The broad excitation and emission spectral profiles exhibited by fluorescent proteins and their color-shifted genetic variants often require specialized considerations when designing live-cell imaging experiments using two or more of these unique probes simultaneously. Of primary concern are potential bleed-through artifacts resulting from the significant degree of emission spectral overlap usually exhibited by fluorescent protein combinations. This interactive tutorial explores matching fluorescent proteins for dual labeling investigations with regards to spectral bandwidth and overlap, excitation efficiency, emission window dimensions, and other parameters necessary to design logical experiments.
The tutorial initializes with the absorption and fluorescence emission spectral profiles from a useful combination of fluorescent proteins (CFP Cerulean and HcRed-Tandem) appearing in the Spectral Profiles window superimposed over the emission spectrum of a mercury arc-discharge lamp. Also included in the window upon initialization are the profiles of dichromatic mirrors appropriate for the fluorophores, as well as the suggested starting points for theEmission Filter Bandwidth profiles. The mouse cursor can be used to drag the grayscale emission filter bandwidth boundaries along the wavelength axis of the Spectral Profiles window to determine the optimum position. The twin slider combination or arrow buttons on the right-hand side of the control for each emission filter can be used to adjust the bandwidth, and the center wavelength can be altered using the left-hand arrow buttons. Dragging the left slider bar also translates the bandwidth profile across the graph, while dragging the right slider bar increases or decreases the bandwidth size. The current center wavelength and bandwidth values for the virtual emission filters are continuously updated and displayed in red numerals above the sliders. The percentage of bleed-through (if any) is continuously updated for each channel and displayed beneath the Spectral Profiles window.
The Dichromatic Mirror slider controls the cut-on wavelength curve position for these elements, and can be toggled between each channel using the color coded radio buttons adjacent to the slider. Likewise, the mercury lamp spectral profile can be turned off by deactivating the Excitation Source check box. Absorption (Ab) and emission (Em) spectral curves can be disabled by removing the checkmark from the boxes above the Illumination Sources yellow display window (default: Mercury Arc Lamp). When a laser source is chosen from the illumination pull-down menu, the excitation efficiency percentage based on the molar extinction coefficient at the laser wavelength is displayed in the green box above the illumination display window. Laser lines, which are drawn with colors approximating the wavelength in the Spectral Profiles window, can be deactivated by disabling the Excitation Source check box. In order to retain the current tutorial parameters (excitation and emission filter profiles displayed, etc.) while toggling through the list of probes or illumination sources, the Maintain State check box can be enabled. New fluorescent protein classes can be selected using the set of radio buttons to the right of the Spectral Profiles window along with the Choose A Probe pull-down menu.
The absorption and fluorescence emission spectra used in calculating the values for this tutorial were recorded under controlled conditions and are normalized for comparison and display purposes only. During actual fluorescence microscopy investigations, spectral profiles may vary due to environmental effects, such as pH, ionic concentration, and solvent polarity, as well as fluctuations in localized probe concentration, and therefore, may differ from those actually observed under experimental conditions.
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.