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Compared with traditional optical microscopes, multiphoton confocal microscopes have higher resolution, enabling simultaneous observation of multiple fluorescence and the ability to form clear three-dimensional images. Therefore, it has been widely used in the field of biological research since its inception. In the observation of biological samples, multiphoton microscopy has its advantages: continuous scanning of living cells and tissues or cell sections can obtain three-dimensional images of fine cytoskeleton, chromosome, organelle and cell membrane system. A higher contrast, high resolution image than conventional fluorescent microscopes, high sensitivity, and excellent sample protection are available. The acquisition of multi-dimensional images, such as 7-dimensional images (XYZaλIt): xyt, xzt, and xt scans, time-series scans, regional scans, spectral scans, and image processing at the same time. Intracellular ion fluorescent labeling, single labeling or multi-labeling, measuring the ratio and dynamic changes of intracellular concentrations such as pH and sodium, calcium and magnesium. Fluorescently labeled probes for living cells or biopsy of live specimens or sliced ​​specimens, membrane markers, immunological substances, immune responses, receptors or ligands, nucleic acids, etc.; simultaneous multi-substance labeling can be performed on the same sample while observing. No damage, accurate, accurate, reliable and excellent repeatability for cell detection; data images can be exported in time or stored for long periods of time.
 At present, the research fields of laser confocal microscopy are widely used: cell biology: such as: cell structure, cytoskeleton, cell membrane structure, fluidity, receptors, organelle structure and distribution changes, apoptosis mechanism; various organelles, Quantitative analysis of the content, composition and distribution of cell-specific structures such as structural proteins, DNA, RNA, enzymes and receptor molecules; DNA and RNA content, observation and quantification of DNA damage caused by UV using specific antibodies; analysis The relationship between normal cells and cancer cell skeleton and nuclear changes; cell adhesion behavior. Biochemistry: such as enzymes, nucleic acids, receptor analysis, fluorescence in situ hybridization, variegated gene mapping, etc., using confocal technology can replace traditional techniques such as nucleic acid imprinting and so on, gene expression detection, gene transcription, Detections such as translation are much simpler and more accurate. Pharmacology: such as the action of drugs on cells and their kinetics; the dynamic process, localization and quantification of drugs entering cells. Physiology, developmental biology: such as membrane receptors, ion channels, ion content, distribution, dynamics; animal development and embryo formation, differentiation behavior of bone marrow stem cells; measurement of cell membrane potential, measurement of fluorescence bleaching recovery (FRAP). Genetics and histomorphology: such as cell growth, differentiation, maturation, cell three-dimensional structure, chromosome analysis, gene expression, gene diagnosis. Neurobiology: such as the structure of nerve cells, the composition of neurotransmitters, transport and transmission. Microbiology and parasitology: such as bacterial, parasitic morphological structures. Pathology and pathology clinical applications: such as rapid diagnosis of biopsy specimens, tumor diagnosis, diagnosis of autoimmune diseases. Immunology, environmental medicine, and nutrition: such as the location of immunofluorescent markers (single, double or triple), distribution of cell membrane receptors or antigens, distribution of microfilaments, microtubules, coexistence of two or three proteins Colocalization with colocalization, protein and organelles; accurate localization and dynamic observation of proteins in living cells can track the temporal and spatial expression of specific proteins in cell growth, division and differentiation in real time.
Principle and application of multiphoton confocal scanning microscopy
Multiphoton confocal microscopy is a major improvement in optical microscopy. It can be used to observe the deep structures of living cells, fixed cells and tissues, and to obtain clear and sharp multi-layer Z-plane structures, ie optical sections, and to construct specimens. The three-dimensional solid structure. Confocal microscopes use a laser source that expands to fill the focal plane of the entire objective and then passes through the lens system of the objective lens to converge into very small points on the focal plane of the specimen. According to the numerical aperture of the objective lens, the brightest illumination spot has a diameter of about 0.25 ~ 0.8μm and a depth of about 0.5 ~ 1.5μm. The size of the co-focus is determined by the microscope design, laser wavelength, objective characteristics, scanning unit state settings, and specimen properties. The illumination range and illumination depth of the field microscope are large, while the illumination of the confocal microscope is concentrated at a precise focus on the focal plane. The most basic advantage of confocal microscopy is the ability to perform fine optical sections on thick fluorescent specimens (up to 50 μm or more) with a thickness of about 0.5 to 1.5 μm. Series optical slice images can be obtained by moving the specimen up and down through a precise microscope Z-axis stepper motor. The acquisition of image information is controlled in a precise plane without being disturbed by signals from other locations on the specimen. After removing the background fluorescence effect and increasing the signal-to-noise ratio, the contrast and resolution of the confocal image is significantly improved compared to the conventional field illumination fluorescent image. In many specimens, many intricate structural components are intertwined to form a complex system. It is difficult to restore the structural features of the specimen itself with only a few optical slices, but once enough optical slices can be collected, we can use software to perform them. Three-dimensional reconstruction. This experimental method has been widely used in biological research to clarify the complex structural and functional relationships between cells or tissues.
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