Filter Sets in Fluorescence Microscopy

The theory of fluorescence was presented in Chapter 6, Labels. To review, fluorescence is the property of a molecule that allows it to absorb one wavelength of light and emit a different higher wavelength. In the fluorescence microscope, the Alter set is the important element, because it separates the excitation and the emission wavelengths. 

Transmitted bulb and condensor lens (not used in fluorescence) 

The excitation filter is located just before the dichroic (Fig. 13.1) and it allows passage of a narrow band of light from just under 450-500 nm (Fig. 13.2a, solid gray line). The graph of the excitation spectra of Alexa Fluor 488 extends beyond the 

A complete view of the filter set shows the excitation filer, dichroic, and the emission filter spectra plotted together (Fig. 13.3a). This filter set is called long pass filter set because it will transmit all excitation photons above 550 nm. This long pass filter set allows maximum detection of the 488 emission. 

Additional fluorophores can be added to experiments and the filter sets selected accordingly. Typically, a green fluorophore (Alexa Fluor 488) and a red fluorophore (Alexa Fluor 555) are commonly used. For Alexa Fluor 555, with excitation maximum at 552 nm and an emission maximum at 568 nm, the filter set (Fig. 13.3c) has corresponding excitation and emission filters. Note, compare the filter set for Alexa Fluor 488 (Fig. 13.3b) with that for Alexa Fluor 555 (Fig. 13.3c), and confirm that the filters and dichroics allow different wavelength to be transmitted. For most samples, these two filter sets will allow collection of images without bleed-through. 

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