How to improve the resolution of microscope?
Microscope is one of the main equipment of inspection equipment, and the important index to judge the performance of microscope is resolution. Resolution refers to the ability to clearly distinguish two small points or small distances between two lines. The human eye itself is a microscope. Under standard lighting conditions, the resolution of the human eye at the photopic distance (25cm is internationally recognized) is approximately equal to 1/10mm. For viewing two straight lines, the resolution of the eye can also be improved because the straight line stimulates a series of nerve cells.
The resolution of the human eye is only 1/10mm, so the distance between an object smaller than 1/10mm or two tiny objects closer than 1/10mm cannot be distinguished by the human eye. So there appeared from simple macroscopic magnifying glass to optical microscope for microscopic observation, and then electron microscope. The definition of microscope resolution is the small distance between two small points that can be clearly resolved on a specimen. Its calculation formula is: D=0.61λ/NA
In the formula: D is the resolution (um); λ is the wavelength of the light source (um); NA is the numerical aperture (also called the lens aperture) of the objective lens.
It can be obtained from the formula that the resolution of the microscope is determined by the wavelength of the incident light source and the numerical aperture of the matched objective lens. It can be seen that the method of improving the optical microscope:
1. Reduce the wavelength of the light source.
The shorter wavelength of visible light is 390nm. If the ultraviolet light of this wavelength is used as the illumination light source, the resolution of the optical microscope can be reduced to 0.2um. However, because most common materials of glass absorb a large amount of light with wavelengths below 340 nm, ultraviolet light cannot form a clear and bright image after a lot of attenuation. Therefore, expensive materials such as quartz (which can transmit ultraviolet light as low as 200 nm) and fluorite (which can transmit ultraviolet light as low as 185 nm) have to be used, and ultraviolet light microscopes cannot be observed with the naked eye, even the observed samples The limitation of , coupled with the high cost, so this way of improving microscope resolution is not widely used due to its own limitations.
2. Increase the numerical aperture NA of the objective lens.
Numerical aperture NA=n*sin(u)
In the formula, n is the refractive index of the medium between the objective lens and the specimen; u is the half aperture angle of the objective lens. Therefore, from the perspective of optical design, appropriately using a larger aperture angle, or increasing the refractive index, has become a common method to improve the resolution of an optical microscope. Generally, low magnification objective lens, such as below 10X, uses air as the medium, with a refractive index of 1, that is, a dry objective lens; the water immersion medium is distilled water, and its refractive index is 1.33; the oil immersion objective lens is cedar oil or other transparent oil, and its refractive index is generally At around 1.52, it is close to the refractive index of lenses and glass slides, such as Olympus' 100X oil lens. Water immersion objectives and oil immersion objectives not only have high magnification, but also improve the resolution of the objective due to the use of high refractive index media
