How a Fluorescence Microscope Works

Fluorescence microscope: Fluorescence microscope uses ultraviolet light as a light source to irradiate the object to be inspected to make it emit fluorescence, and then observe the shape and location of the object under the microscope.
Fluorescence microscopy is used to study the absorption, transport, distribution and localization of chemical substances in cells. Some substances in cells, such as chlorophyll, can fluoresce after being irradiated by ultraviolet rays; some substances themselves cannot fluoresce, but if they are dyed with fluorescent dyes or fluorescent antibodies, they can also fluoresce when irradiated by ultraviolet rays. One of the tools for qualitative and quantitative research on this type of substances.

Identify

Fluorescence microscopes and ordinary microscopes have the following differences:
1. The illumination method is usually epi-illumination, that is, the light source is projected on the sample through the objective lens;
2. 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 light to protect human eyes.
Fluorescence microscope is also a kind of optical microscope, the main difference is that the excitation wavelength of the two is different. This determines the difference in structure and usage between fluorescence microscopes and ordinary optical microscopes.
Fluorescence microscopy is an essential tool for immunofluorescence cytochemistry. It is composed of main components such as light source, filter plate system and optical system. It uses a certain wavelength of light to excite the specimen to emit fluorescence, and magnifies it through the objective lens and eyepiece system to observe the fluorescence image of the specimen.

Fluorescence Microscope Working principle

light source
The 200W ultra-high pressure mercury lamp is mostly used as the light source. It is made of quartz glass, with a spherical shape in the middle, and a certain amount of mercury is filled in it. During operation, the discharge between the two electrodes causes the mercury to evaporate, and the pressure in the ball rises rapidly. When the 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 emission of light quantum during the continuous dissociation and reduction of mercury molecules by the discharge between the electrodes. It emits strong ultraviolet and blue-violet light enough to excite various fluorescent substances, so it is commonly used in fluorescence microscopes.
Ultra-high pressure mercury lamps also emit a lot of thermal energy. Therefore, the lamp room must have good heat dissipation conditions, and the working environment temperature should not be too high.
The new type of ultra-high pressure mercury lamp can be ignited without high voltage at the initial stage of use. After some time of use, it needs to be started with high voltage (about 15000V). After starting, the maintenance working 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 when it is used for 2h each time. Therefore, try to reduce the number of starts when using it. In the process of using the bulb, its light efficiency is gradually reduced. After the light goes out, wait for it to cool down before restarting. Do not turn off the bulb immediately after lighting, so as to avoid incomplete evaporation of mercury and damage to the electrode, generally need to wait 15min. Because the ultra-high pressure mercury lamp has high pressure and strong ultraviolet rays, the bulb must be placed in the lamp chamber before it can be ignited to avoid eye damage and explosion.
The circuit of the light source of the ultra-high pressure mercury lamp (100W or 200W) includes several parts such as voltage transformation, ballast and starting. There is a system for adjusting the light-emitting center of the bulb on the lamp house, an aluminum-coated concave reflector is installed behind the bulb bulb, and a light-collecting lens is installed in the front.

Color filter system
The color filter system is an important part of the fluorescence microscope, which consists of excitation filter plate and pressing filter plate. The filter plate model, the names of various manufacturers are often not unified. The filter plate is generally named after the basic tone, the letters in front represent the tone, the letters in the back represent the glass, and the numbers represent the model characteristics. For example, the German product (Schott) BG12 is a kind of blue glass, B is the first letter of blue, and G is the first letter of glass; there are some manufacturers whose filter plates are named entirely by numbers, such as those of the Corning factory in the United States. NO: 5-58, which is equivalent to BG12.

