What is a Fluorescence Microscope?

Fluorescence microscope: Fluorescence microscope uses ultraviolet light as the light source to illuminate the object under test to make it fluoresce, and then observe the shape and location of the object under the microscope.

Fluorescence microscope

Fluorescence microscope: Fluorescence microscope uses ultraviolet light as the light source to illuminate the object under test to make it fluoresce, and then observe the shape and location of the object under the microscope.

Fluorescence Microscopy

Fluorescence microscope and ordinary microscope have the following areas

Specimen preparation requirements for fluorescence microscopes

1.Slide

The thickness of the slide should be between 0.8 and 1.2 mm. Too thick slopes, on the one hand, absorb more light, on the other hand, they cannot cause excitation light to collect on the specimen. The slide must be smooth and uniform in thickness, without significant autofluorescence. Sometimes a quartz glass slide is required.

Coverslips

The cover glass is about 0.17mm thick and smooth. In order to strengthen the excitation light, an interference cover glass can also be used. This is a special cover glass with a surface coated with several layers of substances that interfere with different wavelengths of light (such as magnesium fluoride). Passes smoothly and reflects the excitation light, and this reflected excitation light can excite the specimen.

3.Specimen

Tissue sections or other specimens should not be too thick. If they are too thick, most of the excitation light is consumed in the lower part of the specimen, while the upper part directly observed by the objective lens is not sufficiently excited. In addition, cell overlap or concealment of impurities affects judgment.

4, sealing agent

Glycerin is commonly used as a mounting agent. It must be free of autofluorescence, colorless and transparent. The fluorescence brightness is brighter at pH 8.5 to 9.5, and it is not easy to fade away quickly.

5.Mirror oil

Generally, when using dark-field fluorescence microscopes and oil specimens to observe specimens, mirror oil must be used. It is best to use a special non-fluorescent mirror oil, which can also be replaced by the above glycerin. Liquid paraffin is also available, but the refractive index is low, and the image quality is slightly influences.

(1) Turn on the light source. The ultra-high pressure mercury lamp needs to be warmed up for 15 minutes to reach the brightest point.

(2) The transmission fluorescence microscope needs to install the required excitation filter between the light source and the dark field condenser, and install the corresponding pressed filter behind the objective lens. The epi-fluorescence microscope needs to insert the required excitation filter, two-color beam splitter, and pressed filter insert into the slot of the optical path.

(3) Observe with a low magnification lens, and adjust the center of the light source so that it is located in the center of the entire illumination spot according to the adjustment device of different types of fluorescence microscopes.

(4) Place the specimen and observe after focusing. Note during use: Do not observe directly with the eye without the filter installed, so as not to cause eye damage. When observing the specimen with an oil microscope, you must use a special mirror oil without fluorescence. After the high-pressure mercury lamp is turned off, it cannot be turned on again immediately. The mercury lamp can only be restarted after it has completely cooled down, otherwise it will be unstable and affect the life of the mercury lamp.

(5) Observe. For example, using a blue-violet filter under a fluorescence microscope, it was observed that cells stained with 0.01% acridine orange fluorescent dye, the nucleus and cytoplasm were excited to produce two different colors of fluorescence (dark green and orange-red).

(1) Operate strictly in accordance with the factory instructions of the fluorescence microscope. Do not change the program at will.

(2) Inspection should be carried out in a dark room. After entering the dark room, connect the power supply and ignite the ultra-high pressure mercury lamp for 5-15 minutes. After the strong light from the light source stabilizes, the eyes fully adapt to the dark room, and then start to observe the specimen.

(3) To prevent damage to the eyes by ultraviolet rays, wear protective glasses when adjusting the light source.

(4) The inspection time is preferably 1 to 2 hours each time, more than 90 minutes, the luminous intensity of the ultra-high-pressure mercury lamp gradually decreases, and the fluorescence weakens; the specimen also has a significant decrease in fluorescence after being exposed to ultraviolet rays for 3 to 5 minutes; 3h.

(5) The lifetime of the light source of the fluorescence microscope is limited. Specimens should be inspected in order to save time and protect the light source. When it is hot, you should add an electric fan to cool down, and you should record the usage time from the beginning when you replace the bulb. If the lamp is to be reused after it is turned off, the lamp must be sufficiently cooled before it can be ignited. Avoid lighting the light source several times during the day.

(6) Observe the specimen immediately after staining, and the fluorescence will gradually weaken as time goes by. If the specimen is stored in a polyethylene plastic bag at 4 ° C, the fluorescence attenuation time can be delayed and the sealant can be prevented from evaporating. Prolonged excitation light irradiates the specimen, which will cause the fluorescence to decay and disappear. Therefore, the irradiation time should be shortened as much as possible. When not observing temporarily, you can cover the excitation light with a light blocking plate.

(7) The specimen should be observed without fluorescent oil, and eyes should be avoided to look directly at the ultraviolet light source.

(8) The power supply should be installed with a voltage regulator. Unstable voltage will reduce the life of the fluorescent lamp.

Difference between fluorescence microscope and optical microscope

There are the following differences between fluorescence microscopes and ordinary microscopes:

1. The illumination method is usually epi-illumination, that is, the light source is projected on the sample through the objective lens;

2. The light source is ultraviolet light, with a shorter wavelength and higher resolution than ordinary microscopes;

3. There are two special filters, the one in front of the light source is used to filter out visible light, and the one between the eyepiece and the objective lens is used to filter out ultraviolet rays to protect people's eyes.

Fluorescence microscope is also a kind of optical microscope. The main difference is that the two have different excitation wavelengths. This determines the differences in the structure and usage of fluorescent microscopes and ordinary optical microscopes.

Fluorescence microscopy is a basic tool for immunofluorescent cytochemistry. It is composed of the main components such as light source, filter plate system and optical system. A certain wavelength of light is used to excite the specimen to emit fluorescence, and it is enlarged through the objective lens and eyepiece system to observe the fluorescence image of the specimen. ^[1]