Principle and structural characteristics of fluorescence microscopy :
Fluorescence microscopy is the use of a high light emission efficiency point light source passes through the color system emit certain wavelengths of light (such as ultraviolet light into the 3650 or 4200 into the blue violet) as excitation light, excites the fluorescent substance in the sample emit different color After fluorescence, it was observed by magnification of the objective lens and the eyepiece. In this way, in the strong contrast background, even if the fluorescence is very weak, it is easy to recognize and has high sensitivity, and is mainly used for research on cell structure and function as well as chemical composition. The basic configuration of the fluorescent microscope by ordinary optical microscope with some accessories (such as fluorescent light, excitation filter, dichroic beam splitters and blocking filters, etc.) based on the composition. Fluorescent light source - as employed high pressure mercury lamp (200W 50 a), which can emit light of various wavelengths, but each has a fluorescent substance generates fluorescence excitation wavelength of the strongest, the need to add with the excitation filter ( Generally, ultraviolet, purple, blue, and green excitation filters are used , and only a certain wavelength of excitation light is transmitted through the specimen, and other light is absorbed. After each substance is irradiated with excitation light, it emits visible fluorescence longer than the irradiation wavelength in a very short time . Fluorescence is specific and generally weaker than the excitation light. In order to observe the specific fluorescence , a blocking ( or suppression ) filter is added after the objective lens . It has two functions: one is to absorb and block the excitation light into the eyepiece to avoid disturbing the fluorescence and damage the eye, and the other is to select and allow the specific fluorescence to pass through, showing a specific fluorescent color. Both filters must be selected for use.
Fluorescence microscopy has two types of light paths:
1 . Transmissive fluorescence microscopy : The excitation source is excited by a concentrating mirror through a sample material. Common dark field concentrators , can also use ordinary concentrators, adjust the mirror to make the excitation light and side-by-side to the specimen. This is a relatively old fluorescence microscope . The advantage is that the fluorescence is strong at low magnification, and the disadvantage is that the fluorescence is weakened with increasing magnification. Therefore, it is better to observe larger specimen materials.
2 . Epi-fluorescence microscope This is a new type of fluorescence microscope developed in modern times. The difference is that the excitation light is directed from the objective lens to the surface of the specimen, that is, the same objective lens is used as the illumination concentrator and the objective lens for collecting fluorescence. A two-color beam splitter is added to the light path, which is 45 with the uranium . The excitation light is reflected into the objective lens and is collected on the sample. The fluorescence generated by the sample and the excitation light reflected from the surface of the objective lens and the surface of the cover glass enter the objective lens at the same time, and return to the two-color beam splitter to make the excitation light. Separated from the fluorescence, the residual excitation light is then absorbed by the blocking filter. For example, a combination of different excitation filter/two-color beam splitter/blocking filter inserts can meet the needs of different fluorescent reaction products. The advantage of such a fluorescence microscope is that the field of view illumination is uniform, the imaging is clear, and the larger the magnification, the stronger the fluorescence.
Fluorescence microscopy is the use of a high light emission efficiency point light source passes through the color system emit certain wavelengths of light (such as ultraviolet light into the 3650 or 4200 into the blue violet) as excitation light, excites the fluorescent substance in the sample emit different color After fluorescence, it was observed by magnification of the objective lens and the eyepiece. In this way, in the strong contrast background, even if the fluorescence is very weak, it is easy to recognize and has high sensitivity, and is mainly used for research on cell structure and function as well as chemical composition. The basic configuration of the fluorescent microscope by ordinary optical microscope with some accessories (such as fluorescent light, excitation filter, dichroic beam splitters and blocking filters, etc.) based on the composition. Fluorescent light source - as employed high pressure mercury lamp (200W 50 a), which can emit light of various wavelengths, but each has a fluorescent substance generates fluorescence excitation wavelength of the strongest, the need to add with the excitation filter ( Generally, ultraviolet, purple, blue, and green excitation filters are used , and only a certain wavelength of excitation light is transmitted through the specimen, and other light is absorbed. After each substance is irradiated with excitation light, it emits visible fluorescence longer than the irradiation wavelength in a very short time . Fluorescence is specific and generally weaker than the excitation light. In order to observe the specific fluorescence , a blocking ( or suppression ) filter is added after the objective lens . It has two functions: one is to absorb and block the excitation light into the eyepiece to avoid disturbing the fluorescence and damage the eye, and the other is to select and allow the specific fluorescence to pass through, showing a specific fluorescent color. Both filters must be selected for use.
Fluorescence microscopy has two types of light paths:
1 . Transmissive fluorescence microscopy : The excitation source is excited by a concentrating mirror through a sample material. Common dark field concentrators , can also use ordinary concentrators, adjust the mirror to make the excitation light and side-by-side to the specimen. This is a relatively old fluorescence microscope . The advantage is that the fluorescence is strong at low magnification, and the disadvantage is that the fluorescence is weakened with increasing magnification. Therefore, it is better to observe larger specimen materials.
2 . Epi-fluorescence microscope This is a new type of fluorescence microscope developed in modern times. The difference is that the excitation light is directed from the objective lens to the surface of the specimen, that is, the same objective lens is used as the illumination concentrator and the objective lens for collecting fluorescence. A two-color beam splitter is added to the light path, which is 45 with the uranium . The excitation light is reflected into the objective lens and is collected on the sample. The fluorescence generated by the sample and the excitation light reflected from the surface of the objective lens and the surface of the cover glass enter the objective lens at the same time, and return to the two-color beam splitter to make the excitation light. Separated from the fluorescence, the residual excitation light is then absorbed by the blocking filter. For example, a combination of different excitation filter/two-color beam splitter/blocking filter inserts can meet the needs of different fluorescent reaction products. The advantage of such a fluorescence microscope is that the field of view illumination is uniform, the imaging is clear, and the larger the magnification, the stronger the fluorescence.
Ganoherb International Inc. , http://www.ganoherb.us