What is the purpose of a confocal microscope?
1. After the efforts and improvements of our great predecessors, the optical microscope has reached the point of perfection. In fact, ordinary microscopes can provide us with beautiful microscopic images simply and quickly. However, an event that brought revolutionary innovation to this nearly perfect microscope world happened, which is the invention of the "laser scanning confocal microscope". The feature 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, and restores the obtained information in the image memory while changing the focus, so that complete 3D information can be obtained. A stark image of intelligence. With this method, it is possible to easily obtain information about the surface shape that cannot be confirmed with a normal microscope. In addition, for ordinary optical microscopes, "increasing resolution" and "deepening depth of focus" are contradictory conditions, especially at high magnifications, this contradiction is more prominent, but in terms of confocal microscopes, this problem is easily solved.
2. Advantages of confocal optical system
Schematic diagram of laser confocal microscope
The confocal optical system performs point illumination on the sample, and the reflected light is also received by the point receptor. When the sample is placed in the focus position, almost all the reflected light can reach the photoreceptor, and when the sample is out of focus, the reflected light cannot reach the photoreceptor. That is to say, in the confocal optical system, only the image that coincides with the focal point 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 by a point photoreceptor. Therefore, a point light source becomes necessary. Lasers are very point light sources. In most cases, laser light sources are used as light sources for confocal microscopes. In addition, the characteristics of monochromaticity, directionality and excellent beam shape of laser are also important reasons for its wide adoption.
4. Real-time observation based on high-speed scanning becomes possible
For laser scanning, the acoustic-activated optical deflection unit (Acoustic Optical Deflector, AO element) is used in the horizontal direction, and the Servo Galvano-mirror is used in the vertical direction. Since the acousto-optical deflection unit has no mechanical vibration part, it can perform high-speed scanning and real-time observation on the monitor screen is possible. This high-speed 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 of the sample is the maximum when the sample is correctly placed in the focal position, and its brightness will decrease sharply before and after it (the solid line in Figure 4). The sensitive selectivity of the focal plane is also the principle of confocal microscope height direction determination and focal depth expansion. In contrast, ordinary optical microscopes do not have significant brightness changes before and after the focus position.
6. High contrast, high resolution
In ordinary optical microscopes, due to the interference of the reflected light off the focus part, it overlaps with the focus imaging part, resulting in a decrease in image contrast. On the other hand, in the confocal optical system, the scattered light outside the focal point and the scattered light inside the objective lens are almost completely removed, so that 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 first, which also improves the resolving power of the microscope.
7. Optical localization function
In the confocal optical system, the reflected light other than the point coincident with the focal point is shielded by the micropore. Therefore, when observing a three-dimensional sample, an image is formed as if the sample is sliced with the focal plane (Figure 5). This effect is known as optical localization and is one of the specialties of confocal optical systems.
8. Focus mobile memory function
The so-called reflected light outside the focal point is shielded by the micropores. On the other hand, it can be considered that all points on the image formed by the confocal optical system coincide with the focal point. Therefore, if the three-dimensional sample is moved along the Z-axis (optical axis), the images are accumulated and stored in the memory, and finally the image formed by the entire sample and the focal point will be obtained. The function of infinitely deepening the depth of focus in this way is called the function of mobile memory.
9. Surface shape measurement function
In terms of focus shifting function, the surface shape of the sample can be measured in a non-contact manner by adding a surface height recording circuit. Based on this function, it is possible to record the Z-axis coordinates formed by the maximum luminance value in each pixel, and based on this information, information related to the shape of the sample surface can be obtained.
10. High-precision micro-size measuring function
The light-receiving unit adopts a 1-dimensional CCD imaging sensor, so it is not affected by the scanning tilt of the scanning device, so that high-precision measurement can be completed. In addition, due to the use of the focus shift memory function with adjustable focus depth (deepening), the measurement error caused by focus shift can be eliminated.
11. Three-dimensional image analysis
Using the surface shape measurement function, you can easily create a three-dimensional image of the sample surface. Not only that, but also can perform a variety of analysis such as: surface roughness measurement, area, volume, surface area, circularity, radius, maximum length, perimeter, center of gravity, tomographic image, FFT transformation, line width measurement, etc.
Laser confocal scanning microscope can be used not only for observing cell morphology, but also for quantitative analysis of intracellular biochemical components, optical density statistics and measurement of cell morphology.