1. Excitation filter plate According to the characteristics of light source and fluorescent pigment, the following three types of excitation filter plate 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 400nm and block visible light above 400nm from passing through. Commonly used models are UG-1 or UG-5, plus a BG-38 to remove the red wake.
Ultraviolet blue light excitation filter plate: This filter plate can pass light in the range of 300-450nm. Commonly used models are ZB-2 or ZB-3, plus BG-38.
Violet blue excitation filter plate: it can pass the light of 350～490nm. The commonly used model is QB24 (BG12).
The fluorescein (such as rhodamine pigment) whose maximum absorption peak is above 500nm can be excited by blue-green filter plate (such as B-7).
The metal film interference filter plate was used. Due to its strong pertinence and appropriate wavelength, the excitation effect is better than that of glass filters. For example, the special KP490 filter plate for FITC from the Leitz factory in West Germany and the S546 green filter plate from Rhodamine are far better than the glass filter plate.
The excitation filter plate is divided into two types, thin and thick. Generally, a thin filter plate is used in the dark field, and a thicker filter plate can be used in the bright field fluorescence microscope. The basic requirement is to obtain the brightest fluorescence and the best background.

2. Pressed filter plate The function of the pressed filter plate is to completely block the passage of excitation light and provide fluorescence in the corresponding wavelength range. Corresponding to the excitation filter plate, the following three types of pressed filter plates are commonly used:
Ultraviolet light pressing filter plate: can pass visible light and block ultraviolet light from passing through. Can be combined with UG-1 or UG-5. Commonly used GG-3K430 or GG-6K460.
Violet blue pressed filter plate: It can pass light with wavelengths above 510nm (green to red), and can be combined with BG-12. Usually OG-4K510 or OG-1K530 are used.
Ultraviolet-violet pressed filter plate: It can pass light with wavelengths above 460nm (blue to red), can be combined with BG-3, commonly used OG-11K470AK 490, K510.

Reflector
The reflective layer of the reflector is generally aluminized, because aluminum absorbs less in the blue-violet region of ultraviolet light and visible light, and the reflection is more than 90%, while the reflection of silver is only 70%; generally, flat reflectors are used.

Condenser
Concentrators designed for fluorescence microscopy are made of quartz glass or other glass that transmits UV light. There are two types of light-field condensers and dark-field condensers. There are also phase contrast fluorescent concentrators.
1. Brightfield Condenser Brightfield Condenser is commonly used in general fluorescence microscopes. It has strong light-gathering power and is easy to use. It is especially suitable for observation of specimens with low and medium magnifications.
2. Darkfield Condensers Darkfield condensers are increasingly used in fluorescence microscopy. Because the excitation light does not directly enter the objective lens, except for scattered light, the excitation light does not enter the eyepiece. A thin excitation filter can be used to enhance the intensity of the excitation light, and the pressing filter can also be very thin. Colorless filters (no UV transmission) while still producing a dark background. Thus, the brightness and contrast of the fluorescent image are enhanced, the quality of the image is improved, and the observation is comfortable, and it is possible to find fine fluorescent particles that are difficult to distinguish in the bright field.
3. Phase Contrast Fluorescence Condenser The phase contrast condenser is used in conjunction with the phase contrast objective lens, which can perform combined phase contrast and fluorescence observation at the same time, and can see both the fluorescence image and the phase contrast image, which is helpful for the accurate positioning of the fluorescence. Such condensers are rarely required for general fluorescence observation.

Objective lens
A variety of objectives can be used, but an achromatic objective is preferred because its autofluorescence is minimal and its transmittance (wavelength range) is suitable for fluorescence. Since the fluorescence brightness of the image in the microscope field of view is proportional to the square of the aperture ratio of the objective lens and inversely proportional to its magnification, in order to improve the brightness of the fluorescence image, an objective lens with a large aperture ratio should be used. Especially at high magnification, its effect is very obvious. Therefore, for specimens with insufficient fluorescence, an objective lens with a large aperture ratio should be used with the eyepiece as low as possible (4×, 5×, 6.3×, etc.).

