Laser abduction microdissection (LCM), additionally alleged Microdissection, Laser MicroDissection (LMD), or Laser-assisted microdissection (LMD or LAM) is a adjustment for isolating specific beef of absorption from diminutive regions of tissue/cells/organisms
Friday 9 December 2011
Extraction process
A laser is accompanying into a microscope and focuses assimilate the tissue on the slide. By movement of the laser by eyes or the date the focus follows a aisle which is predefined by the user. This trajectory, a so alleged Element, is again cut out and afar from the adjoining tissue. After the acid process, an abstraction action has to chase if an abstraction action is desired. More contempo technologies advance non-contact microdissection.
Theoretically, there are several means to abstract tissue from a microscope accelerate with a histopathology sample on it:
Press a adhesive apparent assimilate the sample and breach out. This will abstract the adapted region, but additionally bears the adventitious to backpack particles or exceptionable tissue on the surface, because an allround adhesive apparent is not selective.
Melt a artificial film assimilate the sample and breach out. The calefaction is alien by an, e.g., red or IR laser assimilate a film decrepit with an arresting dye. As this adheres the adapted sample assimilate the membrane, as with any film that is put abutting to the histopathology sample surface, there ability be some bits extracted. Another crisis is the alien heat: Some molecules like DNA, RNA, or protein don't acquiesce to be acrimonious too abundant or at all for the ambition of actuality abandoned as absolutely as possible.
Carriage after contact. There are three altered approaches:
Carriage artlessly by force application an cocked microscope or
Reliable and absolute carriage by Laser Pressure Catapult
The best contempo bearing utilizes a technology based on Laser Induced Forward Transfer (LIFT)
Theoretically, there are several means to abstract tissue from a microscope accelerate with a histopathology sample on it:
Press a adhesive apparent assimilate the sample and breach out. This will abstract the adapted region, but additionally bears the adventitious to backpack particles or exceptionable tissue on the surface, because an allround adhesive apparent is not selective.
Melt a artificial film assimilate the sample and breach out. The calefaction is alien by an, e.g., red or IR laser assimilate a film decrepit with an arresting dye. As this adheres the adapted sample assimilate the membrane, as with any film that is put abutting to the histopathology sample surface, there ability be some bits extracted. Another crisis is the alien heat: Some molecules like DNA, RNA, or protein don't acquiesce to be acrimonious too abundant or at all for the ambition of actuality abandoned as absolutely as possible.
Carriage after contact. There are three altered approaches:
Carriage artlessly by force application an cocked microscope or
Reliable and absolute carriage by Laser Pressure Catapult
The best contempo bearing utilizes a technology based on Laser Induced Forward Transfer (LIFT)
Procedure
Under a microscope application acomputer application interface, a tissue breadth (typically 5-50 micrometres thick) is beheld and alone beef or clusters of beef are articular either manually or in semi-automated or added absolutely automated means acceptance the imaging and again automated alternative of targets for isolation. Currently six primary isolation/collection technologies abide application a microscope and accessory for corpuscle isolation. Four of these about use an ultraviolet pulsed laser (355 nm) for the acid of the tissues anon or the membranes/film, and sometimes in aggregate with an IR laser amenable for heating/melting a adhering polymer for cellular adherence and isolation. IR laser provides a added affable access to microdissection. A fifth ultraviolet laser based technology uses appropriate slides coated with an activity alteration blanket which, back activated by the laser pulse, propels the tissue or beef into a accumulating cap.
The assorted technologies alter in the accumulating process, accessible imaging modalities (Fluorescence microscopy/Bright acreage microscopy/Differential arrest adverse microscopy/Phase adverse microscopy/ etc.) and the types of holders and tissue alertness bare afore the imaging and isolation. Most are primarily committed micro-dissection systems, and some can be acclimated as analysis microscopes as well, alone one technology (#2 here, Leica) uses an cocked microscope, attached some of the sample administration capabilities somewhat, abnormally for alive corpuscle work.
1.) The aboriginal technology cuts about the sample again collects it by a "catapulting" technology. The sample can be catapulted from a accelerate or appropriate ability bowl by a defocused U.V laser beating which generates a photonic force to actuate the actual off the slide/dish, a address sometimes alleged Laser Micro-dissection Pressure Catapulting (LMPC). The dissected mzterial is beatific advancement (up to several millimitres) to a microfuge tube cap or added beneficiary which contains either a absorber or a specialized broken-down actual in the tube cap that the tissue will attach to. This alive catapulting action avoids some of the changeless problems back application membrane-coated slides. 3
2.) Another carefully accompanying LCM action cuts the sample from aloft and the sample drops via force into a abduction accessory beneath the sample. 4
3.) Back the beef (on a accelerate or appropriate ability dish) of best are in the centermost of the acreage of view, the abettor selects the beef of absorption application apparatus software. The breadth to be abandoned back a near-IR laser to actuate alteration blur on a cap placed on the tissue sample, melting the adhering which again fuses the blur with the basal beef of choice; and/or by activating a UV laser to cut out the corpuscle of interest. The beef are again aerial off the attenuate tissue section, abrogation all exceptionable beef behind. The beef of absorption are again beheld and accurate above-mentioned to extraction. (http://www.youtube.com/watch?v=v5L0fV23ThI&feature=related)
4.) The fourth UV based technology offers a slight aberration to the 3rd technology actuality by about creating a sandwich of sorts with slide>sample>and film aloft the sample by the use of a anatomy accelerate whose film apparent is cut by the laser and ultimately best up from aloft by a appropriate adhering cap.
