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The fluorochrome and optical filter lists are updated from time to time. To force download of the latest versions, have your browser clear its cache before visiting FCMDesigner.
Operation
Select the desired fluorochrome combination from the "Fluorochromes" list.
Select optical filters from the "Filters" list, while displaying "Emission".
Select from the laser lines available on your system and display in turn "Excitation" and "Leakage".
Determine by trial and error, the optimal combination for maximal detection, minimal fluorochrome spillover into adjacent filters and minimal leakage of laser light through the optical filters.
Notes
Quantum Dot excitation curves are normalised to 100% at 350 nm, conventional fluorochromes to 100% at peak excitation.
Dichroic mirror curves are plotted for visual selection but their effect on transmission is not calculated.
Most optical filters, unless specified as "fully blocked", will exhibit harmonic transmissions far from the pass band that may produce unexpected spillovers. All spectra should be checked over a wide wavelength range.
Some of the fluorochrome curves designated as "Excitation" curves are actually absorption curves and may therefore exhibit small departures from the true fluorescence excitation spectra.
Optical filter curves must be considered "generic". The transmission curves of individual filters obtained may show slight differences. Take this into account where the exciting laser line is close to the transmission band since a slight shift in filter cuton wavelength can result in a huge change in the degree of blocking.
References
Curves for optical filters beginning with numerals (e.g. 660DF32, 690ALP) obtained by private communication from Roy Kinoshita at Omega Optical Inc, or more recently from the Omega Optical web site.
Curves for optical filters beginning with "D" or "HQ" (e.g. D535_40, HQ650_45) from the Chroma Technology Corp. web site.
Curves for optical filters beginning with "F" or (e.g. F630_30) are theoretical curves only.
Many fluorochrome excitation and emission curves are from the "Spectra" section of the Invitrogen web site.
Emission curves for several tandem conjugates were obtained by private communication from Mario Roederer [see "Conjugation of monoclonal antibodies"].
Many fluorescent protein spectra are from the Clontech web site.
PerCP.Cy5.5, PE.Cy7 and APC.Cy7 spectra were originally featured on the Becton Dickinson BD Biosciences web site.
