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Iron Man User Guide v. 1.4
17
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So for this example we have three laser wavelengths: 405 (DAPI), 488 (FITC) and 543 (Rhodamine)
and would use the 405/488/543/633 dichroic in position (1). This will reflect excitation light down
to the (2) sample and block it on the way back. In position (3) we want to split the lower
wavelength DAPI and FITC from the Rhodamine by using the NFT543, which will reflect all light
below 543nm to the right and allow to pass through all wavelengths greater than 543. The
following plot illustrates the emission spectra of our three fluorophores with a green line at
543nm.
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Now we need to further isolate the DAPI and FITC signals. At position (4) we will use a NFT490
(blue line in graph below) to send the DAPI emission light to the channel 2 path and the FITC
emission light to the channel 3 path. The emission spectra are further selected for using (5, 6)
emission filters: DAPI (BP420-480) and FITC (BP505-530); the shaded regions in the plot below.