The relationship between the total magnification and resolution of microbiological oil mirrors
The objective lenses of microscopes used in microbiology research are usually oil low magnification objective lenses (10 x), high magnification objective lenses (40 x), and oil lenses (100 x). There is also the word "OI" (oil immersion) indicating that it is the one with the highest magnification among the three. According to the use of eyepieces with different magnifications, the inspected object can be magnified by 1000-1600 times. When in use, the difference between the oil lens and other objective lenses is that there is not a layer of air between the slide and the objective lens, but a layer of oil, which is called the oil immersion system. This type of oil often uses cedar oil because its refractive index is n=1.52, which is the same as glass. When light passes through a glass slide, it can directly enter the objective lens through cedar oil without refraction. If the medium between the glass slide and the objective lens is air, it is called a dry system. When light passes through the glass slide, it undergoes scattering due to refraction, and the amount of light entering the objective lens is obviously reduced, which reduces the illumination of the field of view. The use of oil mirrors can not only increase illumination, but also primarily increase numerical aperture, as the magnification efficiency of a microscope is determined by its numerical aperture. The so-called numerical aperture refers to the product of half the sine of the maximum angle at which light is projected onto the objective lens (known as the aperture angle) multiplied by the refractive index of the medium between the glass slide and the objective lens. It can be expressed by the following formula: NA=n x sin α, where NA=numerical aperture; N=refractive index of the medium; A=half of the maximum incident angle, i.e. half of the aperture angle. Therefore, the greater the angle at which light is projected onto the objective lens, the greater the efficiency of the microscope, and the magnitude of this angle is determined by the diameter and focal length of the objective lens. Meanwhile, the theoretical limit of a is 90.. sin90.＝1, Therefore, when using air as the medium (n=1), the numerical aperture cannot exceed 1. For example, when using tar as the medium, n increases, and its numerical aperture also increases. If the incident angle of light is 120o and half of its sine is sin60o=0.87, then: when using air as the medium: NA=1 x 0.87=0.87, when using water as the medium: NA=1.33 x 0.87=1.15, when using tar as the medium: NA=1.52 x 0.87=1.32. The resolution of a microscope refers to its ability to distinguish the minimum distance between two points. It is proportional to the numerical aperture of the objective lens and inversely proportional to the wavelength length. Therefore, the larger the numerical aperture of the objective lens, the shorter the wavelength of the light wave, and the greater the resolution of the microscope. The finer structures of the object being tested can also be clearly distinguished. Therefore, a high resolution means a small distinguishable distance, and these two factors are inversely proportional. Some people often refer to resolution as the number of micrometers or nanometers, which actually confuses resolution with the minimum resolution distance. The resolution of a microscope is represented by the minimum distance that can be resolved. The minimum distance between two points that can be distinguished is λ/2NA. In the formula, λ=wavelength of the light wave, and the average length of the light wave that can be perceived by the naked eye is 0.55 μ m. If a high-power objective with a numerical aperture of 0.65 is used, it can distinguish the distance between two points as 0.42 μ m. However, the distance between two points below 0.42 μ m cannot be distinguished, even with a larger magnification eyepiece, the total magnification of the microscope still cannot be distinguished. Only by using larger objective lenses with larger numerical apertures can their resolution be increased. For example, when using an oil mirror with a numerical aperture of 1.25, the minimum distance between two points that can be distinguished is 0.55/(2 x 1.25)=0.22 μ m. Therefore, we can see that if a high-power objective with a magnification of 40 times (NA=0.65) and an eyepiece with a magnification of 24 times are used, although the total magnification is 960 times, the minimum resolution distance is only 0.42 μ m. If an oil mirror with a magnification of 90 times (NA=1.25) and an eyepiece with a magnification of 9 times are used, although the total magnification is 810 times, a distance of 0.22 μ m can be distinguished.
