Contrast control of microphotography

Optical micrography is best with strong contrast. There are five factors that affect the contrast:

Influence of specimen itself on contrast

The thickness of specimen slice will produce refraction and reflection of light, and the detailed structure will overlap.

The specimens are fresh, brightly colored and in sharp contrast.

The thickness, color and waterline of specimen slide or cover will reduce the contrast.

Influence of illumination on contrast: the illumination should be moderate, otherwise the contrast will be reduced. Too bright will impact the detailed structure of the specimen, and too dark will make the dirt in the light path appear on the screen.

When the beam diameter is the same as the field of view, the contrast is strong, and when the beam diameter is larger than the field of view, the contrast will decrease.

Influence of light collector and aperture on contrast: If the light collector is too low and the aperture and aperture are small, the contrast will decrease, so it is necessary to lift the light collector or open the aperture according to the specific situation. The diameter of the iris must be consistent with the aperture of the objective lens.

The influence of color filter on contrast: the contrast of complementary color filter is strong, and that of uniform color filter is weak.

Although the contrast of optical microscopy is better, it should be handled properly, otherwise the detailed structure will be lost, the resolution will be affected and the perfect effect will not be achieved.

Attachment: Structure, imaging and performance of optical microscope

According to different uses, there are many kinds of modern optical microscopes, such as biological microscope, metallographic microscope and dissecting microscope. , but the basic structure is still optical and mechanical.

Single optical microscope (commonly used magnifying glass, dissecting mirror, etc. ) Because of its simple optical structure, small magnification and obvious aberration, it is not suitable for microphotography, so I won't introduce it here.

The optical system of compound microscope is relatively perfect, and the mechanical device is relatively complex, which is suitable for microscopic work.

1. Microscopic imaging

Imaging optical path of microscope;

It forms an enlarged optical path through optical devices such as eyepiece and objective lens, so that the distance of the obtained virtual image A "B" is exactly equal to the sight distance of human eyes -250 mm, thus obtaining a visible image of the enlarged specimen 10 ~ 200 times.

2. Basic structure of optical microscope

The basic structure of optical microscope is divided into two parts. One part is optical system, such as eyepiece, objective lens, condenser, light source, etc. The other part is mechanical system, such as temple, arm, lens barrel, stage, objective lens converter, large and small screws, propeller, etc.

(1) objective lens

The objective lens is the key component of the optical microscope, which determines the imaging quality and resolution of the whole microscope system. It consists of a set of precision lenses, which is used to enlarge the specimen for the first time. Various aberration corrections have been made in the production of microscope objective. It has a diaphragm-aperture diaphragm, which can control the diameter of the incident beam and reduce or expand the field of view without changing the brightness of the image.

The objective lens has different magnifications, and the drying system is used for observation under normal conditions. In the water immersion system or oil immersion system, water or oil should be added to the joint between the objective lens and the sample during observation.

Because the imaging quality of the objective lens affects the photographic effect and observation effect, the objective lens is also an important part of photomicrography.

The objective lens has three main parameters-numerical aperture, magnification and cover thickness. They are all marked on the lens housing.

① n a value

The numerical aperture N A is an important parameter to characterize the performance of the objective lens, which determines the resolution of the objective lens. N a = n sin θ where n is the refractive index of the medium and q is the incident angle.

The relationship between numerical aperture of objective lens and refractive index and incident angle of medium is given. The theoretical maximum incident angle is 180, and the sine of its half (90) is equal to 1, while the refractive index of air is 1.0, that of water is 1.33, and that of turpentine is 1.5. Therefore, at the same incident angle, the numerical value of the liquid immersion objective is larger than that of the aperture ratio dry objective.

The greater the N A value, the greater the resolution, the smaller the N A value, and the longer the depth of field and focal depth.

The value of N A is still related to the angle of the mirror. The greater the angle of the mirror, the greater the amount of light entering, the clearer the image in the field of vision and the greater the resolution. On the other hand, the resolution is too small to see clearly. Because the mirror angle of the low-power objective is smaller than that of the high-power objective, the resolution of the low-power objective is not as large as that of the high-power objective. (This means that for dry systems, the oil-immersed objective lens has a dielectric refractive index, which can improve the resolution.

Resolution is also affected by illumination intensity and wavelength of light source. In a certain range, the greater the light intensity, the greater the resolution.

② magnification

Magnification refers to the magnification of inspection (i.e. virtual image magnification). The greater the magnification, the closer the working distance. For each specific objective lens, its magnification and working distance are constant. If 10× eyepiece and 40× objective lens are used, the magnification during observation is 400 times, but for photomicrography, the distance from eyepiece to photosensitive plate needs to be increased, so it is not 400 times, and the magnification of some photomicrographs is 0/2 of that of 65438+ microscope.

