A fluorescence microscope is a microscope that uses ultraviolet light as a light source to illuminate an object to be observed to make it emit fluorescence, and able to observe information on the position, shape, and structure of the fluorescent portion of the object.
Some substances in nature can fluoresce themselves when exposed to ultraviolet light. Some substances cannot fluoresce themselves, but they can also fluoresce after being dyed with fluorescent materials. Fluorescence microscope is an important tool for studying cytology. It is widely used in scientific research and teaching fields such as medicine, polymer structure, and luminescent materials research, and so on.
The most important feature of the fluorescence microscope is that it has an illumination source that can emit ultraviolet light, forming a lighting system with the configured fluorescence condenser and filters that are designed for excitation and acceptance of fluorescence.
Fluorescent light sources generally use mercury or metal halide lamps, as well as high-power LEDs or laser sources. The ultraviolet light emitted by the light source has a shorter wavelength and thus has a higher resolution. Generally, the high-pressure mercury lamp has a continuous spectrum and a large irradiation intensity, making it a relatively ideal fluorescent microscope source; the xenon lamp has stable intensity in the visible light spectrum range and higher intensity in the infrared band than the mercury lamp, but has certain defects in the ultraviolet band. High-power LED light source cannot provide continuous band spectrum, but has advantages in the range of specific wavelengths, there is no need to preheat, start up and use, has long service life and low labor maintenance cost. So when choosing a light source, you need to understand the characteristics of the light source to match the specific application. Halogen lamps have a narrower range of excitation light wavelengths and can only be concentrated in certain specific wavelengths, such as blue light, so applications are less.
Fluorescence microscope can use transmitted light and reflection light for illumination. It can also be installed with a dark field device to become a dark field fluorescence microscope. A phase contrast accessory can be added to form a phase contrast fluorescent microscope, or an interference device can be added to become a fluorescent interference microscope.
Reflection light is also commonly called incidence light fluorescence microscope, also known as the EPI-Fluorescence Microscope. The illumination source and the excitation light are passed through the same objective lens. The excitation light path does not pass through the slide as it is directly irradiated onto the specimen, with small excitation light loss and high fluorescence efficiency.
The illumination path of the transmitted light fluorescence microscope needs to pass through the slide. In order to reduce the loss of the excitation light, the transmissive fluorescence microscope should use quartz glass slides and coverslips. Because, as consumables, quartz slides are expensive, people prefer to use an EPI-fluorescence microscope.
The inverted fluorescence microscope is composed of fluorescent accessories and an inverted microscope. The objective lens and the condenser have a long working distance, and can directly observe and study the object in the culture dish, characterized by microscopic observation in a culture flask or petri dish, mainly used for fluorescence of living tissues such as cells, and are suitable for microscopic observation of tissue culture, in vitro cell culture, plankton, food inspection, etc. in the fields of biology and medicine.
Fluorescence microscope systems require the use of two sets of filters for both excitation and emission (Emission) processes.
1. Excitation: After the light source emits light, it first enters the incident light filter, filters out the visible light, and only retains the wavelength portion for exciting the sample, therefore the incident light filter is called an exciter filter.
2. Then, the excitation light is directed vertically to the objective lens through the dichroic lens or a reflector, and then to the specimen through the objective lens, so that the specimen is excited to generate fluorescence and, at this point, the objective lens directly functions as a condenser.
3. The fluorescence, after excitation, is reflected back through the objective lens to the dichroic lens and the barrier filter to block other light except the wavelength of the emitted light from the sample (i.e., the fluorescent portion), so that the observer observes through the eyepiece or the camera to form image. The barrier filter can also filter out the ultraviolet rays between the eyepiece and the objective lens to protect the observer's eyes.
The reflective layer on the reflector of fluorescent microscopes is generally aluminized. Aluminum absorbs less ultraviolet and visible light in the blue-violet region and reflects more than 90%, while silver reflects only 70%.
