What Is Laser Capture Microdissection?
Laser capture microdissection: Laser capture microdissection (LCM) technology is to obtain target cells directly from frozen or paraffin-embedded tissue sections without damaging the tissue structure and preserving the integrity of the cells to be captured and the surrounding tissue. It is usually used to precisely separate a single cell from a tissue.
- Chinese name
- Laser microdissection
- Foreign name
- Laser capture microdissection (LCM)
- profession
- Light and laser biomedical technology
- Laser capture microdissection: Laser capture microdissection (LCM) technology is to obtain target cells directly from frozen or paraffin-embedded tissue sections without damaging the tissue structure and preserving the integrity of the cells to be captured and the surrounding tissue. It is usually used to precisely separate a single cell from a tissue.
Laser Capture Microdissection Background
- Body tissue contains hundreds of different cells, each of which adheres to surrounding cells, stroma, blood vessels, glands, inflammatory cells, or immune cells. In normal or developing tissues and organs, intracellular signals, signals from neighboring cells, and humoral stimuli act on specific cells, causing these cells to express different genes and undergo complex molecular changes. In the pathological state, if the same molecular changes occur in the same type of cells, this molecular change may play a key role in the occurrence of the disease. However, cells with the same molecular changes may only occupy a small part of the total tissue volume; at the same time, the target cells of the study are often surrounded by other tissue components. In order to perform molecular-level analysis of tissue damage during disease development, it is necessary to isolate pure target cells. In 1996, the National Cancer Institute [2] of the National Institutes of Health (NIH) developed the laser capture microdissection (LCM) technology. The following year, the American Arcturus Engineering company successfully developed a laser capture microdissection system. And realize commercial sales. The application of this technology can quickly and accurately obtain the required single cell subpopulations or even single cells under the direct view of a microscope, thereby successfully solving the problem of cell heterogeneity in tissues. This technology has become a supporting technology of the "tumor genome anatomy project" in the United States [1] .
- With an inverted fluorescence microscope and digital imaging system
Laser capture microdissection principle
- The basic principle of LCM is to activate a thermoplastic film through a low-energy infrared laser pulse-ethylene vinyl acetate (EVA) film (its maximum absorption peak is close to the infrared laser wavelength), and selectively target cells under direct vision. Or tissue fragments stick to the membrane [2] . The LCM system includes an inverted microscope, a solid-state infrared laser diode, a laser control device, a joystick that controls the microscope stage (fixed glass slide), an electrically coupled camera, and a color display. The thermoplastic film used to capture the target cells is usually 6mm in diameter and is covered by a transparent plastic cap, which matches the standard 0.5ml centrifuge tube used in subsequent experiments.
- The robotic arm suspends and controls a plastic cap covered with a thermoplastic film and places it on a target site on a dehydrated tissue section. Select the target cells under the microscope's direct view, emit laser pulses, and instantly raise the temperature to make the EVA film partially melt. The molten EVA membrane penetrates into the tiny tissue gaps on the section and rapidly solidifies within a few milliseconds. The adhesion of tissue to the membrane exceeds the adhesion between the tissue and the slide, so that the target cells can be selectively transferred. The laser pulse usually lasts from 0.5 to 5.0 milliseconds, and can be repeated multiple times across the entire surface of the plastic cap, so that a large number of target cells can be quickly separated. The plastic cap was capped on the centrifuge tube containing the buffer solution, and the selected cells were transferred to the centrifuge tube, so that the molecule of interest can be separated for experiments [3] .
- The EVA film is about 100 ~ 200m thick, which can absorb most of the energy generated by the laser, instantly raise the temperature of the laser beam irradiation area to 90 ° C, and maintain rapid cooling after a few milliseconds to ensure that biological macromolecules are not damaged. The use of low-energy infrared lasers can also prevent the occurrence of damaging photochemical reactions.
- Laser microdissection light path diagram
- Touch screen to select cutting area directly
LCD Advantages and disadvantages of laser capture micro-cutting LCD
- The most significant advantage of LCM is its speed, accuracy and versatility. Based on the characteristics of the tissue structure and the required cutting accuracy, a large number of target cells can be quickly obtained by selecting the diameter of the laser beam. Compared with the microdissection technology based on micromanipulator [4] , LCM has the following advantages: (1) fast separation of cells without the need for sophisticated operation skills; (2) capture of morphological characteristics of cells and remaining tissue Both are kept intact, which can better control the specificity of the captured cells; (2) The capture cells are tightly bound to the plastic cap, reducing the risk of tissue loss. In contrast, in addition to laser-cut ejection micro-separation systems [5], stained sections for archiving can also be successfully microdissected.
- Although LCM is widely used, the visual resolution of conventionally stained, fixed, and uncovered tissue sections is greatly limited. For complex tissues (such as lymphoid tissues, extensively infiltrating adenocarcinomas, etc.) that lack certain structural characteristics, it is almost impossible to accurately isolate certain types of cells. Fend et al. [6] solved the above problems by using special staining, especially immunohistochemical methods, to make the target cells or cells to be removed more eye-catching.
- The application of LCM occasionally fails to remove selected cells from the section. There are two reasons for this result: (1) Insufficient adhesion between the cell and the thermoplastic membrane, usually due to incomplete tissue Caused by dehydration or low energy setting of the laser; (2) The adhesion between the tissue section and the slide is too strong, which usually occurs in frozen sections where the microdissection drying time is too long. For different sample tissues (including tissue sections of immunohistochemical staining), some research groups have reported in detail the use of appropriate processing methods to achieve the best microdissection conditions [7] .
Laser capture microdissection applications
- LCM has made breakthrough progress over previous microdissection techniques, and has been widely used in tumor research, including prostate cancer [8] , kidney
- Combined application of laser capture microdissection and biochip
Laser Capture Microdissection Outlook
- LCM has successfully solved the problem of tissue heterogeneity, and has many advantages such as rapidness and accuracy. It has been widely used in the study of tumor gene level and other diseases, and has shown good application prospects [1] . However, in the future, the following major aspects may need to be developed and improved: theoretically, LCD can be used to separate all other tissue cells (such as spleen macrophages, liver Kuffer cells, etc.) in addition to the above tissues and cells, but Their respective methods of section preparation, staining and other technical methods still need to be explored; the development of corresponding applications, only by entering the specific parameters of the target cells or tissues, can realize the computer automatic control of the LCD [12] , thereby greatly reducing the manpower required And time; improve the accuracy of capturing single cells to reduce non-target tissue contamination; further optimize the steps of rapid immunohistochemical staining, improve DNA and RNA extraction technology, and achieve high-quality nucleic acids from a small number of captured cells or tissues .