Principle and construction of fluorescence microscopy:
An important component on the device is to have a light source device capable of providing a sufficient specific wavelength, so that the object to be inspected is ideally excited to emit strong fluorescence. In order to achieve this requirement, the spectrum of the excitation light is limited to this specific wavelength region by means of a filter, and all of the visible light emitted is absorbed or reflected off.
Fluorescence microscopy devices are available in both transmissive and epi-illuminated versions.
(1) Transmissive type: The excitation light comes from the lower side of the object to be inspected, and the condensing mirror is a dark field condensing mirror, so that the excitation light does not enter the objective lens, and the fluorescence enters the objective lens. It is bright at low magnification and dark at high magnification. It is difficult to operate in oil immersion and adjustment. Especially the low-light illumination range is difficult to determine, but it can get a very dark background. The penetrating type is not suitable for non-transparent objects to be inspected.
(2) Projection type: The new type of fluorescence microscope is mostly in the form of a falling beam. It has a beam splitter in the light path. The light source is above the object to be inspected, so it is suitable for transparent objects to be inspected. Since the objective lens functions as a condensing mirror, it is not only easy to operate, but also can achieve uniform illumination of the entire field of view from low to high magnification, but it is dark at low magnification and high at high magnification.
The special structure of the fluorescence microscope includes:
(1) Color filter system
The color blocks are an important part of the fluorescence microscope, the core member by the excitation light filter (first barrier filter), the emitted light filter (second barrier filter) filter and a half (beam-splitting mirror) Composition . The color filter models and names of various manufacturers are often not uniform. ----
1. Excitation light filter and emission filter: According to the characteristics of light source and fluorochrome, the following three types of matching are usually used to provide excitation light in a certain wavelength range, and the fluorescence excited by the sample is transmitted to the eyepiece imaging. .
Ultraviolet light excitation: The excitation light filter can transmit ultraviolet light and block the passage of visible light above 400 nm. A corresponding light-emitting filter allows blue light to pass through, and the light in the field of view is blue, as applied to DAPI staining.
Blue light excitation: The excitation light filter allows blue light to pass through and blocks light in other wavelength bands. A corresponding emission filter can allow green light to pass through, such as GFP staining.
Green light excitation: The excitation light filter passes green light and blocks other bands of light. Corresponding emission filters typically allow red light to pass through, such as Rhodamine staining.
2. Transflective color filter: Its function is to completely block the passage of the excitation light and reflect it; and transmit the light of the corresponding wavelength range. The model number corresponds to the excitation light filter and the emission light filter.
(2) Objective lens and eyepiece
Various objective lenses can be basically applied, but it is preferable to use an objective lens with augmentation and achromaticity because the autofluorescence is extremely small and the light transmission property (wavelength range) is suitable for fluorescence. Since the fluorescence brightness of an image in the field of view of the microscope is proportional to the square of the aperture 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 for specimens that are not sufficiently fluorescent, an objective lens with a large mirror ratio and high light transmission should be used, and the eyepiece should be as low as possible.
(3) Other optical devices
Reflective mirrors, whose reflective layer is generally aluminized, because aluminum absorbs less blue and violet light in the ultraviolet and visible light, reflecting more than 90% (and silver is only 70% reflective). Planar mirrors are commonly used. Condenser, a concentrator designed for fluorescence microscopy, is made of quartz glass or other UV-transparent glass. The epi-optic device, in addition to the function of a transmissive light source, is more suitable for direct observation of opaque and translucent specimens such as slabs, filters, colonies, tissue culture and the like. The new type of fluorescence microscope developed in recent years mostly uses an epi-lighting device, which is called an epi-fluorescence microscope.
(four) light source
Now use 50 or 100W high pressure mercury lamps as the light source. During operation, it is discharged between two electrodes, causing mercury to evaporate, and the gas pressure in the ball rises rapidly (this process generally takes about 5 to 15 minutes), and in this process, the photon is emitted, and the wavelength of the released light is sufficient to excite various types of fluorescence. Substances, therefore, are commonly used for fluorescence microscopy.
Mercury lamps have a short life span of 200 hrs. In view of this life limit, in recent years, a new fluorescent light source X-Cite has an extremely long lamp life of 2000 hrs and flexible use – no preheating required It is widely used right out of the box.
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