Using the Fluorescence SpectraViewer—Note 23.1

The Fluorescence SpectraViewer (thermofisher.com/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 and provides examples of its utility in the experimental design process.

Getting started

The SpectraViewer is built to be both intuitive and efficient, with the graphical image rendered dynamically as you sequentially work through the fields in the form below the plot. We encourage you to jump right in and select a fluorophore by simply beginning to type the name of the dye in the Fluorophores box and then choosing a specific dye from the options presented. The excitation (dotted line) and emission (solid line with filled area) will appear on the plot. Add a second dye using the Add another fluorophore option. There is no limit to the number of fluorophores that can be added, and a fluorophore can be removed by simply clicking on the X at the far right of the dye name. Figure 1 walks you through the general layout of the tool and describes the many display options available at each step.

Figure 1. Opening view and layout of the SpectraViewer.(A) Load, export, and reset tabs. (B) Display options. (C) The Y and X axes. (D) Fluorophore selection menu. (E) Display options for individual fluorophores. (F) Light source menu. (G) Excitation filters. (H) Emission filters. (I) Save and select products options.

A tour of the SpectraViewer layout

A. Load, export, and reset tabs

Load: The load tab enables a saved configuration or an instrument preset to be loaded into the SpectraViewer.
Export: The export tab allows a graphical image or spectral data for the selected fluorophores to be exported to print or saved to a file.
Reset: The last SpectraViewer working session is saved upon exiting. To remove this session and start over, select the reset button to clear the SpectraViewer configuration.

B. Display options

Click the gear icon to open Display options—grid lines, labels, excitation plots, emission plots, light sources, filters, and plot normalization—that can be toggled on or off. To remove individual excitation or emission plots from display, use the selection features to the right of each configuration element. (see D, F, G, and H below).

C. The Y and X axes

The 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. Fluorophore selection menu

To add a fluorophore, begin typing the name (any part of the name can be used) of the dye or probe into the Fluorophores selection box. Once you've typed the first letter, the tool shows a list of matching fluorophores based on the letter added. Continue to type the name of the dye or probe to refine the list, or scroll through the list that is displayed, and select a fluorophore. Choose Add another fluorophore if you want additional fluorophore spectra to be displayed on the same plot. An unlimited number of fluorophores can be displayed. Figure 2 shows a sample plot containing the spectra for DAPI, Alexa Fluor 488, and Alexa Fluor 647 fluorophores.

E. Display options for individual fluorophores

After a dye or probe is selected, its fluorescence spectra are displayed on the plot. Selecting Ex (excitation), Em (emission), and Display on plot to the right of the fluorophore name allows you to control which spectral data to display on or remove from the plot.

F. Light source menu

Based on your instrument, select either Add lasers or Add a lamp as the excitation source. After selecting Add lasers, open the drop-down menu (▼) and select from the list of available lasers; if an alternative laser configuration is required, select Custom and add the laser wavelength. Note that if an instrument has been selected using the preset configuration found in the Load tab (see A), the light sources and filters will be automatically loaded; however, additional light sources and filters can still be added.

After selection, the laser excitation is displayed on the plot (see vertical bars in Figure 3). To normalize the spectra to a specific laser, click on the light bulb icon to the right of the laser, and all emission spectra will then be adjusted to 100% relative intensity. To toggle on and off the display of the laser excitation on the plot, click on the eye icon to the right of the laser.

G. Excitation filters

The Excitation filters menu will only be visible if a lamp is selected as the light source. Upon selection of a lamp as the light source, the selection of excitation filters can proceed. Open the drop-down menu (▼) and scroll to find the preloaded excitation filters. If a custom filter is required, select Custom and supply the Wavelength and Bandwidth. The excitation filters are displayed on the plot and labeled with Ex:[filter name or number] (Figure 4).

H. Emission filters

The Emission filters menu is available when using a lamp (Figure 4) or a laser (Figure 5) as the light source. The selection of emission filters is the same as that of excitation filters. Open the drop-down menu (▼) and scroll to find the preloaded emission filters. If a custom filter is required, select Custom and supply the Wavelength and Bandwidth. The emission filters are displayed on the plot and labeled with Em:[filter name or number] (Figures 4 and 5). Note that if you are using a laser as the light source, the selected emission filters can be paired with the fluorophores to make the Spillover Table functional. In the Fluorophores selection box, there is an Emission target drop-down menu (▼) that allows you to assign an emission filter to the intended fluorophore.

I. Save and select products

The SpectraViewer configuration can be saved by clicking on the Save button and assigning a unique name; this plot can be recalled using the Load tab. Note that the configurations are saved in your browser cache and therefore will not be available on other browsers or other computers. If you delete your browser cache, your configurations will be deleted as well. The products used to generate the current SpectraViewer plot can be viewed by clicking on the Select Products tab.

Figure 2. Fluorescence spectra for the fluophores DAPI, Alexa Fluor 488, and Alexa Fluor 647. The fluorophores DAPI, Alexa Fluor 488, and Alexa Fluor 647 have been selected, and the excitation (dotted line) and emission (solid line with filled area) spectra for all three dyes are plotted on a single graph. Note that the white arrow represents the pointer location; the wavelength (in nm) and percentage intensity at the wavelength where the pointer is located can be reviewed in the lower lefthand corner of the plot.

Figure 3. The addition of three lasers to the three-fluorophore plot in Figure 2. Three lasers (UV, Blue, and Orange) have been selected from the laser drop-down menu (▼) and are displayed as vertical bars along with the excitation and emission spectra for the DAPI, Alexa Fluor 488, and Alexa Fluor 647 fluorophores. (A) The three emission spectra have been normalized to the UV laser. (B) Normalization to a laser has been turned off.

Figure 4.The addition of excitation and emission filters to the three-fluorophore plot in Figure 2. Excitation and emission filters have been selected to capture the fluorescence from the DAPI, Alexa Fluor 488, and Alexa Fluor 647 fluorophores. Excitation and Emission filters are labeled with Ex:fluorophore and Em:fluorophore, respectively, and displayed along with the three fluorescence spectra. In this case, the mercury lamp was selected as the light source.

Figure 5.The addition of emission filters to the three-fluorophore three-laser plot in Figure 3. Emission filters have been selected to capture the fluorescence from the DAPI, Alexa Fluor 488, and Alexa Fluor 647 fluorophores. Emission filters are labeled with Em:fluorophore and displayed along with the three fluorescence spectra and the three lasers (UV, Blue, and Orange, shown as vertical bars). Note that each of the fluorophores has been assigned an emission filter using the Emission target drop-down menu (▼).

Spillover table

In the spillover table, the gray boxes are the percentage emission capture from each fluorophore in the target emission filter, regardless of excitation value. Outside of the gray boxes are the spillover or spectra overlap values that do take level of excitation into account. In the example displayed in Figure 6A, use of the UV laser and the GFP emission filter will result in a DAPI spectra overlap of 572.1% (the DAPI fluorescent signal is 572.1% of the Alexa Fluor 488 fluorescent signal). However, Figure 6B demonstrates that use of the Blue laser and the GFP emission filter will result in no DAPI spectra overlap into the GFP channel.

Figure 6. Spillover tables for fluorophores DAPI, Alexa Fluor 488, and Alexa Fluor 647. The spillover tables for DAPI, Alexa Fluor 488, and Alexa Fluor 647 using laser excitation and assigned emission filters (DAPI, GFP, and Cy5, respectively). The spectral overlap or spillover resulting from using the (A) UV laser or (B) Blue laser are displayed.