Upright biofluorescence microscope
The principle of fluorescence microscopy is to use a highly efficient point light source (such as an ultra-high pressure mercury lamp) to emit a certain wavelength of light (such as ultraviolet light 3650 λ or purple blue light 4200 λ) through a color filter system as excitation light, which excites the fluorescent substances in the specimen to emit various colors of fluorescence. Then, it is filtered by a blocking (or suppressing) filter behind the objective lens and observed through the magnifying effect of the eyepiece.
The blocking filter has two functions: firstly, it absorbs and blocks excitation light from entering the eyepiece to avoid interfering with fluorescence and damaging the eyes; The second is to select and allow specific fluorescence to pass through, exhibiting a specific fluorescent color.
Fluorescence microscopes can be divided into two types based on the principle of light path:
1. Transmission fluorescence microscope
Older fluorescence microscopes use a spotlight to excite fluorescence by passing the excitation light source through the specimen material. Its advantage is strong fluorescence at low magnification, but its disadvantage is that its fluorescence decreases with increasing magnification. So it is only suitable for observing larger specimen materials.
2. Falling light fluorescence microscope
The excitation light falls down from the objective lens onto the surface of the specimen, using the same objective lens as the illumination condenser and the objective lens for collecting fluorescence.
A dual color beam splitter (dichroic mirror) needs to be added to the optical path, which forms a 45 degree angle with the optical axis. The excitation light is reflected into the objective lens and focused on the sample. The fluorescence generated by the sample, as well as the excitation light reflected from the surface of the objective lens and cover glass, enter the objective lens at the same time and return to the dual color beam splitter to separate the excitation light and fluorescence. The residual excitation light is then blocked by the filter absorption. If different combinations of excitation filters, dual color beam separators, and blocking filters are used, they can meet the needs of different fluorescent reaction products.
The advantages of this fluorescence microscope are uniform field illumination, clear imaging, and stronger fluorescence with larger magnification.
