Knowledge related to confocal fluorescence microscopy
The basic principle of confocal fluorescence microscopy is to use a point light source to irradiate the specimen, forming a well-defined small spot on the focal plane. The fluorescence emitted by the spot after irradiation is collected by the objective lens and sent back to the spectrophotometer composed of a bidirectional color mirror along the original irradiation path. The spectrophotometer sends fluorescence directly to the detector. There are two pinholes in front of the light source and detector, respectively called illumination pinholes and detection pinholes. The geometric dimensions of the two are consistent, about 100-200nm; Compared to the light point on the focal plane, the two are conjugate, meaning that the light point passes through a series of lenses and can ultimately focus on both the illumination pinhole and the detection pinhole simultaneously. In this way, light from the focal plane can converge within the range of the detection hole, while scattered light from above or below the focal plane is blocked outside the detection hole and cannot be imaged. By scanning the sample point by point with a laser, the photomultiplier tube after detecting the pinhole also obtains the corresponding confocal image of the light point point by point, which is converted into a digital signal and transmitted to the computer. Finally, a clear confocal image of the entire focal plane is synthesized on the screen.
Each focal plane image is actually an optical cross-section of the specimen, which always has a certain thickness, also known as an optical thin section. Due to the fact that the light intensity at the focal point is much greater than that at the non focal point, and the non focal plane light is filtered out by pinholes, the depth of field of the confocal system is approximately zero. Scanning along the Z-axis direction can achieve optical tomography, forming a two-dimensional optical slice at the focal spot of the observed sample. By combining X-Y plane (focal plane) scanning with Z-axis (optical axis) scanning, a three-dimensional image of the sample can be obtained by accumulating continuous layers of two-dimensional images and processing them with specialized computer software.
The detection pinhole and the light source pinhole are always focused on the same point, so that fluorescence excited outside the focusing plane cannot enter the detection pinhole.
The simple expression of the working principle of laser confocal microscopy is that it uses a laser as the light source, and adds a laser scanning device and a conjugate focusing device on the basis of traditional fluorescence microscopy imaging. It is a system controlled by a computer for digital image acquisition and processing.
