Laser capture microdissection (LCM), also called microdissection, laser microdissection (LMD), or laser-assisted microdissection (LMD or LAM), is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms [1] [2] (dissection on a microscopic scale with the help of a laser).
Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss of heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. The total time required to carry out this protocol is typically 1–1.5 h. [3]
A laser is coupled into a microscope and focuses onto the tissue on the slide. By movement of the laser by optics or the stage the focus follows a trajectory which is predefined by the user. This trajectory, also called element, is then cut out and separated from the adjacent tissue. After the cutting process, an extraction process has to follow if an extraction process is desired. More recent technologies utilize non-contact microdissection.
There are several ways to extract tissue from a microscope slide with a histopathology sample on it. Press a sticky surface onto the sample and tear out. This extracts the desired region, but can also remove particles or unwanted tissue on the surface, because the surface is not selective. Melt a plastic membrane onto the sample and tear out. The heat is introduced, for example, by a red or infrared (IR) laser onto a membrane stained with an absorbing dye. As this adheres the desired sample onto the membrane, as with any membrane that is put close to the histopathology sample surface, there might be some debris extracted. Another danger is the introduced heat: Some molecules like DNA, RNA, or protein don't allow to be heated too much or at all for the goal of being isolated as purely as possible.
For transport without contact. There are three different approaches. Transport by gravity using an upright microscope (called GAM, gravity-assisted microdissection) or transport by laser pressure catapult; the most recent generation utilizes a technology based on laser induced forward transfer (LIFT). With cut-and-capture, a cap coated with an adhesive is positioned directly on the thinly cut (5-8 μm) tissue section, the section itself resting on a thin membrane (polyethylene naphthalene). An IR laser gently heats the adhesive on the cap fusing it to the underlying tissue and an UV laser cuts through tissue and underlying membrane. The membrane-tissue entity now adheres to the cap and the cells on the cap can be used in downstream applications (DNA, RNA, protein analysis). [4]
Under a microscope using a software interface, a tissue section (typically 5-50 micrometres thick) is viewed and individual cells or clusters of cells are identified either manually or in semi-automated or more fully automated ways allowing the imaging and then automatic selection of targets for isolation. Currently six primary isolation/collection technologies exist using a microscope and device for cell isolation. Four of these typically use an ultraviolet pulsed laser (355 nm) for the cutting of the tissues directly or the membranes/film, and sometimes in combination with an IR laser responsible for heating/melting a sticky polymer for cellular adhesion and isolation. IR laser provides a more gentle approach to microdissection. A fifth ultraviolet laser based technology uses special slides coated with an energy transfer coating which, when activated by the laser pulse, propels the tissue or cells into a collection cap.
The laser cutting width is usually less than 1 μm, thus the target cells are not affected by the laser beam. Even live cells are not damaged by the laser cutting and are viable after cutting for cloning and reculturing as appropriate. [5]
The various technologies differ in the collection process, possible imaging methods (fluorescence microscopy/bright field microscopy/differential interference contrast microscopy/phase contrast microscopy/ etc.) and the types of holders and tissue preparation needed before the imaging and isolation. Most are primarily dedicated micro-dissection systems, and some can be used as research microscopes as well, only one technology (#2 here, Leica) uses an upright microscope, limiting some of the sample handling capabilities somewhat, especially for live cell work.
The first technology (used by Carl Zeiss PALM) cuts around the sample then collects it by a "catapulting" technology. The sample can be catapulted from a slide or special culture dish by a defocused U.V laser pulse which generates a photonic force to propel the material off the slide/dish, a technique sometimes called Laser Micro-dissection Pressure Catapulting (LMPC). The dissected material is sent upward (up to several millimetres) to a microfuge tube cap or other collector which contains either a buffer or a specialized tacky material in the tube cap that the tissue will adhere to. This active catapulting process avoids some of the static problems when using membrane-coated slides. [6]
Another process follows gravity-assisted microdissection method that turns on gravity to collect samples in tube cap under the slide used (used by ION LMD system, Jungwoo F&B). In case of this system, it moves the motorized stage to cut the cells of interests, keeping the laser beam fixed. And the system uses a 355 nm Solid-state laser(UV-A) which is the safest way to cut the tissues without RNA or DNA damage. [7] [ failed verification ]
Another closely related LCM process (used by Leica) cuts the sample from above and the sample drops via gravity (gravity-assisted microdissection) into a capture device below the sample. [8] The different point with upper one is, the laser beam here is moving to cut tissue by moving dichroic mirror.
