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), and the relationship is NA=η·sinu/2. It is the main technical parameter of the objective lens and 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 imaging quality. When the objective lens is observed, the aperture angle cannot be changed, and the change of the refractive index of different media can change the NA. Therefore, water immersion objective lens and oil immersion objective lens are derived. Water η=1.333, the NA of the water immersion objective can be 0.1~1.25; cedar oil η=1.515, the NA of the oil immersion objective can be 0.80~1.45; the new ring Bronaphthalene η=1.66, the objective NA≥1.40.
Numerical aperture is proportional to resolution, magnification, image brightness, and inversely proportional to depth of focus. When 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 the 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 optical 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. The visible light source can only resolve two object points at a minimum distance of 0.4 μm.
The improvement of resolution depends on 4 related factors: 1. When a light source with a shorter wavelength is used, λ decreases; 2. When a medium with a higher refractive index is used, η increases and NA increases; 3. Design and manufacture a larger aperture angle of the objective lens ; 4. Increase the contrast of light and dark in the image and improve the image clarity.
gain
Depth of focus
Refers to the depth of focus, that is, the range of the same observation clear range above and below the focal plane of the sample. The greater the depth of focus, the more layers in which the sample will be sharp.
① The depth of focus is inversely proportional to the total magnification, the numerical aperture of the objective lens, and the 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 accommodated in the circular field of view of the microscope, also known as the field of view diameter. The larger it is, the greater the amount of sample information.
① The width of the field of view is proportional to the number of fields of view of the eyepiece. If the magnification of the eyepiece remains unchanged, the larger the number of fields of view, the larger the width of the field of view, which is convenient for observation (Note: the number of fields of view refers to the width of the field of view of the eyepiece, which is represented by FN, and is marked on the eyepiece shell). ②The magnification of the objective lens increases, and the width of the field of view becomes smaller. That is, the whole picture is seen under a low-power lens, and the part is seen under a high-power lens.
poor coverage
The international standard for the thickness of the sample cover glass is 0.17mm, and the objective lens has corrected for this aberration and is marked on the housing. When the light enters the air through a cover glass with a non-standard thickness, it is refracted, and the resulting aberration is called poor coverage.
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 thickness of the coverslip increases, poor coverage increases and focusing becomes difficult.
(2) The oil immersion objective has no 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 thickness of the cover glass, and the stricter the quality requirements for the thickness of the cover glass.
working distance
Refers to the distance between the front lens surface of the objective lens and the sample, also known as the object distance. The sample should be at 1 to 2 of the focal length of the objective lens during observation. 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 remains unchanged, if the working distance is shortened, the aperture angle needs to be increased. The higher the NA of the high-power objective, the smaller the working distance.
Mirror Brightness vs Field Brightness
(1) The brightness of the mirror image is the brightness of the image, which indicates the brightness of the image observed by the eyes. It is required not to be dim, not dazzling, and not fatigued.
(2) The brightness of the field of view is the brightness of the field of view under the microscope, which is affected by various factors such as the objective lens, eyepiece, and light source intensity.
The relationship between the brightness of the mirror image and other technical parameters of the microscope has two main points.
(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.
objective lens
The objective lens is the first imaging optical component of the microscope and consists of multiple groups of lenses cemented together. Focal length is the total focal length of the lens group.
Depending on the degree of correction for chromatic aberrations, aberrations, field curvature, etc., as well as proprietary characteristics, there are various types of objectives: (plan) achromatic objectives, (plan) apochromatic objectives, ultra-plan and specialty objectives, etc.
eyepiece
The eyepiece magnifies the real image of the objective lens, which is the magnification of the intermediate image, which is the second magnification. The eyepiece structure is relatively simple, consisting of several lenses in several groups. The point where the light rays passing through the eyepiece intersect at the top is called the eye point, which is the best position for imaging observation.
Eyepieces have a variety of magnification configurations, 10X is the most commonly used; 5X has higher imaging reproducibility, but the magnification is small; 20X eyepieces have the largest magnification, but the image clarity is reduced. Choose according to actual needs.
condenser
The condenser lens is used to make up for the lack of light quantity, appropriately change the light properties of the light source, focus the sample, and improve the illumination. It is located under the stage and must be matched when using an NA≥0.40 objective. It has a variety of structures, and the requirements for the condenser are also different for the numerical aperture of the objective lens.
1. Abbe condenser: Abbe condenser consists of two lenses, which has better light-gathering ability. When the objective lens of the ordinary microscope is NA ≥ 0.60, the correction of chromatic aberration and spherical aberration is incomplete and needs to be used together.
2. Achromatic aplanatic condenser: Achromatic condenser consists of a series of lenses, which can correct chromatic aberration and spherical aberration and obtain satisfactory imaging. It is the best one in bright field observation, equipped with advanced microscope and low magnification objective lens Not applicable.
3. Other condensers refer to condensers used for other purposes than the above brightfield, such as darkfield condensers, phase contrast condensers, polarized condensers, differential interference condensers, etc.
