Principles of Confocal Microscopy
Confocal microscope is a high-precision imaging instrument that emerged and developed in the 1980s, and is an essential scientific research instrument for studying submicron structures. With the development of computers, image processing software and lasers, confocal microscopes have also undergone great development, and are now widely used in the fields of biology, microsystems and material measurement. Confocal microscope is a new type of microscope integrating confocal principle, scanning technology and computer graphics processing technology. Its main advantages are: high lateral resolution and high axial resolution, and effective suppression of stray light , with high contrast.
A typical confocal microscope setup is to place two small holes on the conjugate plane of the focal plane of the measured object, one of which is placed in front of the light source and the other is placed in front of the detector, as shown in Figure 1. It can be seen from the figure that when the measured sample is in the quasi-focus plane, the light intensity collected by the detection end is the largest; when the measured sample is in the out-of-focus position, the light spot at the detection end diffuses and the light intensity decreases rapidly. Therefore, only the light emitted by points on the focal plane can pass through the exit pinhole, while the light emitted by points outside the focal plane is defocused on the exit pinhole plane, and most of them cannot pass through the central pinhole. Therefore, the observation target point on the focal plane appears bright, and the non-observation point appears black as the background, increasing the contrast and clearing the image. During the imaging process, the two pinholes are confocal, the confocal point is the detected point, and the plane where the detected point is located is the confocal plane.
The size of the pinhole at the detector in confocal microscopy plays a critical role. It directly affects the resolution and signal-to-noise ratio of the system. If the pinhole is too large, the confocal detection effect will not be achieved, which not only reduces the resolution of the system, but also introduces more stray light; if the pinhole is too small, it will reduce the detection efficiency and reduce the microscopic image. brightness. Studies have shown that when the diameter of the pinhole is equal to the diameter of the Airy disk, the confocal requirements are met, and the detection efficiency is not significantly reduced. Since the diameter of the pinhole is generally on the order of microns, if there is a deviation between the focus point of the laser beam and the position of the pinhole, signal distortion will occur. Therefore, confocal microscopes generally use an autofocus system, which virtually increases the measurement time.
Since the laser confocal scanning microscope is a point imaging, in order to obtain a two-dimensional image of the object, it is necessary to use two-dimensional scanning in the x and y directions. Different microscopes use different scanning methods:
(1) Object scanning. That is, the object itself moves according to a certain law, while the light beam remains unchanged. Advantages: stable optical path; Disadvantages: a large scanning table is required, so the scanning speed is greatly limited.
(2) A beam scanning system is formed by using a reflective galvanometer. That is, by controlling the scanning galvanometer, the focused light spot is regularly reflected to a certain layer of the object to complete two-dimensional scanning. Its advantage is that it has high precision and is often used for high-precision measurement. Scanning speed has improved over object scanning, but it's still not fast.
(3) Use the acousto-optic deflection element for scanning, and the scanning is realized by changing the output frequency of the sound wave and then changing the transmission direction of the light wave. Its outstanding advantage is that the scanning speed is very fast. The scanning system developed by the United States uses an acousto-optic deflector to generate real-time video images. It only takes 1/30s to scan a two-dimensional image, and it almost achieves real-time output.
(4) Nipkow disk scanning. The scanning process is completed by rotating the Nipkow disk while keeping other components still. It can be imaged at one time and the speed is very fast. However, since the imaging beam is off-axis light, the off-axis aberration of the lens must be corrected, and the utilization rate of light energy is very low.
