The principle of selecting fluorescent filter is to let the fluorescence/emission light pass through the imaging end as far as possible, and completely block the excitation light at the same time, so as to obtain the highest signal-to-noise ratio. Especially for the application of multiphoton excitation and total internal reflection microscope, the weak noise will also cause great interference to the imaging effect, so the requirement for signal to noise ratio is higher.
Usually, when we look at the parameters of the filter, we usually look at the transmittance spectrum, but the fluorescence intensity is usually weak relative to the excitation intensity. Therefore, when selecting the filter, we need to refer to the blocking depth spectrum – OD value. The OD value can more intuitively reflect the light blocking characteristics of the filter.
When selecting the excitation filter and emission filter, the blocking depth spectral intersection point (OD intersection point) of the two is an important judgment standard. Generally, it is required that the OD value of the intersection point is greater than 5 for wide field light source excitation and greater than OD6 for laser excitation, so as to achieve a better fluorescence signal to noise ratio.