Important optical technical parameters of optical microscope

February 07, 2024

During microscopic examination, people always hope to be able to get clear and bright ideal images, which requires that the optical technical parameters of the microscope reach certain standards, and requires that in use, they must be coordinated according to the purpose of the microscopic examination and the actual situation The relationship between the parameters. Only in this way can we give full play to the performance of the microscope and obtain satisfactory microscopic examination results.

The optical technical parameters of the microscope include: numerical aperture, resolution, magnification, depth of focus, field of view width, poor coverage, working distance, etc. These parameters are not all as high as possible. They are interrelated and mutually restrictive. During 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 prevail. .

1. Numerical Aperture

The numerical aperture is abbreviated as NA. The numerical aperture is the main technical parameter of the objective lens and the condenser lens, and is an important sign to judge the performance of the two (especially for the objective lens). The magnitude of its value is marked on the shell of the objective lens and condenser lens respectively.

The numerical aperture (NA) is the product of the sine of the half of the refractive index (n) of the medium and the aperture angle (u) between the front lens of the objective and the object to be inspected. The formula is as follows: NA = nsinu / 2

Aperture angle, also known as "lens 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 greater the brightness of the light entering the objective lens, which is proportional to the effective diameter of the objective lens and inversely proportional to the focal distance.

During microscope observation, if you want to increase the NA value, the aperture angle cannot be increased. The only way is to increase the refractive index n 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 value of the medium is greater than 1, the NA value can be greater than 1.

The maximum numerical aperture is 1.4, which has reached the limit both theoretically and technically. At present, bromonaphthalene with a high refractive index is used as a medium. The refractive index of bromonaphthalene 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 role of the numerical aperture of the objective lens, the NA value of the condenser lens should be equal to or slightly larger than the NA value of the objective lens during observation.

The numerical aperture has a close relationship with other technical parameters. 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 field of view width and 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 "discrimination rate". The 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 factor of the objective lens and the wavelength of the illumination light source. The larger the NA value and the shorter the wavelength of the illumination light, the smaller the σ value and the higher the resolution.

To increase the resolution, that is to reduce the σ value, the following measures can be taken

(1) Reduce the wavelength λ value and use a short-wavelength light source.

(2) Increase the medium n value to increase the NA value (NA = nsinu / 2).

(3) Increase the aperture value u to increase the NA value.

(4) Increase the contrast of light and dark.

3. Magnification and effective magnification

Due to the two magnifications through the objective and eyepieces, the total magnification Γ of the microscope should be the product of the objective magnification β and the eyepiece magnification Γ1:

Γ = βΓ1

Obviously, compared with a magnifying glass, a microscope can have a much higher magnification, and by changing the objective and eyepieces of different magnifications, the magnification of the microscope can be easily changed.

Magnification is also an important parameter of the microscope, but one cannot blindly believe that the higher the magnification, the better. The limit of microscope magnification is the effective magnification.

Resolution and magnification are two different but related concepts. Related: 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. Even if the magnification is increased excessively, only an image with a large outline but unclear details can be obtained. , Called invalid magnification. Conversely, 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

Depth of focus is the abbreviation of focal depth, 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 this plane To be clear, the thickness of this clear part is the depth of focus. If the depth of focus is large, you can see the entire layer of the inspected object, while if the depth of focus is small, you can only see a thin layer of the inspected object.

(1) The depth of focus is inversely proportional to the total magnification and the numerical aperture of the objective lens.

(2) The depth of focus is large and the resolution is reduced.

Since the depth of field of the low-power objective lens is large, it is difficult to take pictures with the low-power objective lens. It will be described in detail when taking photomicrographs.

5. Field diameter (FieldOfView)

When observing a microscope, the bright circular area you see 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, also called the width of the field of view, refers to the actual range of the object to be inspected 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 a formula F = FN / β

Where F: field diameter, FN: field number (FieldNumber, abbreviated as FN, marked on the outside of the eyepiece barrel), β: objective 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 magnification of the objective lens reduces the field of view diameter. Therefore, if you can see the whole picture of the object under inspection at a low magnification lens, and change to a high magnification objective lens, you can only see a small part of the object under inspection.

6. Poor coverage

The optical system of the microscope also includes coverslips. Because the thickness of the cover glass is not standard, the light path from the cover glass into the air is refracted and changed, resulting in a phase difference. This is the difference in coverage. Poor coverage affects the 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 phase difference of this thickness range has been included in the manufacture of the objective lens. The value of 0.17 on the objective lens casing indicates the thickness of the cover glass required by 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 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. The usual focus adjustment is actually adjusting the working distance.

In the case of a fixed numerical aperture of the objective lens, the short aperture angle of the working distance is large.

High magnification objectives with large numerical apertures have a small working distance.

Article link: China Chemical Instrument Network http: //

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