Microscope common technical parameters and proper nouns
Numerical aperture NA
Numerical aperture NA refers to the refractive index (η) of the medium between the front lens of the objective lens and the sample multiplied by the half of the aperture angle (u). The relationship is NA=η·sinu/2. It is the main technical parameter of the objective lens and the condenser lens. An important indicator for judging the performance of the objective lens is marked on the objective lens housing.
The larger the numerical aperture, the better the image quality. The aperture angle cannot be changed when the objective lens is observed, and the NA can be changed by the change of the refractive index of different media. Therefore, derived water immersion objective lens, oil immersion objective lens. Water η=1.333, water immersion objective lens NA can be 0.1~1.25; cedar oil η=1.515, oil immersion objective lens NA can be 0.80~1.45; new ring bromonaphthalene η=1.66, objective lens NA≥1.40.
Numerical aperture is directly proportional to resolution, magnification, and image brightness, and inversely proportional to depth of focus. As the NA increases, the width of the field of view and the working distance decrease accordingly.
resolution
Resolution refers to the minimum resolution distance at which light spots show differences in the imaging process, expressed as d=λ/NA, where d is the minimum resolution distance, λ is the wavelength of the fiber, and NA is the numerical aperture of the objective lens. It can be seen that the larger the NA, the shorter the λ, the smaller the d, and the higher the resolution. Visible light sources can only resolve two object points at a distance of 0.4 μm.
The improvement of resolution depends on 4 related factors: 1. Using a light source with a shorter wavelength, λ decreases; 2. Using a medium with a higher refractive index, η increases, and NA increases; 3. Design and manufacture a larger aperture angle of the objective lens ; 4. Increase the light and dark contrast of the image and improve the image clarity.
gain
depth of focus
Refers to the depth of the focal point, that is, the interval range above and below the focal plane of the sample that is also clearly observed. The greater the depth of focus, the more layers of the sample will be in focus.
① Depth of focus is inversely proportional to total magnification, numerical aperture of objective lens, and image resolution. The higher the magnification, the larger the NA value, the smaller the depth of focus, and the higher the resolution.
②The refractive index of the surrounding medium such as the mounting agent prepared by the sample increases, and the depth of focus becomes larger.
Field of view width
Refers to the actual range of the sample contained in the circular field of view of the microscope, also known as the diameter of the field of view. The larger it is, the larger the amount of sample information.
①The width of the field of view is proportional to the number of fields of view of the eyepiece. If the magnification of the eyepiece is constant, the larger the number of field of view, the larger the width of the field of view, which is convenient for observation (Note: the number of field of view refers to the width of the field of view of the eyepiece, expressed by FN, and marked on the outer shell of the eyepiece). ②The magnification of the objective lens increases, and the width of the field of view becomes smaller. That is, the whole picture can be seen under the low-power lens, and the part can be seen under the high-power lens.
poor coverage
The international standard for sample cover glass thickness is 0.17mm. The objective lens has been corrected for this phase difference and marked on the casing. When the light passes through the cover glass of non-standard thickness and enters the air, refraction occurs, and the phase difference caused is called coverage difference.
Poor coverage affects the quality of microscopic imaging. When observing samples, you need to understand the following three points:
(1) The higher the magnification, the larger the NA value, and the more obvious the coverage difference. As the coverslip thickness increases, poor coverage increases and focusing becomes difficult.
(2) The oil immersion objective lens does not have the problem of poor coverage, because the refractive index of the oil and the cover glass are both 1.52, forming a uniform optical system.
(3) The larger the NA value of the objective lens, the smaller the allowable error of the cover glass thickness, and the stricter the quality requirements for the cover glass thickness.
working distance
Refers to the distance between the front lens surface of the objective lens and the sample, also known as the object distance. When observing, the sample should be at 1~2 of the focal length of the objective lens. It and the focal length are two concepts. The focusing of the microscope is actually adjusting the working distance.
When the numerical aperture (NA) of the objective lens is constant, if the working distance is shortened, the aperture angle needs to be increased. The NA of the high-magnification objective lens is large, and the working distance becomes small.
Mirror Brightness vs Field Brightness
(1) The brightness of the mirror image is the brightness of the image, which indicates the brightness and darkness of the image observed by the eyes, and it is required not to be dim, dazzling, or fatigued.
(2) The brightness of the field of view is the lightness and darkness of the field of view under the microscope, which is affected by various factors such as the objective lens, eyepiece, and light source intensity.
There are two main points about the relationship between mirror brightness and other technical parameters of the microscope.
(1) The brightness of the mirror image is proportional to the square of the numerical aperture (NA). Under the same conditions, the brightness of the objective lens with a large NA is significantly improved.
(2) The brightness of the mirror image is inversely proportional to the square of the total magnification. Under the same conditions, the magnification of the eyepiece increases and the brightness of the mirror image decreases.
