The special structure of a fluorescence microscope includes:
(I) Colour filter system
Colour filter is an important part of the fluorescence microscope, its core components by the excitation light filter (first barrier filter), emission light filter (second barrier filter) and half-transparent and half-anti-filter (beam-splitting mirror) composition. Various manufacturers of colour filter models, the name is often not uniform.
1. Excitation light filter and emission light filter: according to the light source and the characteristics of the fluorochrome, usually choose the following three types of matching, to provide a certain wavelength range of the excitation light, and make the sample excitation of fluorescence through the eyepiece to the imaging.
UV excitation: The excitation light filter allows the transmission of UV light and blocks the passage of visible light above 400nm. The corresponding emission filter allows the passage of blue light, which appears blue in the field of view, e.g. for DAPI staining.
Blue light excitation: The excitation light filter allows blue light to pass through, blocking other wavelengths of light. The corresponding emission filter allows the passage of green light, e.g. for GFP staining markers.
Green excitation: The excitation filter allows green light to pass through, blocking other wavelengths. The corresponding emission filter usually allows red light to pass through, e.g. Rhodamine staining.
2. Half-transparent and half-reflective filter: its role is to completely block the excitation of light through the reflection; and through the corresponding wavelength range of the emitted light. Its model and the excitation of light filters and emission of light filters correspond to.
(B) objective lens and eyepiece
A variety of objective lenses can be applied, but * good choice of symmetrical, achromatic objective lens, because of its autofluorescence is very small and light transmission properties (wavelength range) suitable for fluorescence. As the fluorescence brightness of the image in the microscope field of view is proportional to the square of the objective's muzzle rate and inversely proportional to its magnification, so in order to improve the brightness of the fluorescence image, the objective with a large muzzle rate should be used. Especially for the fluorescence is not strong enough specimens, should use a large mouth rate, high light transmission objective, with as low as possible with the eyepiece.
(C) other optical devices
Reflector, its reflective layer is generally aluminium-plated, because aluminium on the ultraviolet and visible light absorption of blue-violet region less, reflecting up to 90% or more (while the reflectivity of silver is only 70%). Flat reflectors are generally used. Spotlight mirror, designed and manufactured specifically for fluorescence microscopy spotlight mirror made of quartz glass or other UV-transparent glass. Falling light device, which in addition to the function of transmitted light source, more suitable for opaque and translucent specimens, such as thick film, filter membrane, colony, tissue culture and other samples for direct observation. In recent years, the development of a new type of fluorescence microscope using falling light device, called falling fluorescence microscope.
(D) light source
Nowadays, 50 or 100W high-pressure mercury lamp is used as light source. Work by the two electrodes discharge, caused by mercury evaporation, the ball of air pressure rises rapidly (this process generally takes about 5 ~ 15min), and in the process of emission of light quanta, the wavelength of the released light is sufficient to excite all types of fluorescent substances, therefore, for fluorescence microscopy is commonly used.
Mercury lamps have a relatively short service life, usually 200hrs, and in response to this service life limitation, in recent years, a new fluorescent light source, X-Cite, has been widely used for its long lamp life of 2000hrs and its flexible use - no preheating is required, and it is ready to be used right out of the box.
