Emission Filter - THORLABS OTKB-FL User Manual

Optical Trap Kit Fluorescence Light Unit Chapter 3: OTKB-FL
plot (Figure 2 below), the transition wavelength for the fluorescein isothiocyanate (FITC) Dichroic Mirror (MD499)
is ~500 nm.
Dichroic mirrors are designed to reflect light whose wavelength is below a specific value (i.e. the transition
wavelength) while permitting all other wavelengths to pass through it unaltered.
Figure 2 Transmission vs Wavelength of Dichroic Mirror, Excitation Filter, and Emission Filter. Item
Numbers: Dichroic Mirror, MD499; Excitation Filter, MF475-35; Emission Filter, MF530-43.
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By placing one of these mirrors into the experimental setup at 45 with respect to the incident radiation, the
excitation radiation (shown in blue in Figure 1) is reflected off of the surface of the dichroic mirror and directed
towards the specimen and microscope objective, while the fluorescence emitted from the specimen (shown in red
in Figure 1) passes through the mirror to the detection system.
Although the dichroic mirror plays a crucial role in the fluorescence microscope, they are not perfect when it
comes to blocking unwanted light; typically, ~90% of the light at wavelengths below the transition wavelength
value are reflected and ~90% of the light at wavelengths above this value are transmitted by the dichroic mirror.
Hence, some of the excitation light can be transmitted through the dichroic mirror along with the longer
wavelength fluorescence emitted by the sample. To prevent this unwanted light from reaching the detection
system, an emission filter is used.

3.3.3. Emission Filter

An emission filter serves the purpose of allowing the desirable fluorescence from the sample to reach the detector
while blocking unwanted traces of excitation light. Like the excitation filter, this filter only allows a narrow band of
wavelengths to pass through it. For example, as shown in the transmission spectrum plot (Figure 2), the MF530-
43 emission filter has a bandpass region of 510 - 550 nm; this corresponds to >85% transmission for the
fluorescein isothiocyanate (FITC). Incident radiation outside of this range is either partially (for regions near the
transmission region) or totally (for regions further from the bandpass region) blocked by the filter.
Rev D, May 20, 2020 Page 6