(3) protective layer thickness requirements

Microscope requires the thickness of the cover to be1100 of the lens barrel thickness, reaching 0. 16 ~ 0. 17mm. For every difference of 0.0 1mm, the lens barrel must be changed to 10mm, otherwise its magnification is not the marked magnification.

Oil immersion mirror is to use the refractive index of oil to improve the brightness and resolution of the field of view. After oiling, the objective lens and the sample form an optical group.

Oil immersion mirror commonly used point oil and refractive index are:

Chinese fir oil 1.5 1

Sandalwood oil 1.5 1

Glycerol 1.46

Chewing gum 1.52

Styrax 1.58

The greater the refractive index, the stronger the image brightness and the greater the resolution.

(2) Eyepiece

The eyepiece mainly has the following two functions: First, it magnifies the first magnified image from the objective lens for the second time.

The second is to correct the phase difference, color difference and illumination in objective imaging. The magnification of the eyepiece is generally from × 4 to × 25, and the magnification of the lens barrel length is large, otherwise the magnification is small.

Eye point is a main parameter of eyepiece, which is especially important in photomicrography. The eye point is the exit point formed on the eyepiece after the microscope is focused. Generally, the eye point is about 0.5 cm from the eyepiece. The greater the magnification, the shorter the distance, the smaller the magnification and the longer the distance. During visual observation, the pupil just touches this point. When shooting with a camera with a lens, the lens surface of the front lens of the lens should contact this point. When shooting with a camera without a lens, the extension size of the close-up device (telescopic leather cavity or close-up ring) should be calculated from the eye point, and the actual magnification should be calculated.

(3) Condenser

Microscope condenser includes bright field condenser, dark field condenser and special purpose condenser (such as polarized light and phase difference). ).

① Bright field condenser

Bright field condenser consists of four parts: light collector, rainbow, color filter ring and reflector.

The condenser is composed of two or more lenses, like a simple camera lens, and its numerical aperture (N A) is about 1.20 ~ 1.40. Its main function is to improve the brightness of the image field and the resolution of the objective lens to the specimen.

② Rainbow color

The structure is like the aperture of a camera, which can not only adjust the light intensity, but also change the resolution and contrast, that is, the rainbow is large, the resolution is high, the contrast is weak and the rainbow is narrow. The color filter ring is used to place color filters or ground glass, which can be used to change the color or brightness of light.

The reflector is divided into flat edge and concave edge, and the light beam should be aimed at the center of the reflector.

③ The background of the dark field condenser is dark, and the subject is luminous or bright.

Dark field condenser is usually used to shoot tiny objects, such as chemical gelatin, treponema pallidum or unstained specimens, which cannot be seen in the bright field. It is also commonly used in fluorescence micrography. When shooting with a dark field condenser, the brightness of the light source is stronger, and the color filter cannot be used. In order to prevent the interference of external light, dark field photomicrography is best carried out in a dark room.

3. The main performance of optical microscope

The main performance of optical microscope is reflected in two aspects, namely, magnification and resolution.

(1) magnification

Theoretically, the magnification of an optical microscope is the magnification of the objective lens× eyepiece when the barrel length is 16 ~ 17 cm, but we need to see the object clearly, not only to enlarge it, but also to distinguish its detailed shape, so the magnification should be considered simultaneously with the objective lens and resolution. For example, the eyepiece and objective lens of a microscope with the same magnification of 200 times can be combined in three ways: 4 0×5, 20× 10 and 10×20, but the resolution and depth of focus of the three combinations are very different from the brightness of the imaging surface and the shutter speed to be used when shooting.

Objective lens (1) 20×10 (2) 41× 5 (3)10× 20.

The resolution, depth of focus and brightness of the focal plane of the numerical aperture of the objective lens are 0.40 0.70 higher than the shutter speed of photography? 1.25? 1.0 1/30 seconds 0.65 0.43? 0.77? 0.8 1/200 seconds 0.25 1. 1? 2.0? 0.4 1/40

In photomicrography, the magnification is related to the distance from the exit point to the photosensitive plate, so the formula for calculating the magnification of photomicrography is M= objective magnification × eyepiece magnification × the distance from the exit point of the eyepiece to the negative film//250 mm. This formula shows that only when the distance from the exit point of the eyepiece to the photosensitive plate is 250 mm, the magnification of photomicrography is equal to objective magnification× eyepiece magnification. If the distance from the exit point of the objective lens to the photosensitive plate increases, the magnification will also increase, but this is only a morphological increase and the resolution remains unchanged. When you enlarge a photo with a negative, it's just a form enlargement, and it doesn't improve the resolution.