The difference between laser confocal microscope and traditional optical microscope
Laser Scanning Confocal Fluorescence Microscopy is a relatively advanced molecular and cell biology analysis instrument that uses computer, laser and image processing technology to obtain three-dimensional data of biological samples. It is mainly used to observe the structure of living cells and the biological changes of specific molecules and ions, quantitative analysis, and real-time quantitative determination.
The principle of laser confocal microscopy: use the illumination pinhole placed behind the light source and the detection pinhole placed in front of the detector to realize point illumination and point detection. The light emitted from the light source through the illumination pinhole is focused on a point on the focal plane of the sample, and the emitted fluorescence from this point is imaged on the detection pinhole, and any emitted light outside this point is blocked by the detection pinhole. The illumination pinhole and the detection pinhole are conjugate to the irradiated point or the detected point, so the detected point is the confocal point, and the plane where the detected point is located is the confocal plane.
The computer displays the detected points on the computer screen in the form of image points. In order to generate a complete image, the scanning system in the optical path scans on the focal plane of the sample to generate a complete confocal image. As long as the stage moves up and down along the Z axis, a new layer of the sample is moved to the confocal plane, and the new layer of the sample is imaged on the monitor. With the continuous movement of the Z axis, continuous images of different layers of the sample can be obtained. Light cut image.
The Difference Between Traditional Light Microscopes
Traditional fluorescence microscopes have an insurmountable shortcoming: the fluorescent structures outside the focal plane are blurred and blurred. The reason is that most biological specimens have overlapping structures. If the fluorescently labeled structures are distributed at different levels and overlap, the scattered fluorescence from above or below the focal plane is also received by the objective lens, and the resolution of the fluorescence microscope will be greatly improved. reduce.
On the basis of traditional optical microscopes, laser scanning confocal microscopes use laser light as the light source, adopt the principle and device of conjugate focusing, and use computers to perform digital image processing, observation, analysis and output of the observed objects. The sample can be tomographically scanned and imaged for non-destructive observation and analysis of the three-dimensional spatial structure of cells. At the same time, using immunofluorescent labeling and ion fluorescent labeling probes, this technology can not only observe fixed cells and tissue sections, but also perform real-time dynamic observation and detection of the structure, molecules, ions and life activities of living cells, at the subcellular level Observing physiological signals such as Ca2+, pH value, membrane potential and changes in cell morphology, has become a new generation of powerful research tools in the fields of morphology, molecular cell biology, neuroscience, pharmacology, and genetics.
