots and efficiency of microdissection are critical criteria to be monitored during microdissection. The single most important factor for ensuring consistency between the number of laser shots and nucleic acid or protein yield is the efficiency of microdissection. Efficiency should be estimated by observing the cap after microdissection for empty laser shots in relation to laser shots containing cellular material. Following the above guidelines for tissue fixation, staining and microdissection, typical microdissection efficiency is 70–95%.
Table 1 describes the number of cells required for various downstream analyses. Inconsistency in the literature regarding the number of cells (laser shots) required for analysis can be attributed to the fact that recovery of DNA, RNA and protein is less than 100% efficient. Molecular yield is affected by pre-analytical variables, such as type of tissue fixation and time to preservation, as well as the type of extraction method and isolation conditions.
Table 2 describes the anticipated cellular yield in terms of laser shots and tissue area. UV cutting microdissection instruments measure area of microdissection, and therefore the ability to correlate area with laser shots is useful for determining the amount of tissue required for downstream analysis, regardless of microdissection platform.
In our experience, 30,000 microdissected epithelial cells (6,000 laser shots at 30 m) yield approximately 1.0 g l-
1 total protein. This is sufficient quantity for reverse-phase protein microarrays, western blotting and LC-MS/MS analysis.
