What Are the Special Structural Components of a Fluorescent Microscope?
The color filter block is an important part of a fluorescence microscope, and its core components consist of a first barrier filter for excitation light, a second barrier filter for emission light, and a beam splitting mirror. The color filter models and names of various manufacturers are often inconsistent.
1. Excitation light filter and emission light filter: Based on the characteristics of the light source and fluorescent pigment, the following three types of matching are usually selected to provide excitation light within a certain wavelength range and allow the fluorescence excited by the sample to pass through and reach the eyepiece for imaging.
UV excitation: The excitation light filter can allow UV light to pass through and block visible light above 400nm from passing through. The corresponding emission light filter allows blue light to pass through, and the light in the field of view appears blue, such as when applied to DAPI staining.
Blue light excitation: The excitation light filter can allow blue light to pass through and block light from other wavelengths. The corresponding emission light filter allows green light to pass through, such as GFP staining markers.
Green light excitation: The excitation light filter allows green light to pass through and blocks light from other wavelengths. The corresponding emission light filter usually allows red light to pass through, such as Rhodamine staining.
2. Semi transparent and semi reflective color filter: Its function is to completely block the excitation light from passing through and reflect it; And emit light within the corresponding wavelength range. Its model corresponds to the excitation light filter and the emission light filter.
(2) Objective lens and eyepiece
Various objective lenses can be applied, but it is best to choose lenses with added scale and chromatic aberration reduction, as their self fluorescence is extremely low and their light transmission performance (wavelength range) is suitable for fluorescence. Due to the fact that the fluorescence brightness of an image in the microscope field is directly 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 fluorescence images, an objective lens with a larger aperture ratio should be used. Especially for specimens with insufficient fluorescence, an objective lens with a high aperture ratio and high light transmittance should be used, accompanied by an eyepiece with the lowest magnification possible.
(3) Other optical devices
The reflective layer of a mirror is usually coated with aluminum because aluminum absorbs less ultraviolet and visible light in the blue purple region, reflecting over 90% (while silver has a reflectivity of only 70%). Generally, flat mirrors are used. Focusing lens, specifically designed and manufactured for fluorescence microscopes, is made of quartz glass or other glass that transmits ultraviolet light. The falling light device, in addition to having the function of a transmissive light source, is more suitable for direct observation of opaque and semi transparent specimens, such as thick plates, filter membranes, bacterial colonies, tissue cultures, and other samples. In recent years, many new types of fluorescence microscopes have been developed using a falling light device, known as a falling light fluorescence microscope.
(4) Light source
Nowadays, high-pressure mercury lamps of 50 or 100W are commonly used as light sources. During operation, discharge occurs between two electrodes, causing mercury to evaporate and the pressure inside the sphere to rapidly increase (this process usually takes about 5-15 minutes). During this process, light quanta are emitted, and the wavelength of the released light is sufficient to excite various fluorescent substances. Therefore, it is widely used in fluorescence microscopes.
The service life of mercury lamps is relatively short, usually 200 hours. In response to this limitation in service life, in recent years, a new type of fluorescent light source X-Cite has been widely used with a super long bulb life of 2000 hours and flexible use - no preheating required, ready to use.
