BD Fluorescence Spectrum Viewer A Multicolor Tool

How to use the BD Fluorescence Spectrum Viewer


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 may be disabled by unchecking Show Filter Trace.

Zooming in/out

To zoom in: Click and drag the left mouse button over the desired graph area. To return to the original view, double click the left mouse button over the graph background.

 Varying excitation wavelengths

The default Normalize Emission to Laser Selection preference is to display the normalized emission by the degree of excitation at the indicated laser line (wavelength). This may be varied dynamically by clicking and dragging the right mouse button (or Ctrl + left mouse button) over the excitation curves displayed on the plot. To view all spectra at 100% uncheck 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 efficiency for the primary detector filter (shown with light grey background within parentheses) is displayed along with that into the other detectors. The values correspond to normalized signal levels and may differ from cytometer spillover values, which are gain (PMT voltage)dependent.

Limitations: The representative fluorometer obtained data are from a variety of sources, and the curves are not necessarily predictive of all reagent conjugates, especially tandems conjugates such PE-Cy7. Additionally, keep in mind that not all spectral data were obtained from the same spectrofluorimeter (see below). There are two methods to estimate leakage:

% Measured
The spill estimate is the percent of that measured in the primary detector (varies with primary filter).
% Total
The spill estimate is the percent of the total (measured + unmeasured).

Important information

Band pass filters are specified by central wavelength and Full Width Half Maximum (FWHM), and are rendered by this applet as rectangles. As a result, band pass filters allow varying amounts 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 may vary. Long pass 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) that might be 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 infra-red. 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.