Fluorescence Spectra Viewer User Guide

Windows Users
To use the Fluorescence Spectra Viewer, you will need a recent version of Java installed on your computer. You can download the necessary software from the Sun Microsystems website at http://java.com/en/download/manual.jsp

Macintosh Users
The Fluorescence Spectra Viewer is currently only supported on the Safari browser, which is included in Macintosh OS X and higher. Support for Internet Explorer, Mozilla, and Firefox browsers is currently in development.

Fluorophore
Select a fluorophore from the drop-down list. The fluorophores are listed in alphabetical order. The spectra for some fluorophores can change as a function of the environment—for example as a function of pH (BCECF), ions (fura-2), or solvent (Dapoxyl). Once the spectra have been displayed, the legend on the right-hand side of the Fluorescence Spectra Viewer shows additional information regarding the specific conditions used to generate the spectrum.

Excitation
Laser: Lasers can be chosen from a drop-down list. To add additional lasers, enter the wavelength into the "Filter" box but leave the "Bandpass" box blank, and hit your Enter/Return key. Laser wavelengths will be indicated above the spectra.

Filter / Bandpass: For bandpass filters, enter the midpoint wavelength of the excitation filter into the "Filter" box, and the total bandwidth of the filter into the "Bandpass" box. For example, for a 530 nm filter with a bandwidth of 30 nm, enter 530 into the first box and 30 into the second, and hit your Enter/Return key. The filter attributes will be indicated above the spectra.

Emission
Emission filters can provide either longpass or bandpass wavelength transmission.

For bandpass transmission, enter the midpoint wavelength of the emission filter into the "Filter" box, and the total bandwidth of the filter into the "Bandpass" box. For example, for a 630 nm filter with a bandwidth of 30 nm, enter 630 into the first box and 30 into the second, and hit your Enter/Return key. The filter range will be superimposed on the spectra. Click once within the colored emission band to see a text box that displays the relative percentage of the emission spectrum of each fluorophore that lies within the band.

For longpass transmission, enter into the "Filter" box the wavelength at which the emission filter begins to transmit light, and hit your Enter/Return key. Leave the "Bandpass" box blank. The transmission range will be superimposed on the spectra. Click once within the colored emission area to see a text box that displays the relative percentage of the emission spectrum of each fluorophore that lies within the area.

Toggle Excitation Display
Click to add or remove all excitation spectra.

Toggle Emission Display
Click to add or remove all emission spectra.

Clear All
Click to clear all selections and graphs and reset the laser wavelength to 488 nm (default).

Axis Values
On the x-axis, the minimum wavelength is 297 nm and the maximum wavelength is 915 nm. On the y-axis, excitation and emission are expressed as a percentage of the maximum value for each under the defined conditions described in the legend.

As you move your cursor over the graph, the x and y values are displayed in the upper right-hand corner of the Fluorescence Spectra Viewer.

Normalized Spectra
The spectra have been normalized to 100% using the maximum excitation and emission for each dye.

Changing the Viewing Scale of the X-Axis
Click and drag the cursor to draw a box around a region of the graph that you want to expand (the graph will expand with respect to the x-axis only). To return to the default view, double-click anywhere within the expanded graph.

The Fluorescence Spectra Viewer can provide relative information on the suitability of a fluorophore for your instrumentation. Please note that the data provided by the Fluorescence Spectra Viewer are not quantitative. When investigating various fluorophores, it is important to take into consideration other parameters that lie outside the scope of the data generated by the Fluorescence Spectra Viewer. What is the amount of fluorescence expected by the fluorophore? Is the fluorophore being used to detect a low-abundance target? What is the relative power of the excitation source? Answers to these questions will help you determine whether or not the fluorophore can perform, and, to a certain extent how well one fluorophore may perform relative to another. You will still need to test the actual performance in your system.

Assessment of Fluorophore Compatibility
Overlay any number of dye spectra with your specific instrument values—the Fluorescence Spectra Viewer will help you choose the dyes that are most compatible with each other and your instrumentation. Please note that because the fluorescence spectra are normalized, the relative fluorescence units for a given amount of dye may not be equivalent. Please contact probestech@invitrogen.com for additional information on specific dyes and dye recommendations.

Windows Users

1. Capture the entire screen using the Print Screen key, sometimes abbreviated to Prnt Scrn on some keyboards. Alternately, the contents of the current window can be captured by holding down the Alt key, then hitting the Print Screen key.
2. Paste the resulting screen-shot from the clipboard into the application of your choice using the keyboard combination Ctrl+V. If you have a graphics program this is the best choice, but other applications like Word or Paint (which comes with Windows by default) will work.
3. Once the screen-shot has been pasted into the application, you can use the normal print functionality in that application to print the Spectra. You may also crop the image to only include the spectra and not the entire screen or browser window prior to printing.

Macintosh Users

1. Capture the entire screen using Shift+Apple+3 or capture a selected area using Shift+Apple+4.
2. The entire screen or selected area is captured by default to a PDF document located on your desktop.
3. Open the PDF using Acrobat Reader or application of your choice and print from within that application.