The Fluorescence SpectraViewer is an online tool that allows you to:

  • Assess the spectral compatibility of dyes and probes
  • Design and optimize fluorescence-based experiments for imaging, flow cytometry, or microplate reader instruments
  • Quantitate spectral overlap and determine adjustments needed to reduce spectral overlap
  • Save, share, export, or print experimental designs
     

Go to the SpectraViewer

How to use the fluorescence SpectraViewer

5 steps to use the SpectraViewer

Whether you are using imaging systems, flow cytometry, or microplate readers these 5 simple steps outline how to easily use the SpectraViewer for better fluorescence-based experimental design. The SpectraViewer has been designed to help all levels of experimental complexity, and can be relied upon for generating quality results.
 

Step 1—Instrument configuration

(A) Pre-loaded instrument configurations—instrument configurations have been pre-loaded into the SpectraViewer. These configurations can be accessed by selecting Options (upper right corner), open the Load, and select the instrument preset configuration. Additionally, saved configurations will be displayed in the Load folder.

(B) Manual configurations—if the instrument configuration is not available the Light Sources, Excitation and Emission filters can be entered manually. For Light Source, select Laser or Lamp and then select from the drop-down list or select custom, add wavelength, and name.

(C) Display—Laser and Emission Filter selections are displayed in the chart. After selection, the laser excitation is displayed on the plot.

(D) Light source—instead of a Laser, a Lamp can be added using the Light Source tab. After selection of the lamp, select the excitation and emission filters from the drop-down menus.

(E) Save configuration—if desired, the entered configuration can be saved. Select Options (upper right), from the drop-down menu select Save, name your configuration, and then save. The saved configurations can be accessed using the Load function also located in the Options tab.

Step 2—Fluorophore selection

(A) Adding a fluorophore—press the Add fluorophore button and begin typing the name of the fluorophore. Continue to type in the name of the dye or probe to shorten the list or scroll through the list that is displayed. After a dye or probe is selected it is displayed on the plot. Selecting the Ex (excitation), Em (emission) and Display on plot to the right allows the ability to control which spectra data to remove or display on the plot. The number of Fluorophores added to the SpectraViewer will be displayed by a number located to the left of Fluorophores.

(B) Additional fluorophores—continue to select additional fluorophores by using the Add fluorophore at the bottom of the right-hand column.

Step 3—Analysis and adjustments

(A) Selection of Laser to normalize spectra—under the Light Source function, select the Laser to normalize the spectra. To normalize the spectra to a specific laser, click on the circle under the Normalize column. If no laser is selected to normalize the spectra to and all emission spectra are adjusted to 100% Relatively Intensity. Toggle the lasers on and off the plot using the Show icons to the left of the Laser name.

(B)Initial analysis of experimental design—hover your pointer over the chart area and a vertical white line will be displayed. The line will display the wavelength selected (top of the white line or lower left corner of the chart). The white circles are used as visual clues and correspond to the percentages of excitation and emission for the various fluorophores displayed the chart.

Step 4—Detailed analysis and adjustment using the Spillover Table

(A) Spillover Table—the Spillover Table displays potential spectral overlapping issues and allows you to refine your experimental design. Each of the Lasers selected as Light Sources are displayed as tabs and all fluorophores used in the chart are displayed on the left. Next to the Fluorophores will be the Channel to be used to collect the emission signal. From the drop-down menu select the Channel to be used for each of the fluorophores.

(B) Adjusting spectra overlap—in the Spillover Table, the gray boxes are the percentage emission capture from targeted fluorophore using the specific laser and the emission filter or channel.

Outside of the gray boxes are the spillover or spectra overlap values. In the example, if the target fluorophore is the Alexa Fluor 488, using the Blue Laser and the GFP channel, 52.8% of the Alexa Fluor 488 emission spectra is captured. By reviewing the table horizontally and using the Blue Laser and the GFP channel or emission filter, the fluorescence emission from the GFP fluorophore is also captured with Alexa Fluor 488. In this example, the GFP signal is 76.1% of the Alexa Fluor 488 signal (the target for this channel).

Additionally, the amount of Alexa Fluor 488 spectral overlapping into the row's target fluorophore is displayed when reviewing the table vertically. In the example below the amount of the Alexa Fluor 488 emission signal using the RFP emission filter is captured from the Blue Laser. When using the Blue Laser and the RFP emission filter, the amount of Alexa Fluor 488 signal captured is 40.5% of the intended or Alexa Fluor 555 signal.

Changes to the fluorophores used can be made in the Spillover Table that result in changes in the Chart, resulting in an easy visual confirmation of the changes.
 

Step 5—Additional features

(A) Zoom—zoom-detailed analysis using the Zoom Function can be performed by hovering over the chat until the white line appears. Click and hold the left mouse button, move across the chart to highlight the area of interest, and then release the mouse button.

The highlighted area will be expanded. To reset the zoom, click on the button in the upper left of the chart.
 

(B) Separation of fluorophores or lasers—Fluorescence experiments using multiple adjacent or overlapping excitation and emission spectra can result in an additional level of complexity. To aid in the experimental design using adjacent or overlapping spectra a new feature has been added to the Fluorescence SpectraViewer. Included in the SpectraViewer is the ability to separate the fluorophores or lasers onto individual plots.

To access the Fluorophore or Laser Separation functionality, click on View full screen in the upper right corner.

Absorption and emission spectrum

After entering full screen, select the View button in the uppler left corner to access the drop down menu and then select either Separate lasers or Separate fluorophores. Selecting Separate fluorophores results in each fluorophore being separated on a single plot (see below).

Absorption and emission spectrum

To return to the normal view access the View drop down menu and select Default view. To return to the initial screen, which includes the fluorophore and instrument configurations, select Exit full screen.

(C) Reset—upon exiting, the last SpectraViewer working session will be saved. However, if you chose to reset the application to start over, select the reset button and select if you want to clear the Fluorophores, or the Instrument Configuration, or both. A notice that you are resetting the graph will be displayed.

(D) Settings—click to open and display of grid lines, labels, excitation plots, emission plots, light sources, filters, or plot normalization can be turned or turned off. To remove individual excitation or emission plots from display, use the selection features to the right of each configuration element.

(E) View Full Screen—expands the chart to cover the entire screen. Exit the Full Screen by using the Exit Full Screen button located in the upper right corner of the chart. As described later, the Full Screen can also be used to separate fluorophores or laser setting for detailed analysis.

(F) Expand Menu—the Light Source/Excitation Filter/Emission Filter menu is expanded to allow for visibility of multiple settings.

(G) Save and Select Products—the SpectraViewer configuration can be saved by clicking on the Save button and assigning a unique name. The plot can be loaded by using the Load function (described in Section A). The products used to generate the current SpectraViewer plot can be viewed by clicking on the Select Products tab. Note: the configurations are saved in your browser’s 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.

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