The condenser of the fluorescence microscope is made of quartz glass or glass that can transmit ultraviolet light. It can use bright field condensers and dark field condenser, and there is also the phase contrast fluorescent condensers. The bright field condenser has strong condensing power, convenient to use, and is suitable for low and medium multiple power observation. The dark field condenser can produce a dark background because the excitation light does not directly enter the objective lens, thereby enhancing the brightness and contrast of the fluorescent image, capable of observing the fluorescent fine particles that cannot be distinguished by the bright field. The phase contrast fluorescent condenser needs to be used together with the phase contrast accessories and the phase contrast objective lens to observe the phase contrast and the fluorescence effect at the same time. It can see both the fluorescence image and the phase difference image, which helps the accurate positioning of the fluorescent structure.
Ordinary fluorescence microscopes can adopt various kinds of conventional objective lenses. As the more the number of lenses of the objective lens, the greater the loss of fluorescence, and in particular, the apochromatic objective lens contains quartz and other components, which can spontaneously fluoresce to form interference, therefore, the general use of flat field achromatic objective lens can already achieve very good results. The fluorescence brightness in the field of view of the microscope is proportional to the square of the numerical aperture of the objective lens, and inversely proportional to the magnification. For specimens with insufficient fluorescence, in order to improve the brightness of the fluorescence image, an objective lens with a large numerical aperture should be used, especially when using high power microscope for observation, it is often necessary to use a dedicated high-magnification fluorescent objective to provide excellent chromatic aberration correction and image quality.
Fluorescence Microscope Camera and Image
A fluorescence microscope camera can be connected to a fluorescence microscope to form a fluorescence microscope imaging system. The fluorescence image seen by the fluorescence microscope has both the morphological features, the fluorescent color and brightness and other features, and the two need to be combined for comprehensive judgment.
The brightness of the fluorescence is relatively weak, and it usually requires the camera to have high sensitivity to low light and signal capture ability in order to obtain a good fluorescence image. The camera can adjust the focus with a brighter fluorescent area. Adjust and set the exposure compensation according to the distribution ratio of the fluorescent image in the photometric area and the brightness of the image. Generally, it is necessary to increase the exposure compensation appropriately to obtain a bright and vivid fluorescent image on a dark background. The camera needs to adopt the appropriate fluorescence shooting mode, do the appropriate background subtraction processing, and set the appropriate parameters, such as Binning, Gain, Gamma, etc., so that the exposure time and gain can be maximized by software control.
Fluorescence microscope can also use a fluorescent cold-cooling camera to reduce noise and improve the signal-to-noise ratio of the image. Especially in low light conditions, using dark field illumination, or under chemiluminescence conditions, long time exposure is required. Using a cooling camera can reduce the dark current noise and obtain a clearer image.
In the weaker fluorescence field of view, in order to obtain a better contrast image, appropriate adjustments should be made to reduce the fluorescence aperture diaphragm so as to obtain images with large depth of field, or use a neutral density filter (ND for short).
When shooting with a high power or magnification objective, any vibration should be avoided, and it is best that an anti-vibration table should be configured.
The slides and coverslips used in fluorescence microscope must have a smooth surface, uniform thickness, and no autofluorescence. The thickness of the slide should be between 0.8~1.2mm. The thickness of the standard coverslip is about 0.17mm. If the slide is too thick, it will cause short-wave excitation light and fluorescence loss. If necessary, quartz glass slides can be used to increase the fluorescence transmittance.
The tissue section or other specimen of the fluorescent specimen is usually about ≤10μm, should not be too thick, otherwise it will affect the excitation light penetrating the specimen, and excessive non-essential cell overlap or impurity masking will affect the observation effect of the objective lens on the upper part of the specimen.
The sealant of the specimen must be colorless and transparent, without autofluorescence. The brightness of the fluorescence is brighter at pH 8.5~9.5, and it is not easy to fade quickly. Usually, glycerin can be used. When using dark-field fluorescence microscope or oil lens for observation, lens oil must be used, and it is best to use special non-fluorescent oil. Especially in the U and V-band excitation, the conventional cedar oil will have cyan fluorescence. Glycerin, liquid paraffin, etc. may also be used.
