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 dots or the small distance between two lines. The human eye itself is a microscope. Under standard lighting conditions, the resolution of the human eye at a photopic distance of 25cm is approximately equal to 1/10mm. For observing two straight lines, since the straight line can stimulate a series of nerve cells, the resolution of the eye can be improved.
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 has been a simple macroscopic magnifying glass to an optical microscope for microscopic observation, and then an electron microscope. The definition of microscope resolution refers to the small distance between two small points that can be clearly distinguished on the specimen. The calculation formula is: D=0.61λ/NA where: D is the resolution (um); λ is the wavelength of the light source (um); NA is the numerical aperture of the objective lens, also called the aperture ratio).
According to the formula, 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 ultraviolet light of this wavelength is used as the illumination source, the resolution of the optical microscope can be reduced to 0.2um. However, since most common glass materials absorb a large amount of light with wavelengths below 340nm, the ultraviolet light cannot form a clear and bright image after a large amount of attenuation. Therefore, quartz (which can transmit ultraviolet light as low as 200nm, fluorite (can transmit ultraviolet light as low as 185nm, and other expensive materials) have to be used, and the ultraviolet microscope cannot be observed with the naked eye, even the sample In addition to the high cost, this method of improving the resolution of the microscope 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, where n is the refractive index of the medium between the objective lens and the specimen; u is one-half the aperture angle of the objective lens. Therefore, a larger aperture angle should be appropriately used in optical design. Or increasing the refractive index has become a common method to improve the resolution of optical microscopes. Generally, low-power objectives such as 10X or less use air as the medium, with a refractive index of 1, which is a dry objective lens; the water immersion medium is distilled water with a refractive index of 1.33 ; The medium of the oil immersion objective lens is cedar oil or other transparent oil, and its refractive index is generally about 1.52, which is close to the refractive index of the lens and glass slide, such as Olympus 100X oil lens. Water immersion objective lens and oil immersion objective lens not only have high magnification but also Due to the use of high refractive index media, the resolution of the objective lens is improved.