When the cells (on a slide or special culture dish) of choice are in the center of the field of view, the operator selects the cells of interest using instrument software. The area to be isolated when a near-IR laser to activate transfer film on a cap placed on the tissue sample, melting the adhesive which then fuses the film with the underlying cells of choice (see Arcturus systems); and/or by activating a UV laser to cut out the cell of interest. The cells are then lifted off the thin tissue section, leaving all unwanted cells behind. The cells of interest are then viewed and documented prior to extraction. [9]
The fourth UV based technology (used by Molecular Machines and Industries AG) offers a slight difference to the 3rd technology here by essentially creating a sandwich of sorts with slide>sample>and membrane overlying the sample by the use of a frame slide whose membrane surface is cut by the laser and ultimately picked up from above by a special adhesive cap. [10]
A fifth UV based technology uses standard glass slides coated with an inert energy transfer coating and a UV based laser microdissection system (typically a Leica LMD or PALM Zeiss machine). Tissue sections are mounted on top of the energy transfer coating. The energy from a UV laser is converted to kinetic energy upon striking the coating, vaporizing it, instantly propelling selected tissue features into the collection tube. The energy transfer coated slides, commercialized under the trade name DIRECTOR slides by Expression Pathology Inc. (Rockville, MD), offer several advantages for proteomic work. They also do not autofluoresce, so they can be used for applications using fluorescent stains, DIC or polarized light. [11]
In addition to tissue sections, LCM can be performed on living cells/organisms, cell smears, chromosome preparations, and plant tissue.
The laser capture microdissection process does not alter or damage the morphology and chemistry of the sample collected, nor the surrounding cells. For this reason, LCM is a useful method of collecting selected cells for DNA, RNA and/or protein analyses. LCM has also been used to isolate acellular structures, such as amyloid plaques. [12] LCM can be performed on a variety of tissue samples including blood smears, cytologic preparations, [13] cell cultures and aliquots of solid tissue. Frozen and paraffin embedded archival tissue may also be used. [14]
Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.
A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of biological interactions. It is a two-dimensional array on a solid substrate—usually a glass slide or silicon thin-film cell—that assays (tests) large amounts of biological material using high-throughput screening miniaturized, multiplexed and parallel processing and detection methods. The concept and methodology of microarrays was first introduced and illustrated in antibody microarrays by Tse Wen Chang in 1983 in a scientific publication and a series of patents. The "gene chip" industry started to grow significantly after the 1995 Science Magazine article by the Ron Davis and Pat Brown labs at Stanford University. With the establishment of companies, such as Affymetrix, Agilent, Applied Microarrays, Arrayjet, Illumina, and others, the technology of DNA microarrays has become the most sophisticated and the most widely used, while the use of protein, peptide and carbohydrate microarrays is expanding.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
A microtome is a cutting tool used to produce extremely thin slices of material known as sections, with the process being termed microsectioning. Important in science, microtomes are used in microscopy for the preparation of samples for observation under transmitted light or electron radiation.
A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.
Microdissection refers to a variety of techniques where a microscope is used to assist in dissection.
Chromosome microdissection is a technique that physically removes a large section of DNA from a complete chromosome. The smallest portion of DNA that can be isolated using this method comprises 10 million base pairs - hundreds or thousands of individual genes.
A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available.
Microfluidic whole genome haplotyping is a technique for the physical separation of individual chromosomes from a metaphase cell followed by direct resolution of the haplotype for each allele.
Gravity-assisted microdissection (GAM) is one of the laser microdissection methods. The dissected material is allowed to fall by gravity into a cap and may thereafter be used for isolating proteins or genetic material. Two manufacturers in the world have developed their own device based on GAM method.
In the field of cellular biology, single-cell analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the 1970s. Before the discovery of heterogeneity, single-cell analysis mainly referred to the analysis or manipulation of an individual cell in a bulk population of cells at a particular condition using optical or electronic microscope. To date, due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) allow the precise isolation of selected single cells from complex samples, while high throughput single cell partitioning technologies, enable the simultaneous molecular analysis of hundreds or thousands of single unsorted cells; this is particularly useful for the analysis of transcriptome variation in genotypically identical cells, allowing the definition of otherwise undetectable cell subtypes. The development of new technologies is increasing our ability to analyze the genome and transcriptome of single cells, as well as to quantify their proteome and metabolome. Mass spectrometry techniques have become important analytical tools for proteomic and metabolomic analysis of single cells. Recent advances have enabled quantifying thousands of protein across hundreds of single cells, and thus make possible new types of analysis. In situ sequencing and fluorescence in situ hybridization (FISH) do not require that cells be isolated and are increasingly being used for analysis of tissues.
A transcriptome in vivo analysis tag is a multifunctional, photoactivatable mRNA-capture molecule designed for isolating mRNA from a single cell in complex tissues.
Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. This method can also be used to determine subcellular localization of mRNA molecules. The term is a variation of Spatial Genomics, first described by Doyle, et al., in 2000 and then expanded upon by Ståhl et al. in a technique developed in 2016, which has since undergone a variety of improvements and modifications.
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
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