Working principle of the laser confocal microscope
Laser confocal microscopy is based on fluorescence microscope imaging with the addition of laser scanning device, the use of computer image processing, the resolution of optical imaging increased by 30% - 40%, the use of ultraviolet or visible light excitation of fluorescent probes, so as to obtain the fluorescence image of the internal microstructure of cells or tissues, at the subcellular level to observe physiological signals and cellular morphology changes, such as Ca2+, PH, membrane potential, etc., has become a new generation of powerful research tools in morphology, molecular biology, neuroscience, pharmacology, genetics and other fields. The laser confocal imaging system is a powerful new generation of research tools in the fields of morphology, molecular biology, neuroscience, pharmacology, genetics and so on. The laser confocal imaging system can be used to observe a variety of stained, non-stained and fluorescently labelled tissues and cells, etc., to observe and study the growth and development of tissue sections and cells in vivo, and to study and measure intracellular transport of substances and energy conversion. It is capable of carrying out the study of ion and PH changes in living cells (RATIO), neurotransmitter research, differential interference and fluorescence tomography, multiple fluorescence tomography and overlap, fluorescence spectroscopy analysis of fluorescence indicators of quantitative analysis of fluorescence samples of time-delayed scanning and dynamic components of three-dimensional dynamic structure of tissues and cells, analysis of the transfer of fluorescence resonance energy, fluorescence in-situ hybridisation research (FISH), cytoskeletal research (FISH), and the study of cytoskeleton. FISH), cytoskeleton research, gene localisation research, in situ real-time PCR product analysis, fluorescence bleaching recovery research (FRAP), intercellular communication research, inter-protein research, research on membrane potential and membrane fluidity, etc., to complete the analysis of image analysis and three-dimensional reconstruction and other analyses.
Laser confocal microscope system application areas:
Involving medicine, animal and plant scientific research, biochemistry, **ology, cell biology, tissue embryo, food science, genetics, pharmacology, physiology, optics, pathology, botany, neuroscience, marine biology, materials science, electronic science, mechanics, petroleum geology, mineralogy.
Basic Principles
The traditional optical microscope uses a field light source, the image of each point on the specimen will be interfered by the diffraction or scattering of light from the neighbouring points; the laser confocal microscope uses a laser beam through the illuminating pinhole to form a point light source to scan each point on the focal plane of the specimen, the irradiated point on the specimen will be imaged at the detection pinhole, and then be received by the detection pinhole after the point-multiplying tube (PMT) or the cold electrocoupling device (cCCD), point by point or line by line, and then be rapidly displayed on the computer monitor. Received point-by-point or line-by-line by the PMT or cCCD behind the probe pinhole, the fluorescent image is rapidly formed on the computer monitor screen. The illumination pinhole and the detection pinhole are conjugate with respect to the focal plane of the objective lens, the points on the focal plane are focused on the illumination pinhole and the emission pinhole at the same time, and the points outside the focal plane will not be imaged at the detection pinhole, so that the confocal image obtained is an optical cross-section of the specimen, which overcomes the disadvantage of the blurring of the image of the ordinary microscope.
