How to define and calculate the efficiency of optical microscope
1. Numerical Aperture
Numerical aperture is abbreviated as NA. Numerical aperture is the main technical parameter of objective lens and condenser lens, and is an important indicator to judge the performance of both (especially for objective lens). The size of its numerical value is marked on the shell of the objective lens and the condenser lens respectively.
Numerical aperture (NA) is the product of the refractive index (n) of the medium between the front lens of the objective and the object to be inspected and the sine of half the aperture angle (u). The formula is expressed as follows: NA=nsinu/2
Aperture angle, also known as "mirror angle", is the angle formed by the object point on the optical axis of the objective lens and the effective diameter of the front lens of the objective lens. The larger the aperture angle, the brighter the light entering the objective, which is proportional to the effective diameter of the objective and inversely proportional to the distance from the focal point.
In microscope observation, if you want to increase the NA value, the aperture angle cannot be increased, and the only way is to increase the refractive index n value of the medium. Based on this principle, the water immersion objective lens and the oil immersion objective lens are produced. Since the refractive index n of the medium is greater than 1, the NA value can be greater than 1.
The maximum numerical aperture is 1.4, which is theoretically and technically the limit. At present, bronaphthalene with high refractive index is used as the medium. The refractive index of bronaphthalene is 1.66, so the NA value can be greater than 1.4.
It must be pointed out here that in order to give full play to the effect of the numerical aperture of the objective lens, the NA value of the condenser should be equal to or slightly larger than the NA value of the objective lens during observation.
Numerical aperture has a close relationship with other technical parameters, and it almost determines and affects other technical parameters. It is proportional to the resolution, proportional to the magnification, and inversely proportional to the depth of focus. As the NA value increases, the width of the field of view and the working distance will decrease accordingly.
2. Resolution
The resolution of the microscope refers to the minimum distance between two object points that can be clearly distinguished by the microscope, also known as the "discrimination rate". Its calculation formula is σ=λ/NA
where σ is the minimum resolution distance; λ is the wavelength of light; NA is the numerical aperture of the objective lens. The resolution of the visible objective lens is determined by the NA value of the objective lens and the wavelength of the illumination light source. The larger the NA value, the shorter the wavelength of the illumination light, the smaller the σ value, and the higher the resolution.
To increase the resolution, i.e. reduce the value of σ, the following measures can be taken:
1. Reduce the wavelength λ value and use a short wavelength light source.
2. Increase the n value of the medium to increase the NA value (NA=nsinu/2).
3. Increase the aperture angle u value to increase the NA value.
4. Increase the contrast between light and dark.
3. Magnification and effective magnification
Due to the two magnifications of the objective lens and the eyepiece, the total magnification Γ of the microscope should be the product of the objective lens magnification β and the eyepiece magnification Γ1:
Γ=βΓ1
Obviously, the microscope can have much higher magnification than the magnifying glass, and the magnification of the microscope can be easily changed by exchanging objective lenses and eyepieces with different magnifications.
Magnification is also an important parameter of the microscope, but you cannot blindly believe that the higher the magnification, the better. The limit of microscope magnification is the effective magnification.
Resolution and magnification are two distinct but related concepts. There is a relational formula: 500NA<Γ<1000NA
When the numerical aperture of the selected objective lens is not large enough, that is, the resolution is not high enough, the microscope cannot distinguish the fine structure of the object. At this time, even if the magnification is increased excessively, only an image with a large outline but unclear details can be obtained. , called the ineffective magnification. On the other hand, if the resolution has met the requirements and the magnification is insufficient, the microscope has the ability to resolve, but the image is too small to be clearly seen by the human eye. Therefore, in order to give full play to the resolving power of the microscope, the numerical aperture should be reasonably matched with the total magnification of the microscope.
4. Depth of focus
The depth of focus is the abbreviation of the depth of focus, that is, when using a microscope, when the focus is on an object, not only the points on the plane of the point can be seen clearly, but also within a certain thickness above and below the plane. Clearly, the thickness of this clear part is the depth of focus. When the depth of focus is large, the entire layer of the object to be inspected can be seen, while when the depth of focus is small, only a thin layer of the object to be inspected can be seen. The depth of focus has the following relationship with other technical parameters:
1. The depth of focus is inversely proportional to the total magnification and the numerical aperture of the objective.
2. The depth of focus is large and the resolution is reduced.
Due to the large depth of field of the low magnification objective lens, it is difficult to take pictures with the low magnification objective lens. Details will be described in the photomicrographs.
Five, the diameter of the field of view (FieldOfView)
When viewing a microscope, the bright circular area seen is called the field of view, and its size is determined by the field diaphragm in the eyepiece.
The diameter of the field of view is also called the width of the field of view, which refers to the actual range of the object under inspection that can be accommodated in the circular field of view seen under the microscope. The larger the diameter of the field of view, the easier it is to observe.
There is formula:
F=FN/β
where F is the diameter of the field of view;
FN-field number (FieldNumber, abbreviated as FN, marked on the outside of the lens barrel of the eyepiece);
β - objective lens magnification.
It can be seen from the formula:
1. The diameter of the field of view is proportional to the number of fields of view.
2. Increasing the multiple of the objective lens reduces the diameter of the field of view. Therefore, if you can see the whole picture of the inspected object under a low-power lens, and replace it with a high-power objective lens, you can only see a small part of the inspected object.
6. Poor coverage
The optical system of the microscope also includes the coverslip. Due to the non-standard thickness of the cover glass, the light path after the light enters the air from the cover glass and is refracted changes, resulting in a phase difference, which is poor coverage. Poor coverage affects the sound quality of the microscope.
Internationally, the standard thickness of the cover glass is 0.17mm, and the allowable range is 0.16-0.18mm. The difference in this thickness range has been calculated in the manufacture of the objective lens. The 0.17 marked on the objective lens housing indicates the required thickness of the cover glass for the objective lens.
7. Working distance WD
The working distance is also called the object distance, which refers to the distance between the surface of the front lens of the objective lens and the object to be inspected. During microscope inspection, the object to be inspected should be between one and two times the focal length of the objective lens. Therefore, it and the focal length are two concepts. What we usually call focusing is actually adjusting the working distance.
When the numerical aperture of the objective lens is constant, the working distance is short and the aperture angle is large.
High magnification objective with large numerical aperture and small working distance
