(a) light source
Nowadays, a 100W high-pressure mercury lamp is used as the light source. It is made of quartz glass and has a spherical shape in the middle. It is filled with a certain amount of mercury. During operation, it is discharged between two electrodes, causing mercury to evaporate and the gas pressure in the ball rises rapidly. When mercury is completely evaporated, it can reach 50 to 70 standard atmospheric pressures, and this process generally takes about 5 to 15 minutes. The luminescence of the ultra-high pressure mercury lamp is the result of the discharge of the light during the continuous dissociation and reduction of the mercury molecules by the discharge between the electrodes. It emits strong ultraviolet and blue-violet light, which is sufficient to excite various types of fluorescent substances, and is therefore commonly used for fluorescence microscopy.
Ultra-high pressure mercury lamps also emit a lot of heat. Therefore, the lamp room must have good heat dissipation conditions, and the working environment temperature should not be too high.
The new ultra-high pressure mercury lamp can be ignited at the initial stage without high voltage. After some time, it needs high voltage start (about 15000V). After startup, the operating voltage is generally 50-60V, and the working current is about 4A. The average life of a 200W ultra-high pressure mercury lamp is about 200h in each case of 2h. The shorter the working time is, the shorter the life is. If the working time is only 20min, the life is reduced by 50%. Therefore, minimize the number of starts when using. When the bulb is in use, its light efficiency is gradually reduced. Wait for cooling after the light is off to restart. Do not turn off the lamp immediately after igniting the lamp, so as to prevent the mercury from evaporating incompletely and damaging the electrode. It usually takes 15 minutes. Due to the high pressure of the ultra-high pressure mercury lamp and the strong ultraviolet light, the bulb must be placed in the lamp chamber to ignite, so as not to cause eye damage and operation when an explosion occurs.
Ultra high pressure mercury lamp (100W or 200W) light source circuit and includes several parts of transformer, ballast, and start-up. There is a system for adjusting the light-emitting center of the bulb on the lamp chamber, and an aluminized concave mirror is mounted behind the bulb portion, and a collecting lens is mounted on the front side.
LED fluorescent light source has recently been introduced on the market, and the reaction is good. The power of LF-LED is 5w, the life is very long, 5-10w hours, the switch does not need to be preheated, which brings a better experience for users. The current product will be more The more and more accepted by the market.
(2) Color filter system
The color filter system is an important part of the fluorescence microscope and consists of an excitation filter plate and a press filter plate. The filter plate model, the name of each manufacturer is often not uniform. Filter plates are generally named after the basic color, the front letters represent the hue, the back letters represent the glass, and the numbers represent the model features. For example, German product (Schott) BG12 is a kind of blue glass, B is the first letter of blue, G is the first letter of glass; the name of our product has been uniformly represented by pinyin letters, such as blue equivalent to BG12 The color filter plate is named QB24, Q is the first letter of cyan (blue) pinyin, and B is the first letter of the glass pinyin. However, some filter plates can also be named by the light-transmissive filter length, such as K530, which means that the light having a filter length of 530 nm or less is passed through the light of 530 nm or more. Other manufacturers' filter plates are completely named by numbers, such as NO: 5-58 of the Corning plant in the United States, which is equivalent to BG12.
1. Excitation filter plate According to the characteristics of light source and fluorochrome, the following three types of excitation filter plates can be selected to provide excitation light in a certain wavelength range.
Ultraviolet light excitation filter plate: This filter plate can transmit ultraviolet light below 400 nm and block visible light above 400 nm. The commonly used model is UG-1 or UG-5, plus a BG-38 to remove the red tail wave.
Ultraviolet blue excitation filter plate: This filter plate can pass light in the range of 300 to 450 nm. Commonly used models are ZB-2 or ZB-3, plus BG-38.
Violet blue excitation filter plate: it can pass light from 350 to 490 nm. The commonly used model is QB24 (BG12).
Fluorescein (such as rhodamine) with a maximum absorption peak above 500 nm can be excited with a blue-green filter plate (such as B-7).
In recent years, metal membrane interference filter plates have been used. Because of their strong pertinence and proper wavelength, the excitation effect is better than that of glass filters. For example, the FITC-specific KP490 filter plate from the West German Leitz plant and the S546 green filter plate from Roda Ming are far better than the glass filter plate.
The excitation filter plate is divided into two types: a thin filter plate for a dark field and a thicker for a bright field fluorescence microscope. The basic requirement is to get the brightest fluorescence and the best background.
2. Pressing the filter plate The function of the press filter plate is to completely block the passage of the excitation light, providing fluorescence of the corresponding filter length range. Corresponding to the excitation filter plate, the following three kinds of pressed filter plates are commonly used:
UV-light filter plate: can pass visible light, block UV light. Can be combined with UG-1 or UG-5. Commonly used GG-3K430 or GG-6K460.
