At the time of microscopic examination, people always hope for clear and bright ideal images. This requires that all optical technical parameters of the microscope reach a certain standard, and it must be coordinated according to the purpose of the microscopic examination and the actual situation when it is used. The relationship between various parameters. Only in this way can the full performance of the microscope be achieved and a satisfactory microscopic examination result can be obtained.
Optical technical parameters of the microscope include: numerical aperture, resolution, magnification, depth of focus, field of view width, coverage difference, working distance, and the like. Not all of these parameters are better, they are interrelated and mutually restrictive. In use, the relationship between the parameters should be coordinated according to the purpose of the microscopic examination and the actual situation, but the resolution should be guaranteed. .
one. Numerical aperture numerical aperture abbreviated NA, numerical aperture is the main technical parameters of the objective lens and condenser lens, is an important indicator to determine the performance of both (especially for the objective lens). The size of the values ​​are marked on the objective lens and condenser lens housing, respectively.
The numerical aperture (NA) is the product of the refractive index (h) of the medium and the half angle of the aperture angle (u) between the objective lens and the object under inspection. Formulated as follows:
NA=hsinu/2
The aperture angle, also known as the "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 lens is, and it is proportional to the effective diameter of the objective lens and inversely proportional to the distance to the focal point.
In the microscope observation, if the NA value is to be increased, the aperture angle cannot be increased. The only way is to increase the refractive index value h of the medium. Based on this principle, a water immersion lens and an oil immersion lens are produced. Since the refractive index h of the medium is greater than one, the NA value can be greater than one.
The maximum value of the numerical aperture is 1.4, which is theoretically and technically reaching the limit. Currently, bromonaphthalene, which has a high refractive index, is used as a medium and bromonaphthalene has a refractive index of 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 numerical aperture of the objective lens, the NA of the condenser should be equal to or slightly larger than the NA of the objective lens during observation.
Numerical aperture is closely related to other technical parameters. It almost determines and affects other technical parameters. It is proportional to the resolution, is proportional to the magnification, and is inversely proportional to the depth of focus, the NA value increases, the field of view width and the working distance will be correspondingly smaller.
two. The resolution resolution is also called "recognition rate" and "resolution." It is another important technical parameter to measure the performance of the microscope.
The resolution of the microscope is formulated as: d=l/NA
Where d is the minimum resolution distance; l is the wavelength of the light; NA is the numerical aperture of the objective lens. It can be seen that the resolution of the objective lens is determined by the NA value of the objective lens and the wavelength of the illumination light source. The larger the value of NA, the shorter the wavelength of the illumination light, and the smaller the value of d, the higher the resolution.
To increase the resolution, that is to reduce the d value, take the following measures
1. Decrease the wavelength l value and use a short wavelength light source.
2. Had a large medium h value and increased NA value (NA = hsinu / 2).
3. Increase the aperture angle.
4. Increase the contrast between light and shade.
three. Magnification magnification is magnification, refers to the ratio of the size of the final image seen by the human eye to the size of the original object after being enlarged by the objective lens and then enlarged by the eyepiece, and is the product of the magnification of the objective lens and the eyepiece.
Magnification is also an important parameter of the microscope, but it cannot be blindly believed that the higher the magnification, the better. In the selection, the numerical aperture of the objective lens should be considered first.
four. Depth of focus is the abbreviation of focal depth, that is, when using a microscope, when focusing on an object, not only the points on the plane of the point can be seen clearly, but also within a certain thickness of the plane above and below, It can be seen clearly that the thickness of this clear part is the depth of focus. When the depth of focus is large, the full layer of the object under inspection can be seen. When the depth of focus is small, only a thin layer of the object under inspection can be seen. The depth of focus and other technical parameters have the following relationship:
1. The depth of focus is inversely proportional to the total magnification and the numerical aperture of the objective lens.
2. Depth of focus, reduced resolution.
Since the depth of field of the low-power objective lens is large, it poses a difficulty in photographing the low-power objective lens. It will be described in detail when photomicrographing.
Fives. Field of view
When you look at the microscope, the range of bright shapes you see is called the field of view. Its size is determined by the field of view in the eyepiece.
The field of view diameter, also known as the field of view width, refers to the actual range of objects that can be inspected within the circular field of view seen under the microscope. The larger the diameter of the field of view, the easier it is to observe.
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 object of the object under inspection at low magnification, you can only see a very small part of the object under inspection when replacing it with a high-power objective lens.
six. The optical system covering the difference microscope also includes coverslips. Due to the non-standard thickness of the cover glass, the optical path of the light refracted from the cover glass into the air is changed, resulting in a phase difference, which is poor coverage. The poor coverage affects the quality of the microscope.
Internationally, the standard thickness of coverslips is 0.17mm, and the allowable range is 0.16-0.18mm. The phase difference of this thickness range has been included in the manufacture of the objective lens. The objective section on the objective lens housing is indeed 0.17, which means that the objective lens requires the thickness of the cover glass.
Seven. Working Distance The working distance is also called the object distance, which means the distance between the surface of the front lens of the objective lens and the object under inspection. During microscopic examination, 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. Usually used to adjust the focus is actually to adjust the working distance.
