How to improve microscope resolution?
Microscopes are one of the main equipment for testing, and the important indicator for evaluating the performance of microscopes is resolution. Resolution refers to the ability to clearly distinguish between two small points or the smaller distance between two lines. The human eye itself is a microscope, and under standard lighting conditions, the resolution of the human eye at a visual distance (internationally recognized as 25cm) is approximately 1/10mm. For observing two straight lines, as they can stimulate a series of nerve cells, the resolution of the eyes can also be improved.
The resolution of the human eye is only 1/10mm, so the distance between objects smaller than 1/10mm or two tiny objects closer than 1/10mm cannot be distinguished by the human eye. So there has been a shift from simple macroscopic magnifying glasses to optical microscopes for microscopic observation, followed by electron microscopes. The resolution of a microscope is defined as the smaller distance between two small points that can be clearly distinguished on a specimen. The calculation formula is: D=0.61 λ/NA
In the formula: D is the resolution (um); λ is the wavelength of the light source (um); NA is the numerical aperture (also known as aperture ratio) of the objective lens.
According to the formula, the resolution of a microscope depends on the wavelength of the incident light source and the numerical aperture of the matched objective lens. From this, it can be seen that the method to improve optical microscopy is:
1. Reduce the wavelength of the light source.
The visible light has a shorter wavelength of 390nm. If ultraviolet light of this wavelength is used as an illumination light source, the resolution of the optical microscope can be reduced to 0.2um. However, due to the fact that most ordinary glass materials absorb a large amount of light with wavelengths below 340nm, ultraviolet light cannot form clear and bright images after significant attenuation. Therefore, expensive materials such as quartz (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 ultraviolet microscopes cannot be observed with the naked eye, and are even limited by the observed samples, coupled with expensive costs. Therefore, this method 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, adopting a larger aperture angle or increasing the refractive index in optical design has become a common method to improve the resolution of optical microscopes. Generally, low magnification objective lenses below 10X use air as the medium and have a refractive index of 1, which is a dry type objective; The water immersion medium is distilled water, with a refractive index of 1.33; The medium for oil immersed lenses is tar or other transparent oil, with a refractive index generally around 1.52, which is close to the refractive index of lenses and glass slides, such as Olympus's 100X oil lens. Water immersion objective and oil immersion objective not only have high magnification, but also improve the resolution of the objective due to the use of high refractive index media.
