Why do we need a confocal microscope?
1. After the efforts and improvements of our great predecessors, the optical microscope has reached a perfect level. In fact, ordinary microscopes can provide us with beautiful microscopic images simply and quickly. However, the event that brought revolutionary innovation to this almost perfect microscope world 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 from the surface where the focus is concentrated. By changing the focus while restoring the obtained information in the image memory, it can obtain a vivid image with complete three-dimensional 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, "improving 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
Schematic diagram of laser confocal microscope
The confocal optical system is used to illuminate the sample with points, while the reflected light is also received by a point sensor. When the sample is placed at the focal point, almost all of the reflected light can reach the photosensitive device. When the sample deviates from the focal point, the reflected light cannot reach the photosensitive device. That is to say, in a confocal optical system, only images that coincide with the focal point will be output, and the light spot and useless scattered light will be shielded.
Why use laser?
In a confocal optical system, the sample is illuminated and the reflected light is also received by a point sensor. Therefore, point light sources become necessary. Laser is a very point light source. In most cases, the light source of a confocal microscope is a laser light source. 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 Deflector (AO prime) 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 nature of this type of camera is a very important item that directly affects the speed of focusing and position retrieval.
5. Relationship between focal position and brightness
In a confocal optical system, the brightness of the sample is high when it is correctly placed in the focal position, and its 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
Usually, in optical microscopes, the reflected light that deviates from the focal point will occur
