Using the Fluorescence SpectraViewer—Note 23.1
The Fluorescence SpectraViewer (www.lifetechnologies.com/handbook/spectraviewer) is an online tool that allows researchers to assess the spectral compatibility of dyes and probes in the course of designing experiments that utilize fluorescence detection techniques. This note outlines the functionality of the SpectraViewer (Figure 1) and examples of its utility in the experimental design process (Figure 2, Figure 3).
Figure 1. SpectraViewer features:
A. Fluorophore selection menu. Up to 5 fluorophore data sets may be displayed simultaneously. Open the drop-down menu (▼) and select from the list of available excitation/emission data sets. The number of available data sets is currently (December 2009) 286, encompassing organic dyes, fluorescent proteins and Qdot nanocrystals. Note that data sets may be added, deleted or modified at our editorial discretion and without notice, although in practice we aim to keep such changes to a minimum to maintain stability of the database. These data sets may be downloaded from our website as 4-column text files for importing into other plotting and calculation utilities. For each selected fluorophore data set, a legend containing sample context information is displayed on the panel to the right of the plot, though we have displayed it on the top in these screenshots for space considerations.
B. For each fluorophore data set, excitation (ex) and emission (em) data may be displayed or hidden by checking or unchecking the respective boxes.
C. Y-axis scaling for excitation and emission spectra is in terms of percentage of peak intensity value. For Qdot nanocrystals, which exhibit quasi-continuous excitation profiles, the 100% intensity value has been arbitrarily defined as that at 300 nm. X-axis values on all plots are wavelengths in nanometers (nm).
D. Excitation source spectral characteristics may be superimposed on the plot in the form of laser lines selected from a drop-down menu or filter characteristics input as numeric center wavelength (CWL) and bandpass (BP) values in nm in the boxes provided. In this example, laser excitation at 488 nm is indicated.
E. Emission filter spectral characteristics may be superimposed on the plot in the form of numeric CWL and BP values in nm entered in the boxes provided. In this example, a typical FITC emission filter with CWL = 535 nm and BP = 50 nm is indicated. The transmission window of the filter is shown on the plot as a green-shaded rectangle.
F. Mouse-controlled X,Y cursor. The crosshairs may be moved to any user-selected location within the plot window and are coupled to a numeric display of the corresponding (X,Y) values.
Figure 2. Evaluating fluorophores for multiplex detection experiments: Excitation spectra. This overlay of the fluorescence excitation spectra of Alexa Fluor 488 and Alexa Fluor 568 dyes provides a useful initial assessment of their suitability for use in a multiplex detection experiment. It also serves to highlight scaling factors that are critical determinants of the final detected signal levels but that are excluded from the SpectraViewer comparison. The plot indicates that excitation of Alexa Fluor 568 at 488 nm is relatively inefficient (~5% of maximum indicated by the cursor). However, this consideration takes no account of the molar absorptivities (extinction coefficient, EC) of the fluorophores. The maximum EC values of Alexa Fluor 488 and Alexa Fluor 568 dyes are actually quite similar (75,000 cm-1 M-1 and 93,000 cm-1 M-1, respectively, as listed in the Molecular Probes Handbook data tables). An even more significant weighting factor is the relative abundances of the molecular targets of the fluorophores in the detector field of view; in general, cellular abundances of proteins vary over more orders of magnitude than the extinction coefficients of fluorophores. In the situation illustrated with excitation limited to 488 nm, the preferred course would be to use Alexa Fluor 568 to detect the more abundant of the two molecular targets and Alexa Fluor 488 to detect the less abundant target, thereby offsetting the absorptivity and target abundance factors.
Figure 3. Evaluating fluorophores for multiplex detection experiments: Emission spectra. The fluorescence emission spectra of Alexa Fluor 488 and Alexa Fluor 568 dyes are shown with the spectral characteristics of typical emission filters superimposed. In this case, the main practical concern is the extent of overspill of Alexa Fluor 488 fluorescence into the Alexa Fluor 568 detection channel (CWL = 645 nm, BP = 75 nm), which can lead to false indications of molecular target colocalization in imaging applications. As in the excitation spectra comparison (Figure 2), this peak-normalized overlay provides an initial assessment of the suitability of fluorophore combinations for multiplex detection, but weighting of fluorescence signals by the relative abundances of molecular targets and other factors will heavily influence the final experimentally observed outcome.