Eyepiece
Use low-power eyepieces, such as 5× and 6.3×, in fluorescence microscopy. In the past, monocular eyepieces were often used because their brightness was more than double that of binocular eyepieces, but research-type fluorescence microscopes mostly use binocular eyepieces, which are very convenient for observation.
Epi light device
The new type of epi-light device is that after the light from the light source hits the interference spectroscopic filter, the short wavelength part (ultraviolet and violet-blue) is reflected due to the nature of the coating on the filter. When the filter is facing the light source, it is 45 degrees. When it is tilted, it shoots vertically to the objective lens, and then shoots to the specimen through the objective lens, so that the specimen is excited, and the objective lens directly acts as a condenser. At the same time, the long part of the filter (green, yellow, red, etc.) is transparent to the filter. Therefore, it does not reflect toward the objective lens, and the filter acts as an excitation filter. Since the fluorescence of the specimen is in the visible light wavelength region , can be observed through the filter to reach the eyepiece, the brightness of the fluorescent image increases with the magnification, and is stronger than the transmitted light source at high magnification. In addition to the function of a transmission light source, it is more suitable for direct observation of opaque and translucent specimens, such as thick sheets, filters, colonies, and tissue culture specimens. The developed new fluorescence microscopes mostly use epi-light devices, which are called epi-fluorescence microscopes.

CCD

Introduction
Fluorescence microscope CCD is a digital camera product closely related to fluorescence microscope. On the one hand, it can transmit the photomicrography product taken by fluorescence microscope to computer through usb interface, which is convenient for image collection and research. On the other hand, through fluorescence microscope CCD We can take better pictures than we can with fluorescence microscopy alone. Fluorescence microscope CCD can be connected to a fluorescence microscope to form a microscopic imaging system.

Scope of use
In general, the fluorescence microscope alone can achieve the desired imaging effect, but in some cases, such as when the fluorescence is relatively weak, the fluorescence microscope alone cannot achieve the ideal shooting effect, or we hope You can upload the photographed fluorescence images to the computer for previewing, revising and even publishing academic papers. At this time, it is impossible to meet the requirements without a fluorescence microscope CCD.

Features
Fluorescence microscope CCD generally has good weak light capture ability, can capture extremely weak fluorescence, so the imaging ability is good. In addition, many fluorescent cold CCD production companies have made such CCD refrigeration treatment, which makes the noise of this type of CCD greatly increased. decrease, the signal-to-noise ratio can be greatly improved. Due to its convenient application effect, such CCD cameras are widely used in fluorescence microscopy.

Specimen Preparation Requirements for Fluorescence Microscope

Specimen Preparation Requirements for Fluorescence Microscopy
1. Glass slides
The thickness of the glass slide should be between 0.8 and 1.2 mm. On the one hand, if the slope is too thick, it absorbs more light, and on the other hand, the excitation light cannot be concentrated on the specimen. The slides must be smooth, uniform in thickness, and free of obvious autofluorescence. Quartz glass slides are sometimes used.

2. Cover glass
The thickness of the cover glass is about 0.17mm, and it is smooth and clean. In order to strengthen the excitation light, an interference cover glass can also be used, which is a special cover glass coated with several layers of substances (such as magnesium fluoride) that have different interference effects on light of different wavelengths. Passing smoothly through, while reflecting the excitation light, this reflected excitation light can excite the specimen.

3. Specimen
Tissue sections or other specimens should not be too thick, if too thick, most of the excitation light is consumed in the lower part of the specimen, and the upper part directly observed by the objective lens is not sufficiently excited. In addition, the overlapping of cells or the masking of impurities affects the judgment.

4. Mounting agent
Glycerol is commonly used as a mounting agent. It must be colorless and transparent without autofluorescence. The brightness of the fluorescence is brighter at pH 8.5 to 9.5, and it is not easy to fade away quickly.