5.) A fifth UV based technology uses accepted bottle slides coated with an apathetic activity alteration blanket and a UV based laser microdissection arrangement (typically a Leica LMD or PALM Zeiss machine). Tissue sections are army on top of the activity alteration coating. The activity from a UV laser is adapted to active activity aloft arresting the coating, vaporizing it, instantly affective alleged tissue appearance into the accumulating tube. The activity alteration coated slides, commercialized beneath the barter name DIRECTOR slides by Expression Pathology Inc. (Rockville, MD), action several advantages for proteomic work. They additionally do not autofluoresce, so they can be acclimated for applications application beaming stains, DIC or polarized light. (http://materials.web.psi.ch/Research/Thin_Films/Methods/LIFT.htm)
NON-LASER BASED MICRODISSECTION/ISOLATION
5.) This aftermost address cannot absolutely be alleged Laser Abduction Microdissection but is carefully related. For abridgement of a more good appellation is micro-chiseling; by application a piezoelectric apprenticed micro-chisel that vibrates accelerated frequencies. This allows for accomplished carving or chiseling of beef and particles in baby areas and for accumulating via a cautiously adjustable aspirator to a cat-and-mouse tube collector. (http://www.intracel.co.uk/eppendorfppmd.htm) Another accompanying address for isolating via a micropositioned architect is available. (http://www.aura-optik.de/Homepage.1.0.html?&L=1)
In accession to tissue sections, LCM can be performed on active cells/organisms, corpuscle smears, chromosome preparations, and bulb tissue
The assorted technologies alter in the accumulating process, accessible imaging modalities (Fluorescence microscopy/Bright acreage microscopy/Differential arrest adverse microscopy/Phase adverse microscopy/ etc.) and the types of holders and tissue alertness bare afore the imaging and isolation. Most are primarily committed micro-dissection systems, and some can be acclimated as analysis microscopes as well, alone one technology (#2 here, Leica) uses an cocked microscope, attached some of the sample administration capabilities somewhat, abnormally for alive corpuscle work.
1.) The aboriginal technology cuts about the sample again collects it by a "catapulting" technology. The sample can be catapulted from a accelerate or appropriate ability bowl by a defocused U.V laser beating which generates a photonic force to actuate the actual off the slide/dish, a address sometimes alleged Laser Micro-dissection Pressure Catapulting (LMPC). The dissected mzterial is beatific advancement (up to several millimitres) to a microfuge tube cap or added beneficiary which contains either a absorber or a specialized broken-down actual in the tube cap that the tissue will attach to. This alive catapulting action avoids some of the changeless problems back application membrane-coated slides. 3
2.) Another carefully accompanying LCM action cuts the sample from aloft and the sample drops via force into a abduction accessory beneath the sample. 4
3.) Back the beef (on a accelerate or appropriate ability dish) of best are in the centermost of the acreage of view, the abettor selects the beef of absorption application apparatus software. The breadth to be abandoned back a near-IR laser to actuate alteration blur on a cap placed on the tissue sample, melting the adhering which again fuses the blur with the basal beef of choice; and/or by activating a UV laser to cut out the corpuscle of interest. The beef are again aerial off the attenuate tissue section, abrogation all exceptionable beef behind. The beef of absorption are again beheld and accurate above-mentioned to extraction. (http://www.youtube.com/watch?v=v5L0fV23ThI&feature=related)
4.) The fourth UV based technology offers a slight aberration to the 3rd technology actuality by about creating a sandwich of sorts with slide>sample>and film aloft the sample by the use of a anatomy accelerate whose film apparent is cut by the laser and ultimately best up from aloft by a appropriate adhering cap.
5.) A fifth UV based technology uses accepted bottle slides coated with an apathetic activity alteration blanket and a UV based laser microdissection arrangement (typically a Leica LMD or PALM Zeiss machine). Tissue sections are army on top of the activity alteration coating. The activity from a UV laser is adapted to active activity aloft arresting the coating, vaporizing it, instantly affective alleged tissue appearance into the accumulating tube. The activity alteration coated slides, commercialized beneath the barter name DIRECTOR slides by Expression Pathology Inc. (Rockville, MD), action several advantages for proteomic work. They additionally do not autofluoresce, so they can be acclimated for applications application beaming stains, DIC or polarized light. (http://materials.web.psi.ch/Research/Thin_Films/Methods/LIFT.htm)
NON-LASER BASED MICRODISSECTION/ISOLATION
5.) This aftermost address cannot absolutely be alleged Laser Abduction Microdissection but is carefully related. For abridgement of a more good appellation is micro-chiseling; by application a piezoelectric apprenticed micro-chisel that vibrates accelerated frequencies. This allows for accomplished carving or chiseling of beef and particles in baby areas and for accumulating via a cautiously adjustable aspirator to a cat-and-mouse tube collector. (http://www.intracel.co.uk/eppendorfppmd.htm) Another accompanying address for isolating via a micropositioned architect is available. (http://www.aura-optik.de/Homepage.1.0.html?&L=1)
In accession to tissue sections, LCM can be performed on active cells/organisms, corpuscle smears, chromosome preparations, and bulb tissue
Applications
The laser abduction microdissection action does not adapt or accident the assay and allure of the sample collected, nor the surrounding cells. For this reason, LCM is a advantageous adjustment of accession called beef for DNA, RNA and/or protein analyses. LCM can be performed on a array of tissue samples including claret smears, cytologic preparations,5 corpuscle cultures and aliquots of solid tissue. Arctic and paraffin anchored archival tissue may additionally be used.6 On formalin or booze anchored paraffin anchored tissues, DNA and RNA retrieval has been successful, but protein assay is not accessible (requires arctic section)
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