Fluorescence Microscope Operation Precautions
As the fluorescence is relatively dark, so the fluorescence microscope is best placed in a dark room, which also allows the human eye to adapt to the darkness for better observation effect. When lighting required, local lighting can be used. In addition, because some mercury lamps produce a great deal of heat, and some xenon lamps can generate a lot of ozone, and therefore good ventilation is required.
Before use, open the packing list first, check the integrity and status of the parts and accessories, and operate according to the instructions.
Before performing fluorescence observation, first check the condition of the lighting equipment of the fluorescent device, turn on the halogen light for illumination first, place a sample film, and do the lens focusing and centering the filament in advance. Then, check whether the fluorescence excitation filter and the emission filter are installed in the nosepiece, and whether the objective lens configuration is proper. If there is a phase contrast observation of transmitted light in the system, check the convergence axis of the condenser lens in advance, and whether the phase contrast ring plate and objective lens are matched correspondingly.
Inspect the slides, coverslips, and other sample vessels, whether the thickness of the sample is within the range of the calibrated working distance of the objective lens, and whether there are liquid, smudges, dust and other interference.
Because the illumination source contains ultraviolet light, avoid looking directly at the ultraviolet light source when the light source is adjusted. The fluorescence microscope should be equipped with a brown UV protection visor to prevent UV damage to the retina of the eyes.
After the above inspection, place the specimen to be tested and turn on the high-pressure lamp source. When the high-pressure mercury lamp is fully lit, stop the excitation and then observe.
Generally, after the high-pressure mercury lamp is turned on, the excitation light intensity tends to be stable in about 5-10 minutes, and reaches the brightest point in 15 minutes. The working time when the mercury lamp is turned on is preferably 1 hour each time. Exceeding 90 minutes, the excitation light intensity will gradually decrease and the fluorescence will be weakened. After 15 minutes of excitation of the specimen, the fluorescence will also be significantly attenuated.
After the high-pressure mercury lamp is turned off, it cannot be re-opened immediately. I can be started again only after it is completely cooled. Otherwise, the mercury lamp will be unstable and affect the service life.
After the mercury lamp is turned off, it takes at least 10 minutes to start again, so that the mercury vapor is cooled to the original state, otherwise the service life of the bulb will be affected.
The power supply should be equipped with a voltage regulator, which will reduce the service life if the voltage is unstable.
The excitation device of the fluorescence microscope and the high-pressure mercury lamp have a limited life span, and the specimens should be collectively inspected in batches so as to reduce the number and time of mercury lamp activation.
First, observe with a low power objective lens, ensure that the specimen is located in the center of the entire illumination spot, and then gradually changed to high power observation. Under the premise of not affecting the resolution, the adjustment ring of the field diaphragm and of the numerical aperture diaphragm of the objective lens can be minimized as much as possible to reduce the excitation area, avoid the influence of stray light, and improve the depth of field.
When observing the sample, in order to prevent the fluorescence of the sample from quenching due to excessive excitation of light illumination during the process of focusing and searching for the image, it is best to adjust the excitation light to a moderate intensity by first reducing the aperture diaphragm or using the ND filter. After finding the key feature points, adjust the fluorescence to the optimal brightness, and finally observe and take pictures.
Long-time excitation light illumination of the specimen will cause the fluorescence to attenuate and disappear. Therefore, the specimen should be observed immediately after dying. If stored for too long, the fluorescence will gradually weaken until quenching. When not observed for a while, the excitation light path should be blocked by a visor. The dyed specimens can be wrapped in black paper, placed in a polyethylene plastic bag, and stored at a low temperature of about 4 °C, which can delay the quenching time of the fluorescence and prevent the sealing agent from evaporating.
Before and after use, the grease and dust contaminating the lenses in front of the objective lens should be inspected and removed. Gently brush it with a soft brush. In the place where fingerprints and oil stains are present, use a soft, clean absorbent cotton, lens rubbing paper dipped in anhydrous ethanol (or methanol) to gently wipe clean, and wipe the oil stain on the surface of the objective lens with gasoline
For more information on the use of fluorescence microscope, please refer to the Biological Microscope on the BoliOptics website.
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