In planning multiple label fluorescence staining protocols for widefield and laser scanning confocal fluorescence microscopy experiments, the judicious choice of probes is paramount in obtaining the best target signal while simultaneously minimizing bleed-through artifacts. This interactive tutorial is designed to explore the matching of dual fluorophores with efficient laser excitation lines, calculation of emission spectral overlap values, and determination of the approximate bleed-through level that can be expected as a function of the detection window wavelength profiles.

The tutorial initializes with the absorption and emission spectra of two commonly matched fluorophores, fluorescein and tetramethylrhodamine, appearing on the Spectral Profiles graph. Superimposed over the absorption spectra of the fluorophores are the 488-nanometer and 543-nanometer laser spectral lines from the argon-ion and helium-neon lasers selected in the Choose A Laser pull-down menu. The overlap region between the emission spectra of the two probes is indicated by a dark gray area where the overlap occurs, and the percentage is indicated in a yellow box positioned beneath the Spectral Profiles graph. Two independent channels (Channel 1 and Channel 2) are used to define specific fluorophores, laser spectral lines, and emission filter bandwidths in the tutorial.

The absorption and fluorescence emission spectra illustrated in this tutorial were recorded under controlled conditions and are normalized for comparison and display purposes. In actual fluorescence microscopy investigations, spectral profiles may differ due to differences in the extinction coefficient, quantum yield, probe concentration, and variations in the localized environment.

In order to operate the tutorial, first select a probe class for each channel using the radio buttons in the upper right-hand portion of the tutorial. Classes include Alexa Fluors, cyanine dyes, tracker dyes (MitoTracker and LysoTracker), SYTO nucleic acid probes, fluorescent proteins, quantum dots, Spectrum dyes, BODIPY probes, monomeric and dimeric cyanine nucleic acid stains, and conventional dyes. The tutorial database contains absorption and emission spectral profiles from over 250 individual fluorophores distributed among these classes. After the probe class has been chosen, use the Choose A Dye pull-down menu to select a dye for each channel. In the pull-down menu, the dyes available from each class are limited to those that will be efficiently excited by the selected laser, which is indicated in a yellow box appearing above the Choose A Laser pull-down menu.

The fluorophore spectra and laser line for each channel can be toggled on an off together using the Channel checkbox, and individual spectral profiles can also be similarly controlled with Ex and Em checkboxes. Note that the peak absorption and emission wavelengths for the selected fluorophores are displayed above these checkboxes. The excitation efficiency of the target laser for each channel is indicated in a green box appearing to the left of the spectral display control checkboxes. If crossover excitation occurs from the laser in the adjacent channel, the percentage is displayed in the red box (Crossover) to the right of the spectral display control checkboxes. Parameters for both channels can be controlled in tandem using the Excitation Curves, Laser Lines, and Emission Curves checkboxes on the right-hand side of the tutorial. The Maintain State checkbox is designed to maintain the current tutorial settings when selecting a new fluorophores or lasers.

The Emission Filter Bandwidth control panel sliders are used to control the spectral bandwidth window size and wavelength region of virtual emission filters (or slit widths) for the channel detectors. For example, the default values for channel one are a 30-nanometer bandpass region starting at 504 nanometers and ending at 534 nanometers. The emission filter for each channel can be activated or deactivated independently using the checkboxes within the control panel. Activated emission filters appear in semi-transparent grayscale regions superimposed over the fluorophore spectral profiles. The grayscale filter windows can be translated back and forth in the Spectral Profile window by dragging with the mouse cursor.