How to optimize the resolution of the microscope

microscopeAs one of the main equipment of testing equipment, and judgemicroscopeAn important indicator of performance is resolution. Resolution refers to the smallest distance between two small dots or two lines that can be clearly distinguished. The human eye itself is a microscope. Under standard lighting conditions, the resolution of the human eye at the photopic distance (the internationally recognized 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 human eye cannot distinguish the distance between an object smaller than 1/10mm or two tiny objects closer than 1/10mm. 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 minimum distance between two small points that can be clearly distinguished on a 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 known as the lens aperture ratio).

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. It can be seen that the method of improving the optical microscope:

1. 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 one-half the aperture angle of the objective lens. Therefore, it is the most common method to increase the resolution of optical microscopes by appropriately adopting a larger aperture angle or increasing the refractive index from the optical design. Generally, low magnification objectives such as below 10X use air as the medium, with a refractive index of 1, that is, a dry objective; the water immersion medium is distilled water, and its refractive index is 1.33; the oil immersion objective medium is cedar oil or other transparent oil, and its refractive index is average Around 1.52, close to the refractive index of the lens and glass slide, such as Olympus 100X oil mirror. The water immersion objective lens and the oil immersion objective lens not only have high magnification, but also increase the resolution of the objective lens due to the use of a high refractive index medium.

2. Reduce the wavelength of the light source.

The shortest 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 a wavelength below 340nm, the ultraviolet light cannot form a clear and bright image after a large amount of attenuation. Therefore, expensive materials such as quartz (which can transmit ultraviolet light as low as 200nm) and fluorite (which can transmit ultraviolet light as low as 185nm) have to be used, and ultraviolet light microscopes cannot be observed with the naked eye, and even samples under observation In addition to the high cost, this method of improving the resolution of the microscope is not widely used due to its own limitations.