Methods of improving the resolution of microscope
Microscope is one of the main detection equipment, and the resolution is an important index to judge the performance of microscope. Resolution refers to being able to clearly distinguish the smaller distance between two dots or two lines. The human eye itself is a microscope. Under standard lighting conditions, the resolution of the human eye at the apparent distance (internationally recognized as 25cm) is about 1/10 mm. For observing two straight lines, the resolution of the eyes can be improved because the straight lines can stimulate a series of nerve cells.
The human eye's resolution is only 1/ 10mm, so the human eye can't distinguish the distance between an object smaller than 1/ 10mm or two tiny objects closer than 1/10 mm. Therefore, from a simple macro magnifying glass to an optical microscope for microscopic observation, and then an electron microscope appeared. 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 light source (um); NA is the numerical aperture (also called aperture ratio) of the objective lens.
According to the formula, the resolution of the microscope depends on the wavelength of the incident light source and the numerical aperture of the matched objective lens. Therefore, the methods to improve the optical microscope are as follows:
1. Reduce the wavelength of light source.
The shorter wavelength of visible light is 390nm. If ultraviolet light with this wavelength is used as illumination source, the resolution of optical microscope can be reduced to 0.2um. However, because most ordinary glass materials will absorb a large amount of light with a wavelength below 340nm, ultraviolet light cannot form a clear and bright image after a large amount of attenuation. Therefore, expensive materials such as Shi Ying (which can transmit ultraviolet light as low as 200nm) and fluorspar (which can transmit ultraviolet light as low as 185nm) have to be used, and the ultraviolet microscope can't be observed with naked eyes, even limited by the observed samples, plus the expensive cost, so this way to improve the resolution of the microscope is not widely used because of 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 the half aperture angle of the objective lens. Therefore, it is a common method to improve the resolution of optical microscope by adopting a larger aperture angle or increasing the refractive index in optical design. Generally, the medium of a low-power objective lens, such as below 10X, is air with a refractive index of 1, that is, a dry objective lens; The immersion medium is distilled water with a refractive index of 1.33; The medium of oil-immersed objective lens is fragrant tar or other transparent oil, and its refractive index is generally around 1.52, which is close to the refractive index of lens and slide, such as 100X oil mirror of Olympus. The water immersion objective lens and the oil immersion objective lens not only have high magnification, but also improve the resolution of the objective lens because of the medium with high refractive index.
