Introduction to commonly used fluorescent light sources
Fluorescent light sources are mainly used in fluorescence microscopes and are fundamental tools in cell biology. They consist of optical systems such as light sources, filters, lenses, etc. They use a certain wavelength of light to excite the specimen to emit fluorescence, and through the objective lens and eyepiece system, magnify and observe the fluorescence image of the specimen to achieve qualitative and quantitative research on the fluorescent sample.
Excitation, filtering, imaging, quenching. The adjustment of fluorescence experiments is essentially to coordinate the above four factors to achieve the best results. One often overlooked aspect is the use of light sources. The commonly used light sources for fluorescence microscopes include white light sources, high-pressure mercury lamps, xenon lamps, and metal halide lamps LED， And the latest diode laser light source.
1. Mercury lamp
The ultra-high pressure mercury lamp (50≤ 200 W) is made of quartz glass, with a spherical shape in the middle and containing a certain amount of mercury. It is discharged between two electrodes, causing mercury evaporation. The pressure inside the sphere rapidly increases, and when mercury is completely evaporated, it can reach 50× 70 standard atmospheric pressure. This process usually takes about 5 minutes. The luminescence of ultra-high pressure mercury lamps is the result of photon emission during the dissociation and reduction of mercury molecules caused by discharge between electrodes. It emits strong ultraviolet and blue violet light, which is sufficient to stimulate various fluorescent substances. Therefore, it has been widely used in fluorescence microscopes.
2. Xenon lamp
Xenon lamps and mercury lamps, as white light sources, can provide a range of wavelengths from ultraviolet to near-infrared, but they have different excitation spectra. Mercury lamps focus on near-infrared, blue, and green light, producing bright fluorescence signals with high excitation energy peaks, but have strong phototoxicity. Therefore, high-pressure oxygen is used to fix samples or weak fluorescence imaging. In contrast, xenon light sources have relatively gentle excitation and can be used to compare the intensity between different wavelengths, such as measuring calcium ion concentration. The strong excitation of xenon lamp is in the near-infrared range of 800-1000 nm.
Characteristics of XBO compared to HBO:
1) More uniform spectral intensity
2) There is still strong spectral intensity in infrared and mid infrared
3) Strong energy makes it easier to reach the aperture behind the objective lens
Recommended products: XBO75W, 100W xenon lamp, general service life: 400H, 1200h.
3. Metal halide lamp
This is a type of light source that has emerged in recent years. It is very similar to the excitation spectrum of mercury lamps, but connected to a microscope through optical fibers, it can dissipate heat and have a longer lifespan. Connecting the optical fibers from the light source to the microscope can greatly reduce the heat generated by light. Another advantage of metal halide lamps used for live cell imaging is the built-in intensity design device, which can easily adjust the laser exposure.
Recommended product: XPO metal halide lamp light source, with a power of around 120W, suitable for high-end fluorescence microscopes from major manufacturers, with a service life of over 2500 hours.
4 LED。 The light source switch is measured in milliseconds, which shortens the exposure time of the sample under light and extends its lifespan. In addition, the attenuation of LED light is rapid and accurate, which can greatly reduce phototoxicity in long-term live cell testing. Compared with white light, LED can only be excited in a narrow spectral range, and multiple LED bands enable LED light sources to provide multi-color fluorescence applications.
Recommended product: Zeiss Colibri, with a lifespan of several thousand hours.
5. Laser light source. It is a light source that uses a diode to emit a single wavelength laser. The advantage of this light source is that the excitation wavelength is extremely narrow, and imaging is not prone to noise. The limitation is that the excitation wavelength of fluorescent dyes provided by various dye manufacturers may vary. If a single wavelength laser is used for excitation, it may sometimes lead to insufficient excitation energy.
Excitation, filtering, imaging, quenching. The adjustment of fluorescence experiments is essentially to coordinate the above four factors to achieve the best results. One often overlooked aspect is the use of light sources. The commonly used light sources for fluorescence microscopes include white light sources, high-pressure mercury lamps, xenon lamps, and metal halide lamps LED， And the latest diode laser light source.
1. Mercury lamp
The ultra-high pressure mercury lamp (50≤ 200 W) is made of quartz glass, with a spherical shape in the middle and containing a certain amount of mercury. It is discharged between two electrodes, causing mercury evaporation. The pressure inside the sphere rapidly increases, and when mercury is completely evaporated, it can reach 50× 70 standard atmospheric pressure. This process usually takes about 5 minutes. The luminescence of ultra-high pressure mercury lamps is the result of photon emission during the dissociation and reduction of mercury molecules caused by discharge between electrodes. It emits strong ultraviolet and blue violet light, which is sufficient to stimulate various fluorescent substances. Therefore, it has been widely used in fluorescence microscopes.
2. Xenon lamp
Xenon lamps and mercury lamps, as white light sources, can provide a range of wavelengths from ultraviolet to near-infrared, but they have different excitation spectra. Mercury lamps focus on near-infrared, blue, and green light, producing bright fluorescence signals with high excitation energy peaks, but have strong phototoxicity. Therefore, high-pressure oxygen is used to fix samples or weak fluorescence imaging. In contrast, xenon light sources have relatively gentle excitation and can be used to compare the intensity between different wavelengths, such as measuring calcium ion concentration. The strong excitation of xenon lamp is in the near-infrared range of 800-1000 nm.
Characteristics of XBO compared to HBO:
1) More uniform spectral intensity
2) There is still strong spectral intensity in infrared and mid infrared
3) Strong energy makes it easier to reach the aperture behind the objective lens
Recommended products: XBO75W, 100W xenon lamp, general service life: 400H, 1200h.
3. Metal halide lamp
This is a type of light source that has emerged in recent years. It is very similar to the excitation spectrum of mercury lamps, but connected to a microscope through optical fibers, it can dissipate heat and have a longer lifespan. Connecting the optical fibers from the light source to the microscope can greatly reduce the heat generated by light. Another advantage of metal halide lamps used for live cell imaging is the built-in intensity design device, which can easily adjust the laser exposure.
Recommended product: XPO metal halide lamp light source, with a power of around 120W, suitable for high-end fluorescence microscopes from major manufacturers, with a service life of over 2500 hours.
4 LED。 The light source switch is measured in milliseconds, which shortens the exposure time of the sample under light and extends its lifespan. In addition, the attenuation of LED light is rapid and accurate, which can greatly reduce phototoxicity in long-term live cell testing. Compared with white light, LED can only be excited in a narrow spectral range, and multiple LED bands enable LED light sources to provide multi-color fluorescence applications.
Recommended product: Zeiss Colibri, with a lifespan of several thousand hours.
5. Laser light source. It is a light source that uses a diode to emit a single wavelength laser. The advantage of this light source is that the excitation wavelength is extremely narrow, and imaging is not prone to noise. The limitation is that the excitation wavelength of fluorescent dyes provided by various dye manufacturers may vary. If a single wavelength laser is used for excitation, it may sometimes lead to insufficient excitation energy.