Why do we need confocal microscopy?
1. After the efforts and improvements of our great predecessors, the optical microscope has reached a perfect level. In fact, regular microscopes can provide us with beautiful microscopic images simply and quickly. However, an event that brought revolutionary innovation to this almost perfect world of microscopes occurred, which was the invention of the "laser scanning confocal microscope". The characteristic of this new type of microscope is that it adopts an optical system that only extracts image information on the surface where the focus is concentrated. By changing the focus and restoring the obtained information in the image memory, it can obtain vivid images with complete 3D information intelligence. Through this method, information about surface shape that cannot be confirmed by conventional microscopes can be easily obtained. In addition, for typical optical microscopes, "increasing resolution" and "deepening focal depth" are contradictory conditions, especially at high magnification. However, in confocal microscopes, this problem is easily solved.
2. Advantages of confocal optical systems
The confocal optical system is used for point illumination of the sample, and the reflected light is also received by a point sensor. When the sample is placed in the focal position, almost all of the reflected light can reach the sensor. When the sample deviates from the focal point, the reflected light cannot reach the sensor. That is to say, in a confocal optical system, only the image that coincides with the focus will be output, and the spot and useless scattered light will be shielded.
Why use laser?
In confocal optical systems, point illumination is applied to the sample, and the reflected light is also received by a point sensor. Therefore, point light sources have become necessary. Laser is a very point light source. In most cases, the light source of confocal microscopy is laser. In addition, the monochromaticity, directionality, and excellent beam shape of lasers are also important reasons for their widespread adoption.
4. Real time observation based on high-speed scanning becomes possible
The laser scanning uses Acoustic Optical Reflector (AO) in the horizontal direction and Servo Galvano mirror in the vertical direction. Due to the absence of mechanical vibrations in the audio optical bias unit, it is possible to perform high-speed scanning and observe in real-time on the monitoring screen. The high speed of this type of camera is a very important factor that directly affects the focusing and position retrieval speed.
5. The relationship between focal position and brightness
In a confocal optical system, when the sample is correctly placed in the focal position, the brightness is high, and the brightness decreases sharply before and after it (solid line in Figure 4). The sensitive selectivity of this focal plane is precisely the principle behind the determination of height direction and the expansion of focal depth in confocal microscopy. Compared to this, typical optical microscopes do not show significant brightness changes before and after the focal position (dotted line in Figure 4).
6. High contrast and resolution
A typical optical microscope, due to the interference caused by the reflected light deviating from the focal point, overlaps with the focal imaging part, resulting in a decrease in image contrast. In contrast, in confocal optical systems, scattered light outside the focus and scattered light inside the objective lens are almost completely removed, thus obtaining images with very high contrast. In addition, due to the fact that light passes through the objective lens twice, the point image becomes sharper first, which also improves the resolution of the microscope.
