Functions of Each Filter in a Fluorescence Microscope
The most critical issue with fluorescence microscopy is the combination of optical filters. Both the excitation filter and the shielding filter must be suitable for the observed fluorescent material and matched with each other. For some specific applications, if there are errors in selecting filters, it may lead to misunderstandings and even incorrect results. The achievements of fluorescence microscopy technology are inseparable from high-quality filters and cleverly matched filter groups. There are several types of filters for fluorescence microscopy.
1. Heat absorbing filter
Heat absorbing filter is a necessary filter to prevent thermal radiation in the light source spectrum from damaging the optical system. Early advanced microscopes were equipped with drum shaped bottles that could hold distilled water as heat absorbing devices. Modern large-scale research microscopes are equipped with glass heat absorbing filters in light source light boxes or lamp chambers, which are transparent and slightly yellow glass sheets. Leitz's KG1 (2 mm) and BG38 (4 mm) filters can transmit almost all ultraviolet and visible light spectra, with a light transmission rate of up to 98%. It only selectively absorbs infrared photothermal radiation.
2. Excitation filter
Excitation filters can selectively absorb long wavelength spectral lines and only transmit ultraviolet light. Purple, blue, and green light filters are excitation filters. Zeiss, Reichert, Olympus' B series, G series, BG series, Shott's UG series, Opton's H, G, BP series, Leitz's BP series and other optical filters are all excitation filters.
3. Blocking filter
A blocking filter is a filter that selectively absorbs short wave spectral lines and infrared rays while allowing longer waves to be seen. Its function is to enable the observer to see the fluorescence excited by the object being inspected, while protecting the observer's cornea from UV damage.
4. Color separation filter
Color separation filter is a filter mirror that reflects excitation light onto the object being inspected, causing the object to emit fluorescence, and then transmits the fluorescence to the eyepiece. This type of filter can only be used in a falling light condenser, while a transmitted light fluorescence microscope does not require color separation.
5. Interference filter
Interference filter is a type of high-performance excitation filter. It is a filter made by stacking several thin metal films between two polished glass sheets. The refractive index of each thin metal film is different, so the spectral lines of various wavelengths of the illumination light source are repeatedly reflected on each metal film. Some spectral lines of certain wavelengths are cancelled out by destructive interference, while others are strengthened by additive interference and transmitted through. This results in a filter with a narrow transmission spectrum and a half peak width of only 6-20nm. The transmittance can reach 60% -70%.
