Why do you need to use a confocal microscope?
1. Optical microscopes have reached a state of perfection through the efforts and improvements of our great predecessors. In fact, ordinary microscopes can provide us with beautiful microscopic images simply and quickly. However, an event occurred that brought revolutionary innovation to this nearly perfect world of microscopy. This was the invention of the "laser scanning confocal microscope." The characteristic of this new type of microscope is that it uses an optical system that extracts image information only on the surface where the focus is focused, and restores the obtained information in the image memory while changing the focus, thereby obtaining complete 3D information. A vivid image of intelligence. Through this method, information about the surface shape that cannot be confirmed with a normal microscope can be easily obtained. In addition, for ordinary optical microscopes, "increasing resolution" and "deepening depth of focus" are conflicting conditions, especially at high magnifications. However, in confocal microscopy, this problem is easily solved.
2. Advantages of confocal optical system
The confocal optical system performs point illumination on the sample, and the reflected light also uses a point receptor to receive the light. When the sample is placed at the focus position, almost all of the reflected light can reach the photoreceptor. When the sample is out of focus, the reflected light cannot reach the photoreceptor. In other words, in the confocal optical system, only the image that coincides with the focus will be output, and the light spots and useless scattered light will be shielded.
3. Why use laser?
In the confocal optical system, the sample is illuminated at a point, and the reflected light is also received using a point photoreceptor. Therefore, point light sources become necessary. Lasers are very point light sources. In most cases, the light source of confocal microscopes uses laser light sources. In addition, the characteristics of laser such as monochromaticity, directivity, and excellent beam shape are also important reasons for its widespread adoption.
4. Real-time observation based on high-speed scanning becomes possible
For laser scanning, an acoustic optical deflector (AO element) is used in the horizontal direction and a servo electrically controlled beam scanning mirror (Servo Galvano-mirror) is used in the vertical direction. Since the acoustic-optical deflection unit does not have a mechanical vibration part, it can scan at high speed, making real-time observation possible on the monitoring screen. The high speed of this kind of imaging is a very important item that directly affects the speed of focusing and position retrieval.
5. The relationship between focus position and brightness
In the confocal optical system, the brightness is maximum when the sample is correctly placed at the focus position, and its brightness will decrease sharply in front and behind it (solid line in Figure 4). This sensitive selectivity of the focal plane is also the principle behind the height direction determination and focus depth expansion of the confocal microscope. In contrast, a normal optical microscope does not have obvious brightness changes before and after the focus position (dotted line in Figure 4).
6. High contrast and high resolution
In ordinary optical microscopes, the reflected light from the out-of-focus part will interfere and overlap with the focus imaging part, resulting in a reduction in image contrast. In contrast, in the confocal optical system, stray light outside the focus and stray light inside the objective lens are almost completely removed, so an image with very high contrast can be obtained. In addition, because the light passes through the objective lens twice, the point image is sharpened, which also improves the resolving power of the microscope.
