## 7 important optical parameters of the microscope

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

The optical parameters of the microscope include numerical aperture, resolution, magnification, depth of focus, field width, coverage difference, working distance, and so on. These parameters are not always the higher the better, they are interrelated and mutually restricted, in use, should be based on the purpose of microscopic examination and the actual situation to coordinate the relationship between the parameters. Let’s take a look at the important optical parameters of the microscope.

## 7 important optical parameters of the microscope

### 1. Numerical aperture

Numerical aperture is short for NA. Numerical aperture is the main technical parameter of objective lens and condenser lens, and it is an important symbol to judge the performance of both (especially for objective lens) (i.e. the ability to eliminate chromatic aberration. Zeiss numerical aperture represents the ability to eliminate chromatic aberration and magnification chromatic aberration). The size of the value is marked on the shell of the objective lens and condenser lens respectively.

The numerical aperture (NA) is the product of the refractive index (η) of the medium between the lens in front of the objective lens and the object to be detected and the positive root of half of the aperture Angle (U). It can be expressed as follows: NA=ηsinu/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 lens in front of the objective lens. The larger the aperture Angle, the greater the light into the objective lens, which is proportional to the effective diameter of the objective lens, and inversely proportional to the distance of the focus.

The aperture Angle cannot be increased if the NA value is to be increased under a microscope. The only way is to increase the refractive index η of the medium. Based on this principle, water-immersed objective lens and oil-immersed objective lens are produced. Since the refractive index η of the medium is greater than one, the NA value can be greater than one. The maximum numerical aperture is 1.4, which is a theoretical and technical limit.

At present, brominaphthalene with a high refractive index is used as the medium. Brominaphthalene has a refractive index of 1.66, so the NA value can be greater than 1.4. It must be pointed out that in order to give full play to the numerical aperture of the objective lens, the NA value of the condenser lens should be equal to or slightly greater than that of the objective lens during observation.

The numerical aperture is closely related to other technical parameters, which almost determines and influences other technical parameters. It is proportional to the resolution, proportional to the magnification, and inversely proportional to the depth of focus. When the NA value increases, the field width, and working distance will decrease correspondingly.

### 2. Resolution

Resolution is also known as “discrimination rate”, “image resolution force”. It is another important technical parameter to measure the performance of a microscope. The resolution of the microscope can be expressed as: d=0.61λ/NA where D 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 source. The higher the NA value, the shorter the illumination wavelength, the smaller the D value, and the higher the resolution.

To improve the resolution, that is, to reduce the D value, the following measures can be taken.

First, reduce the wavelength λ value and use a short wavelength light source.

Second, the greater the η of the medium and the higher the NA (NA=ηsinu/2).

Third, achromatic.

Fourth, increase the contrast between light and dark.

### 3. Magnification

Magnification refers to the ratio of the size of the final image seen by human eyes to the size of the original object after the object is magnified by the objective lens and then by the eyepiece, which is the product of the magnification of the objective lens and the eyepiece. Magnification is also an important parameter of the microscope, but we should not blindly believe that the higher the magnification is the better, and the numerical aperture of the objective lens should be considered first in the selection.

### 4. The depth of focus

Focal depth is the abbreviation of focal depth, that is, in the use of a microscope, when the focal point is aligned with an object, not only the points on the point plane can be seen clearly, but also within a certain thickness of the plane, the thickness of the clear part is the focal depth. When the focal depth is large, the whole layer of the detected object can be seen, while when the focal depth is small, only a thin layer of the detected object can be seen. The focal depth has the following relationship with other technical parameters:

L The focal depth is inversely proportional to the total magnification and the numerical aperture of the objective lens.

The large focal depth and reduced resolution.

It is difficult to take pictures with low-power objective lenses because of their large depth of field.

### 5. Field diameter

When you look at a microscope, the bright protoform you see is called the field of view, and its size is determined by the aperture of the field in the eyepiece. Field diameter, also known as field width, refers to the actual scope of the object to be detected in the circular field of view seen under the microscope. A field of view diameter of 23 is the most scientific, a large field of view is easy to cause field curvature. F=FN/Mob F: field diameter, FN: field number, Mob: magnification of the objective lens. The Field Number (FN) is marked on the outside of the tube of the eyepiece.

It can be seen from the formula:

First, the field diameter is proportional to the number of fields.

Second, increase the multiple of the objective lens, the field of view diameter decreases. Therefore, if you see the whole object at low power, you can see only a small part of the object at high power.

### 6. Poor coverage

The optical system of the microscope also includes cover glass. As the thickness of the cover glass is not standard, the light path refracted from the cover glass into the air changes, resulting in aberration, which is the coverage difference. Poor coverage affects the imaging quality of the microscope. According to international regulations, the standard thickness of the cover glass is 0.17mm, and the permissible range is 0.16-0.18mm. The aberration of this thickness range has been taken into account in the manufacture of the objective lens. The 0.17 mark on the objective lens housing indicates the required cover glass thickness of the objective lens.

### 7. Working distance

The working distance is also called object distance, which refers to the distance between the surface of the lens in front of the objective lens and the object being examined. During the microscopic examination, the object to be examined should be between one and two focal lengths of the objective lens. Therefore, it and focal length are two concepts, usually used to say the focus, in fact, is to adjust 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 power objective lens with a large numerical aperture has a small working distance.