5. Mirror oil
Generally, when observing specimens with dark field fluorescence microscope and oil lens, lens oil must be used. It is best to use special non-fluorescent lens oil. The above-mentioned glycerin can also be used instead. Liquid paraffin can also be used, but the refractive index is low and the image quality is slightly affected. influences.

Instructions

(1) Turn on the light source, the ultra-high pressure mercury lamp needs to be preheated for 15 minutes to reach the brightest point.
(2) The required excitation filter should be installed between the light source and the dark field condenser for the transmission type fluorescence microscope, and the corresponding pressing filter should be installed behind the objective lens. For epi-fluorescence microscopes, the required excitation filters, dichromatic beam splitters, and inserts for pressing filters need to be inserted into the slots in 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 sheet and observe it after focusing. Attention should be paid during use: do not observe directly with the eyes without the filter installed, so as not to cause eye damage; when observing the specimen with the oil lens, special lens oil without fluorescence must be used; after the high-pressure mercury lamp is turned off, it cannot be turned on again immediately, and it needs to be waited for. The mercury lamp cannot be restarted until it is completely cooled, otherwise it will be unstable and affect the life of the mercury lamp.
(5) Observation. For example, using a blue-violet filter under a fluorescence microscope, cells stained with 0.01% acridine orange fluorescent dye were observed, and the nucleus and cytoplasm were excited to produce two different colors of fluorescence (dark green and orange-red).

Precautions

(1) Operate in strict accordance with the requirements of the fluorescence microscope factory instructions, and do not arbitrarily change the program.
(2) The inspection should be carried out in a dark room. After entering the dark room, connect the power supply and light the ultra-high pressure mercury lamp for 5-15 minutes. After the strong light from the light source is stable, the eyes are fully adapted to the dark room, and then start to observe the specimen.
(3) To prevent the damage of ultraviolet rays to the eyes, wear protective glasses when adjusting the light source.
(4) The inspection time is preferably 1 to 2 hours each time. If it exceeds 90 minutes, the luminous intensity of the ultra-high pressure mercury lamp will gradually decrease, and the fluorescence will be weakened; after the specimen is irradiated with ultraviolet light for 3 to 5 minutes, the fluorescence will also be significantly weakened; therefore, it should not exceed 2 to 3h.
(5) The life of the light source of the fluorescence microscope is limited, and the specimen should be inspected centrally to save time and protect the light source. When the weather is hot, fans should be added to cool down, and the use time should be recorded from the beginning when the new bulb is replaced. When you want to use it again after the lamp is extinguished, you must wait for the bulb to cool down before lighting it. Lighting the light source several times a day should be avoided.
(6) Observe the specimen immediately after staining, as the fluorescence will gradually weaken over time. If the specimen is stored in a polyethylene plastic bag at 4°C, the fluorescence weakening time can be delayed and the mounting agent can be prevented from evaporating. Long-term excitation light irradiation on the specimen will cause the fluorescence attenuation and disappearance, so the irradiation time should be shortened as much as possible. The excitation light can be blocked by a light shield when not being observed temporarily.
(7) Non-fluorescent oil should be used when the specimen is observed, and the eyes should be avoided to look directly at the ultraviolet light source.
(8) The power supply should be equipped with a voltage stabilizer, and the voltage instability will reduce the life of the fluorescent lamp.

Fluorescence microscopes and ordinary microscopes have the following differences:
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, the wavelength is shorter, and the resolution is higher than that of 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 light to protect human eyes.
Fluorescence microscope is also a kind of optical microscope, the main difference is that the excitation wavelength of the two is different. This determines the difference in structure and usage between fluorescence microscopes and ordinary optical microscopes.
Fluorescence microscopy is an essential tool for immunofluorescence cytochemistry. It is composed of main components such as light source, filter plate system and optical system. It uses a certain wavelength of light to excite the specimen to emit fluorescence, and magnifies it through the objective lens and eyepiece system to observe the fluorescence image of the specimen.