Comparison between confocal microscope and ordinary optical microscope
General optical microscope
The general biological microscope consists of three parts, namely: ① illumination system, including light source and condenser; ② Optical amplification system, which consists of objective lens and eyepiece, is the main body of microscope. In order to eliminate spherical aberration and chromatic aberration, both eyepiece and objective lens are composed of complex lens groups; (3) mechanical device, used for fixing materials and convenient observation.
Whether the microscope image is clear or not depends not only on the magnification, but also on the resolution of the microscope. Resolution refers to the ability of the microscope (or the place where human eyes are 25cm away from the target) to distinguish the small interval of the object zui. The resolution depends on the wavelength of light, the aperture ratio and the refractive index of the medium, which is expressed by the formula:
R=0.61λ /N．A． N．A．＝ｎsinα/2
Where: n= refractive index of medium; α = mirror angle (the opening angle of the specimen to the lens aperture), and N.A.= numeric aperture. The mirror angle is always less than 180? Therefore, the zui value of sina/2 must be less than 1.
The refractive index of the glass used for making optical lenses is 1.65~1.78, and the refractive index of the medium used is closer to that of the glass, the better. For the dry objective lens, the medium is air, and the aperture ratio is generally 0.05 ~ 0.95; The oil lens uses fragrant asphalt as the medium, and the lens opening rate can be close to 1.5.
The wavelength of ordinary light is 400~700nm, so the resolution of microscope is not less than 0.2μm, and the resolution of human eye is 0.2mm, so the large magnification of zui designed by general microscope is usually 1000X x.
Why do you need a confocal microscope?
1. The optical microscope has been perfected through the efforts and improvement of our great predecessors. In fact, ordinary microscopes can provide us with beautiful microscopic images simply and quickly. However, an event that brought revolutionary innovation to this almost perfect microscope world happened, which is the invention of "laser scanning confocal microscope". This new microscope is characterized by adopting an optical system that only extracts the image information on the plane where the focus is concentrated, and restoring the obtained information in the image memory while changing the focus, so that a vivid image with complete three-dimensional information can be obtained. By this method, information about the surface shape that can't be confirmed by ordinary microscopes can be simply obtained. In addition, for ordinary optical microscopes, "improving the resolution" and "deepening the depth of focus" are contradictory conditions, especially at high magnification, but for confocal microscopes, this problem is solved.
2. Advantages of confocal optical system
Confocal optical system illuminates the sample point, and the reflected light is also received by point receptors. When the sample is placed in the focus position, almost all the reflected light can reach the photoreceptor, but when the sample deviates from the focus, the reflected light cannot reach the photoreceptor. That is to say, in the confocal optical system, only the image that coincides with the focus will be output, and the facula and useless scattered light will be shielded.
3. Why use a laser?
In the confocal optical system, the sample is illuminated and the reflected light is also received by a point photoreceptor. Therefore, point light source becomes necessary. Laser belongs to a very point light source. In most cases, the light source of confocal microscope adopts laser light source. In addition, the characteristics of laser, such as monochromaticity, directivity and excellent beam shape, are also important reasons for its wide use.
4. Real-time observation based on high-speed scanning becomes possible.
In the laser scanning, the Acoustic Optical Deflector (AO prime element) is used in the horizontal direction and the Servo Galvano-mirror is used in the vertical direction. Because there is no mechanical vibration part in the acoustic optical deflection unit, it can scan at high speed, and it is possible to observe in real time on the monitoring screen. The high speed of this camera is a very important project that directly affects the speed of focusing and position retrieval.
5. Relationship between focus position and brightness
In the confocal optical system, when the sample is correctly placed in the focus position, the brightness is large, and before and after it, its brightness will drop sharply (solid line in Figure 4). This sensitive selectivity of the focal plane is also the principle of measuring the height direction of confocal microscope and expanding the focal depth. In contrast, the ordinary optical microscope does not have obvious brightness change before and after the focus position (dotted line in Figure 4).
6. High contrast and high resolution
In general optical microscope, the reflected light deviating from the focus will interfere, and it will overlap with the focus imaging part, thus reducing the image contrast. In contrast, in the confocal optical system, the scattered light outside the focus and the scattered 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 first, and the resolution of the microscope is also improved.
7. Optical localization function
In the confocal optical system, the reflected light of the part other than the focal point is shielded by micropores. Therefore, when observing a three-dimensional sample, an image like that formed after slicing the sample with the focus is formed (Figure 5). This effect is called optical localization, which belongs to one of the specialties of confocal optical system.
8. Focus moving memory function
The so-called reflected light outside the focus 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 focus. Therefore, if the three-dimensional sample is moved along the Z-axis (optical axis) direction, the image will be accumulated and stored in the memory, and zui will eventually get the image formed by the coincidence of the whole sample and the focus. In this way, the function of infinite depth of focus is called mobile memory function.
9. Surface shape measuring function
In the focus moving function, the surface shape of the sample can be measured in a non-contact way by adding a height recording loop. Based on this function, it is possible to record the Z-axis coordinates formed by the large brightness value of zui in each pixel, and based on this information, the information related to the surface shape of the sample can be obtained.
10. High-precision micro-size measurement function
The light receiving unit adopts a one-dimensional CCD imaging sensor, so it can be unaffected by the scanning tilt of the scanning device, so that high-precision measurement can be completed. In addition, because the focus moving memory function with adjustable focus depth is adopted at the same time, the measurement error caused by focus offset can be eliminated.
11. Three-dimensional image analysis
Using the surface shape measuring function, the three-dimensional image of the sample surface can be easily made. Not only that, but also many kinds of analysis can be carried out, such as: surface roughness measurement, area, volume, surface area, circularity, radius, length of zui, perimeter, center of gravity, tomographic image, FFT transform, line width measurement and so on.
Laser confocal scanning microscope can be used not only to observe cell morphology, but also for quantitative analysis of biochemical components in cells, optical density statistics and cell morphology measurement.