In the case of a certain numerical aperture of the objective lens, the working distance is short and the aperture angle is large.
High magnification objectives with large numerical aperture have a small working distance.
Optical technical parameters of the microscope include: numerical aperture, resolution, magnification, depth of focus, field of view width, coverage difference, working distance, and the like. Not all of these parameters are better, they are interrelated and mutually restrictive. In use, the relationship between the parameters should be coordinated according to the purpose of the microscopic examination and the actual situation, but the resolution should be guaranteed. .
one. Numerical aperture numerical aperture abbreviated NA, numerical aperture is the main technical parameters of the objective lens and condenser lens, is an important indicator to determine the performance of both (especially for the objective lens). The size of the values ​​are marked on the objective lens and condenser lens housing, respectively.
The numerical aperture (NA) is the product of the refractive index (h) of the medium and the half angle of the aperture angle (u) between the objective lens and the object under inspection. Formulated as follows:
NA=hsinu/2
The aperture angle, also known as the "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 lens is, and it is proportional to the effective diameter of the objective lens and inversely proportional to the distance to the focal point.
In the microscope observation, if the NA value is to be increased, the aperture angle cannot be increased. The only way is to increase the refractive index value h of the medium. Based on this principle, a water immersion lens and an oil immersion lens are produced. Since the refractive index h of the medium is greater than one, the NA value can be greater than one.
The maximum value of the numerical aperture is 1.4, which is theoretically and technically reaching the limit. Currently, bromonaphthalene, which has a high refractive index, is used as a medium and bromonaphthalene has a refractive index of 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 numerical aperture of the objective lens, the NA of the condenser should be equal to or slightly larger than the NA of the objective lens during observation.
Numerical aperture is closely related to other technical parameters. It almost determines and affects other technical parameters. It is proportional to the resolution, is proportional to the magnification, and is inversely proportional to the depth of focus, the NA value increases, the field of view width and the working distance will be correspondingly smaller.
two. The resolution resolution is also called "recognition rate" and "resolution." It is another important technical parameter to measure the performance of the microscope.
The resolution of the microscope is formulated as: d=l/NA
Where d is the minimum resolution distance; l is the wavelength of the light; NA is the numerical aperture of the objective lens. It can be seen that the resolution of the objective lens is determined by the NA value of the objective lens and the wavelength of the illumination light source. The larger the value of NA, the shorter the wavelength of the illumination light, and the smaller the value of d, the higher the resolution.
To increase the resolution, that is to reduce the d value, take the following measures
1. Decrease the wavelength l value and use a short wavelength light source.
2. Had a large medium h value and increased NA value (NA = hsinu / 2).
3. Increase the aperture angle.
4. Increase the contrast between light and shade.
three. Magnification magnification is magnification, refers to the ratio of the size of the final image seen by the human eye to the size of the original object after being enlarged by the objective lens and then enlarged by the eyepiece, and is the product of the magnification of the objective lens and the eyepiece.
Magnification is also an important parameter of the microscope, but it cannot be blindly believed that the higher the magnification, the better. In the selection, the numerical aperture of the objective lens should be considered first.
four. Depth of focus is the abbreviation of focal depth, that is, when using a microscope, when focusing on an object, not only the points on the plane of the point can be seen clearly, but also within a certain thickness of the plane above and below, It can be seen clearly that the thickness of this clear part is the depth of focus. When the depth of focus is large, the full layer of the object under inspection can be seen. When the depth of focus is small, only a thin layer of the object under inspection can be seen. The depth of focus and other technical parameters have the following relationship:
1. The depth of focus is inversely proportional to the total magnification and the numerical aperture of the objective lens.
2. Depth of focus, reduced resolution.
Since the depth of field of the low-power objective lens is large, it poses a difficulty in photographing the low-power objective lens. It will be described in detail when photomicrographing.
Fives. Field of view
When you look at the microscope, the range of bright shapes you see is called the field of view. Its size is determined by the field of view in the eyepiece.
The field of view diameter, also known as the field of view width, refers to the actual range of objects that can be inspected within the circular field of view seen under the microscope. The larger the diameter of the field of view, the easier it is to observe.
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 object of the object under inspection at low magnification, you can only see a very small part of the object under inspection when replacing it with a high-power objective lens.
six. The optical system covering the difference microscope also includes coverslips. Due to the non-standard thickness of the cover glass, the optical path of the light refracted from the cover glass into the air is changed, resulting in a phase difference, which is poor coverage. The poor coverage affects the quality of the microscope.
Internationally, the standard thickness of coverslips is 0.17mm, and the allowable range is 0.16-0.18mm. The phase difference of this thickness range has been included in the manufacture of the objective lens. The objective section on the objective lens housing is indeed 0.17, which means that the objective lens requires the thickness of the cover glass.
Seven. Working Distance The working distance is also called the object distance, which means the distance between the surface of the front lens of the objective lens and the object under inspection. During microscopic examination, 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. Usually used to adjust the focus is actually to adjust the working distance.
In the case of a certain numerical aperture of the objective lens, the working distance is short and the aperture angle is large.
High magnification objectives with large numerical aperture have a small working distance.