The following steps should be followed when selecting fluorescent filters and mirrors.
Fluorophore (fluorescent substance) wavelength
Ascertain the wavelength of the fluorophore in use. This information is noted in catalogues, but the wavelength properties of fluorophores also change slightly depending on solution conditions such as salinity, pH and intracellular conditions. Therefore, we recommend using a fluorometer to measure both excitation and fluorescence wavelengths. Then, use this information to select the most appropriate optical elements.
Dichroic mirror selection
Barrier filter selection
Select a filter that allows fluorescence wavelengths from the specimen to pass. Usually, longpass filters that transmit long wavelengths are chosen over bandpass filters. However, a bandpass filter that does not transmit these longer wavelengths is often used as a barrier filter when separating wavelengths from a multiple-stained specimen or when using a camera sensitive to longer wavelengths.
Excitation filter selection
Combinations of barrier filters and excitation filters
The ideal combination of barrier filters and excitation filters is one that lets no light pass when combined. The fluorescence emitted is very weak, so any light that leaks through the filters will reduce image quality.
Obtaining bright fluorescence images
Sometimes it is recommended to remove the ND filter in the excitation optics and increase the strength of the excitation light source. However, increasing the strength of the excitation light source will bleach the fluorophore quickly and damage the specimen, as well as increasing autofluorescence (Note 2) in the cell. For these reasons, the light source should be kept as weak as possible and the observation optics made as efficient as possible in picking up the fluorescence signal (by widening the objective aperture, increasing the wavelength band of the fluorescence filter, using a more sensitive camera, etc.).
[Note 1] Stoke's shift refers to the energy difference between excitation energy and fluorescent energy arising from partial loss of energy as heat as electrons fall back from the excited state to their base state in fluorescing materials. (From Iwanami Shoten's Physical and Chemical Dictionary)
[Note 2] Autofluorescence refers to the presence of fluorescing substances in substances other than those generally defined as fluorescent. For example, cell components such as NAADPH or riboflavin give off relatively strong fluorescence in the short wavelength range (ultraviolet to visible) in unstained cells.