BD Fluorescence Spectrum Viewer A Multicolor Tool

The Options menu

Explore the Options menu in the menu bar to select help, various display options, or to copy image data to the system clipboard.

For example: Where available, representative filter performance traces are displayed by default, but can be disabled by clearing Show Filter Trace.

Zooming in/out

To zoom in: Drag the left mouse button over the graph from left to right. To return to the original view, double click the left mouse button over the graph.

Varying excitation wavelengths

The default Normalize Emission to Laser Excitation preference displays the normalized emission by the degree of excitation at the indicated laser line (wavelength). This may be varied dynamically by dragging the right mouse button over the excitation curves displayed on the plot. To view all spectra at their 100% Ex max, clear Normalize Emission to Laser Selection in the Options menu.

Assessing filter performance

This applet estimates filter performance (leakage of fluorescence signal) by summing the normalized emission signal that falls within a filter specification by wavelength. For any given filter/fluorophore combination, click over a filter on the graph (where data permits) and the filter efficiency for the primary detector (shown bold within curly brackets { }) is displayed along with that of the other detectors. The values correspond to normalized signal levels and might differ from cytometer spillover values, which are gain (PMT voltage) dependent. Filter performance estimates from tandem fluorophores are indicated with an asterisk (*).

Limitations: The representative fluorometer data have been obtained from a variety of sources, and the curves are not necessarily predictive of all reagent conjugates, especially tandem conjugates such as PE-Cy™7. Additionally, not all spectral data were obtained from the same spectrofluorometer (see below). There are two methods to estimate leakage:

% Measured: The spill estimate is the percentage measured in the primary detector (varies with primary filter).

% Total: The spill estimate is the percentage of the total (measured + unmeasured).

Important information

This online tool is designed to aid customer understanding of important principles, dependencies, and relationships involved in selecting fluorochromes for multicolor flow cytometry. Users of this tool should ultimately rely on their own judgement when selecting fluorochromes for multicolor panels and experiments.

Bandpass filters are specified by central wavelength and Full Width Half Maximum (FWHM), and are rendered by this applet as rectangles. As a result, bandpass filters allow varying amounts of light to pass beyond the indicated extents until the blocking is complete. As a result, filter performance estimates from this applet and the actual spillover percentages used on cytometer data might vary. Longpass filters are shown over their emission range.

The normalized spectra presented here have been collected on various spectrofluorometers, and are not necessarily corrected for the photomultiplier tube (PMT) in your flow cytometer. Neither are they representative of the actual brightness of various fluorochromes. Actual brightness is a function of the Molar Extinction Coefficient of the dye (how much energy is absorbed), the quantum yield (photons out/photons in), quenching effects, the amount of the fluorochrome in the sample, and several other instrument detection efficiency factors such as light source power density.

The wavelength-dependent sensitivity of the spectrofluorometer PMT has a significant effect on the measured relative fluorescence intensities accross the spectrum. A typical spectrofluorometer has a "good" red sensitive PMT, but can often be more sensitive (approximately 3-fold or more depending on the spectrofluorometer) in the yellow than in the near infrared. This is especially relevant to evaluating signals from tandem dyes such as PE-Cy7, where the PE spillover component is typically not as strong as the emission curves might suggest, because the detector is more efficient in the PE region of the spectrum than in the Cy7 region of the spectrum.