The role of each filter in fluorescence microscopy
The most crucial issue with fluorescence microscopy is the combination of filters, and both the excitation filter and the shielding filter must be suitable for the observed fluorescent body and matched with each other. For certain specific purposes, if there is an error in selecting a filter, it may lead to misunderstandings and even incorrect results. The achievements of fluorescence microscopy technology and the use of high-quality filters and ingenious filter groups are inseparable. 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 device group. 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 chambers, which are transparent, slightly yellow glass sheets. Leitz's KG1 (2 mm) and BG38 (4 mm) filters can transmit almost all ultraviolet and visible light spectra, making them filters with a light flux of up to 98%. It only selectively absorbs infrared photothermal radiation lines.
2. Excitation filter
Excitation filters can selectively absorb long wavelength spectral lines and only transmit ultraviolet light. Filters for purple, blue, and green light are excitation filters. Zeiss, Reichert, Olympus B-series, G-series, BG series, Shott UG series, Opton H, G, BP series, Leitz BP series and other filters are all excitation filters.
3. Blocking filter
Blocking filter is a filter that selectively absorbs short wavelength spectral lines and infrared radiation, allowing the observer to see the fluorescence emitted by the object being tested while protecting the cornea from UV damage.
4. Color light separation filter
Color light separation filter is a filter that reflects the excitation light onto the object being tested, causing the object to emit fluorescence, and then transmits the fluorescence to the eyepiece. This type of filter can only be used in falling beam concentrators, while transmission light fluorescence microscopy 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 different wavelengths of the illumination light source are repeatedly reflected on each metal film, causing some wavelengths of spectral lines to cancel out due to destructive interference, while others are strengthened by additive interference and transmitted through. This results in a filter with a narrow transmission spectrum, a half peak width of only 6~20nm, and a transmittance of 60%~70%.
