Knowledge of the Confocal Fluorescence Microscope
The basic principle of confocal fluorescence microscopy: a point light source is used to irradiate the specimen, forming a small, well-defined point of light on the focal plane; the fluorescence emitted from this point after irradiation is collected by the objective lens and sent back along the original irradiation light path to the beamsplitter consisting of a bi-directional chromatic mirror. The beam splitter sends the fluorescence directly to the detector. Both the light source and the detector each have a pinhole in front of them, called the illumination pinhole and the detection pinhole, respectively. Both have the same geometry, about 100-200 nm, and are conjugate with respect to the point of light on the focal plane, i.e., the point of light passes through a series of lenses and can ultimately be focussed on both the illuminating and detector pinholes. In this way, light from the focal plane can converge within the probe aperture, while scattered light from above or below the focal plane is blocked outside the probe aperture and cannot be imaged. The laser scans the sample point by point, and the photomultiplier tube after detecting the pinhole also obtains the confocal image of the corresponding light point point by point, which is converted into a digital signal and transmitted to the computer, and finally converges into a clear confocal image of the entire focal plane on the screen.
Each focal plane image is actually an optical cross-section of the specimen, and this optical cross-section always has a certain thickness, also known as an optical sheet. Since 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 the pinhole, the depth of field of the confocal system is approximated to be zero, and scanning along the Z-axis direction enables optical tomography to form a two-dimensional optical slice of the specimen to be observed at the focal spot. 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 successive layers of two-dimensional images, which are processed by specialised computer software.
This means that the detection pinhole and the light source pinhole are always focused at the same point, so that fluorescence excited outside the focal plane cannot enter the detection pinhole.
The working principle of laser confocal is simply expressed that it uses laser as a light source, on the basis of traditional fluorescence microscope imaging, additional laser scanning device and conjugate focusing device, through computer control to carry out digital image acquisition and processing system.
