Laser capture microdissection
Encyclopedia
Laser capture microdissection (LCM), also called Microdissection, Laser MicroDissection (LMD), or Laser-assisted microdissection (LMD or LAM) is a method for isolating specific cell
s of interest from microscopic regions of tissue/cells/organisms.
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, a so 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.
Theoretically, there are several ways to extract tissue from a microscope slide with a histopathology
sample on it:
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 various technologies differ in the collection process, possible imaging modalities (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.
1.) The first technology 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 mzterial is sent upward (up to several millimitres) 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.
2.) Another closely related LCM process cuts the sample from above and the sample drops via gravity into a capture device below the sample.
3.) 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; 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.
(http://www.youtube.com/watch?v=v5L0fV23ThI&feature=related)
4.) The fourth UV based technology 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.
5.) 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.
(http://materials.web.psi.ch/Research/Thin_Films/Methods/LIFT.htm)
NON-LASER BASED MICRODISSECTION/ISOLATION
5.) This last technique cannot really be called Laser Capture Microdissection but is closely related.
For lack of a better term is micro-chiseling; by using a piezoelectric driven micro-chisel that vibrates ultrasonic frequencies. This allows for fine etching or chiseling of cells and particles in small areas and for collection via a finely adjustable aspirator to a waiting tube collector. (http://www.intracel.co.uk/eppendorfppmd.htm)
Another related technique for isolating via a micropositioned manipulator is available. (http://www.aura-optik.de/Homepage.1.0.html?&L=1)
In addition to tissue sections, LCM can be performed on living cells/organisms, cell smears, chromosome preparations, and plant tissue.
, RNA
and/or protein
analyses. LCM can be performed on a variety of tissue
samples including blood smears, cytologic preparations, cell cultures and aliquots of solid tissue. Frozen and paraffin embedded archival tissue may also be used. On formalin or alcohol fixed paraffin embedded tissues, DNA and RNA retrieval has been successful, but protein analysis is not possible (requires frozen section).
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s of interest from microscopic regions of tissue/cells/organisms.
Extraction process
A laserLaser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
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, a so 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.
Theoretically, there are several ways to extract tissue from a microscope slide with a histopathology
Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...
sample on it:
- Press a sticky surface onto the sample and tear out. This will extract the desired region, but also bears the chance to carry particles or unwanted tissue on the surface, because an allround sticky surface is not selective.
- Melt a plastic membrane onto the sample and tear out. The heat is introduced by an, e.g., red or 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.
- Transport without contact. There are three different approaches:
- Transport simply by gravity using an upright microscope or
- Reliable and precise transport by Laser Pressure Catapult
- The most recent generation utilizes a technology based on Laser Induced Forward Transfer (LIFT)
Procedure
Under a microscopeMicroscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
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
Infrared
Infrared light is electromagnetic radiation with a wavelength longer than that of visible light, measured from the nominal edge of visible red light at 0.74 micrometres , and extending conventionally to 300 µm...
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 various technologies differ in the collection process, possible imaging modalities (Fluorescence microscopy
Fluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
/Bright field microscopy
Bright field microscopy
Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample...
/Differential interference contrast microscopy
Differential interference contrast microscopy
Differential interference contrast microscopy , also known as Nomarski Interference Contrast or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples...
/Phase contrast microscopy
Phase contrast microscopy
Phase contrast microscopy is an optical microscopy illumination technique of great importance to biologists in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.A phase contrast microscope does not require...
/ 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.
1.) The first technology 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 mzterial is sent upward (up to several millimitres) 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.
2.) Another closely related LCM process cuts the sample from above and the sample drops via gravity into a capture device below the sample.
3.) 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; 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.
(http://www.youtube.com/watch?v=v5L0fV23ThI&feature=related)
4.) The fourth UV based technology 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.
5.) 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.
(http://materials.web.psi.ch/Research/Thin_Films/Methods/LIFT.htm)
NON-LASER BASED MICRODISSECTION/ISOLATION
5.) This last technique cannot really be called Laser Capture Microdissection but is closely related.
For lack of a better term is micro-chiseling; by using a piezoelectric driven micro-chisel that vibrates ultrasonic frequencies. This allows for fine etching or chiseling of cells and particles in small areas and for collection via a finely adjustable aspirator to a waiting tube collector. (http://www.intracel.co.uk/eppendorfppmd.htm)
Another related technique for isolating via a micropositioned manipulator is available. (http://www.aura-optik.de/Homepage.1.0.html?&L=1)
In addition to tissue sections, LCM can be performed on living cells/organisms, cell smears, chromosome preparations, and plant tissue.
Applications
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 DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and/or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
analyses. LCM can be performed on a variety of tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
samples including blood smears, cytologic preparations, cell cultures and aliquots of solid tissue. Frozen and paraffin embedded archival tissue may also be used. On formalin or alcohol fixed paraffin embedded tissues, DNA and RNA retrieval has been successful, but protein analysis is not possible (requires frozen section).
External links
- National Institutes of Health Laser Microdissection Core Facility
- East Carolina University: LCM for "Dummies"
- National Institutes of Health Laser Capture Microdissection Core Facility
- Yale Rice Transcriptional Atlas Project employing Laser Capture Microdissection
- Core Unit Chip Applications, Jena employing Laser Capture Microdissection
- East Carolina University Laser Capture Microdissection Core Facility
- Technology Summary BioCompare: http://www.biocompare.com/Articles/FeaturedArticle/1049/Laser-Capture-Microdissection-LCM--Beam-Me-Up.html