Violet blue-pressed filter plate: It can be combined with BG-12 by fluorescence (green to red) with a filter length of 510 nm or more. Usually OG-4K510 or OG-1K530 is used.
UV-violet pressed filter plate: It can pass fluorescence (blue to red) with wavelength above 460nm, and can be combined with BG-3. OG-11K470AK 490, K510 is commonly used.
(3) Mirror
The reflective layer of the mirror is generally aluminized because aluminum absorbs less blue and violet light in the ultraviolet and visible light, and the reflection is over 90%, while the reflection of silver is only 70%. The flat mirror is generally used, with the development of technology. The mirror has gradually been eliminated.
(four) concentrating mirror
The concentrator designed for fluorescence microscopy is made of quartz glass or other UV-transparent glass. There are two types of dark field concentrators for the clear field concentrator. There is also a phase difference fluorescent concentrator.
1. Bright field concentrator In the general fluorescence microscope, the bright field concentrator is used, which has strong concentrating power and is convenient to use, and is particularly suitable for low- and medium-magnification specimen observation.
2. Dark field concentrators Dark field concentrators are increasingly used in fluorescence microscopy. Because the excitation light does not directly enter the objective lens, in addition to the scattered light, the excitation light does not enter the eyepiece. A thin excitation filter plate can be used to enhance the intensity of the excitation light. The pressed filter plate can also be thin, which can be used when excited by ultraviolet light. A colorless filter plate (without UV) still produces a dark background. Thereby, the brightness and contrast of the fluorescent image are enhanced, the quality of the image is improved, the observation is comfortable, and fine fluorescent particles which are difficult to distinguish bright field of view may be found.
3. Phase contrast fluorescent concentrator The phase difference concentrator is used in combination with the phase difference objective lens to simultaneously observe the phase difference and fluorescence. It can see both the fluorescence image and the phase difference image, which helps the fluorescence to be positioned accurately. This concentrator is rarely needed for general fluorescence observation.
(5) Objective lens
Various objective lenses can be used, but it is preferable to use an achromatic objective lens because its autofluorescence is extremely small and the light transmission property (wavelength range) is suitable for fluorescence. Since the fluorescence brightness of the image in the field of view of the microscope is proportional to the square of the aperture ratio of the objective lens, and inversely proportional to its magnification, in order to increase the brightness of the fluorescent image, an objective lens having a large mirror ratio should be used. Especially at high magnification, the effect is very obvious. Therefore, for specimens that are not sufficiently fluorescent, an objective lens with a large mirror ratio should be used, with the lowest possible eyepiece (4×, 5×, 6.3×, etc.).
(6) Eyepiece
Low magnification eyepieces such as 10x and 6.3x are often used in fluorescence microscopy. In the past, monocular eyepieces were used because their brightness was more than twice that of binoculars. However, the research-type fluorescence microscopes are often used with binoculars for easy observation.
(7) Falling light device
The new type of epi-lighting device is such that after the light from the light source is incident on the interference spectroscopic filter, the short-wavelength portions (ultraviolet and violet-blue) are reflected by the nature of the coating on the filter, and the filter is opposite to the light source. When tilted, it is directed perpendicularly to the objective lens, and is directed toward the specimen through the objective lens to cause the specimen to be excited. At this time, the objective lens directly functions as a concentrator. At the same time, the long part of the filter (green, yellow, red, etc.) is permeable to the filter. Therefore, it does not reflect toward the objective lens. The filter acts as an excitation filter plate, because the fluorescence of the specimen is in the visible long-wavelength region. The filter can be passed through the filter to the eyepiece, and the brightness of the fluorescent image increases as the magnification increases, and is stronger than the transmitted light source at high magnification. In addition to the function of a transmissive light source, it is more suitable for direct observation of opaque and translucent specimens such as slabs, filters, colonies, and tissue culture specimens. The new type of fluorescence microscope developed in recent years mostly uses an epi-lighting device, which is called an epi-fluorescence microscope.
(8) Photographing system
Fluorescence belongs to the dark field. Most of the excited fluorescence is weak. It needs to be photographed with a high-sensitivity and cooled CCD. The cooling function is mainly used to eliminate dark current and noise. Like the Olympus DP73, it is 17.5 million pixels of cooling at room temperature of 10 degrees, and Nanjing's MCX2 is 12.5 million pixels cooled to 20 degrees at room temperature. With the development of CCD technology, more and more essence photomicrographs will appear in front of us.
Hand Held Jade Heating Projector adopt Jade as the thermal massager, when heating the jade, it can do thermal therapy and FIR therapy to any part of the body according to user's demand.
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